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Trastuzumab reverses letrozole resistance and amplifies the sensitivity of breast cancer cells to estrogen

pmcid: 2644349

doi: 10.1158/0008-5472.CAN-08-0857

abstract: In this study we investigated adaptive mechanisms associated with aromatase inhibitor (AI) resistance in breast cancer cells and show that sensitivity to AIs can be extended through dual inhibition of Estrogen Receptor (ER) and Human Epidermal Receptor-2 (Her-2) signaling. We utilized human ER-positive breast cancer cells stably transfected with the aromatase gene (MCF-7Ca). These cells grow as tumors in nude mice and are inhibited by AIs. Despite continued treatment, tumors eventually become insensitive to AI letrozole. The cells isolated from these Long-Term Letrozole Treated tumors (LTLT-Ca) were found to have decreased ERα levels. Our results suggest that LTLT-Ca cells survive estrogen deprivation by activation of Her-2/Mitogen Activated Protein Kinase (MAPK) pathway. Here, we demonstrate that trastuzumab (antibody against Her-2; IC50=0.4mg/ml) was very effective in restoring the ERα levels and sensitivity of LTLT-Ca cells to endocrine therapy by down-regulation of Her-2/MAPK pathway and upregulation of ERα. In contrast, trastuzumab was ineffective in the parental hormone responsive MCF-7Ca cells (IC50=4.28mg/ml) and xenografts. By blocking Her-2, trastuzumab also up-regulates ERα, aromatase expression and hyper-sensitized MCF-7Ca cells to E2. We show that trastuzumab is beneficial in hormone refractory cells and xenografts by restoring ER, implicating Her-2 as a negative regulator of ERα. In xenograft studies the combination of trastuzumab plus letrozole is equally effective in inhibiting growth of MCF-7Ca tumors as letrozole alone. However, upon the acquisition of resistance and increased Her-2 expression the combination of letrozole plus trastuzumab provided superior benefit over letrozole or trastuzumab alone.

pmcid: 2644349

Figure 1C - LTLT-Ca xenografts were grown in female OVX nude mice as described in Materials and Methods. The mice in the control and letrozole treated group exhibited similar rate of tumor growth. The difference in the exponential parameter governing growth was -0.027 (p = 0.71) over first eight weeks and 0.001 (p value = 0.97) over 19 weeks. Four doses of trastuzumab were tested. All of the tested doses caused a marked regression of LTLT-Ca xenografts. The growth rate of tumors of mice treated with trastuzumab (5mg/kg/week) was significantly different from mice in the control (p =0.0008) and letrozole treated (p = 0.0002) mice. The difference in the exponential parameter governing growth between control and trastuzumab was 0.24 (p = 0.0008). The difference in the exponential parameter governing growth between letrozole and trastuzumab was 0.27 (p value = 0.0002).

The ErbB inhibitors, trastuzumab and erlotinib, inhibit growth of vestibular schwannoma xenografts in nude micea preliminary study

pmcid: 2652856

doi: 10.1097/MAO.0b013e31817f7398

abstract: ObjectiveTo determine the ability of ErbB inhibitors to reduce the growth of vestibular schwannoma (VS) xenografts.MethodsVS xenografts were established in the interscapular fat pad in nude mice for 4 weeks. Initially, a small cohort of animals was treated with the ErbB2 inhibitor, trastuzumab, or saline for 2 weeks. Animals also received BrdU injections to label proliferating cells. In a longer-term experiment, animals were randomized to receive trastuzumab, erlotinib (an ErbB kinase inhibitor), or placebo for 12 weeks. Tumor growth was monitored by magnetic resonance imaging (MRI) over the treatment period. Cell death was analyzed by terminal dUTP nick end labeling (TUNEL).ResultsTumors could be distinguished with T2 weighted MRI sequences. Trastuzumab significantly reduced the proliferation of VS cells compared to control (p<0.01) as determined by BrdU uptake. Control tumors demonstrated slight growth over the 12 week treatment period. Both trastuzumab and erlotinib significantly reduced the growth of VS xenografts (p<0.05). Erlotinib, but not trastuzumab, resulted in a significant increase in the percent of TUNEL-positive VS cells (p<0.01).ConclusionsIn this preliminary study, the ErbB inhibitors trastuzumab and erlotinib decreased growth of VS xenografts in nude mice raising the possibility of using ErbB inhibitors in the management of patients with schwannomas, particularly those with neurofibromatosis type 2.

pmcid: 2652856

Figure 3 - Trastuzumab and Erlotinib reduce the growth of VS xenografts. A. The change in relative tumor volume was determined by subtracting the initial tumor volume (Vi) from the final tumor volume (Vf) and dividing the difference by the initial tumor volume according to the formula: (Vf-Vi)/Vi and is plotted for the 12 week interval for each xenograft. Dashed lines in control group represent animals receiving saline while solid lines represent animals receiving erlotinib vehicle. B. The average relative growth of the xenografts for each condition is plotted.. Differences among the means for the treatment groups were determined by one-way ANOVA followed by a Kruskal-Wallis test. Error bars represent standard error. Both trastuzumab (p=0.025) and erlotinib (p=0.017) reduced the growth of the VS xenografts compared with controls.

Specific Blockade of VEGF and HER2 Pathways Results in Greater Growth Inhibition of Breast Cancer Xenografts that Overexpress HER2*

pmcid: 2757147

doi:

abstract: We have previously reported that breast cancer cells which overexpress HER2 produce higher levels of VEGF than cells with low levels of HER2. This study tested the hypothesis that dual targeting of the VEGF (with VEGF-Trap) and HER2 (with trastuzumab) pathways would result in greater growth inhibition of HER2-overexpressing breast cancer xenografts than either agent alone. In this study we found that human and murine endothelial cells expressed high levels of VEGF receptors (VEGFR1, VEGFR2, & VEGFR3). VEGF-Trap decreased levels of secreted VEGF derived from both human and murine cells and effectively blocked VEGF-induced tyrosine phosphorylation of VEGFR2. VEGF-Trap as a single treatment inhibited tumor microvessel density (MVD), tumor vasculature, cell proliferation, and tumor growth of BT474 xenografts in a dose-dependent manner from 2.5 mg/kg to 25 mg/kg. VEGF-Trap decreased levels of both human VEGF and PlGF protein in vivo. Trastuzumab as a single agent effectively inhibited BT474 tumor growth in a dose-dependent manner, associated with a decrease in human VEGF, tumor MVD and tumor cell proliferation. Treatment with a combination of VEGF-Trap (2.5-10 mg/kg) and trastuzumab (1 mg/kg) produced significantly greater inhibition of BT474tumor growth than either individual agent, associated with greater inhibition of tumor MVD and tumor cell proliferation. Thus, VEGF-Trap in combination with trastuzumab produces superior growth inhibition of tumor xenografts which overexpress HER2, which may result from inhibition of both tumor angiogenesis and proliferation. Similar mechanisms may contribute to the clinical anti-tumor activity of trastuzumab in combination with inhibitors of VEGF signaling pathway in women with breast cancers which overexpress HER2.

pmcid: 2757147

Fig. 6 - (A), Growth inhibition of BT474 cells by trastuzumab in vitro. BT474 cells were seeded in 96-well plates in triplicate and treated for 72 hrs with different concentrations of trastuzumab as indicated. A crystal violet assay was used to assess the cell viability. *, p < 0.05, compared to the untreated control. (B), Trastuzumab suppresses tumor growth of BT474 breast cancer xenografts in vivo. BT474 xenografts were established in nu/nu mice as described in Materials and Methods. Mice were treated intraperitoneally twice a week with different doses of trastuzumab (1 and 10 mg/kg) or control hIgG (10 mg/kg). *, p < 0.05, versus the hIgG control at day 47. **, p < 0.01, versus the hIgG control at day 47. (C), Measurement of human-specific VEGF isoforms by QRTPCR in samples from in vitro cell culture (upper panel) and from in vivo BT474 xenografts (lower panel). *, P < 0.05, versus control hIgG. (D) - (F), CD31 and Ki67 staining in xenograft tumors. CD31 and Ki67 IHC staining (D) were performed as described in Materials and Methods. CD31- (E) and Ki67- (F) positive cells were counted in hFc- and VEGF-Trap-treated tumors. An asterisk denotes p = 0.01. ** denotes p = 0.02.

pmcid: 2757147

Fig. 7 - (A), Trastuzumab in combination with VEGF-Trap suppresses tumor growth of BT474 xenografts more effectively than either agent used alone. Mice were treated twice a week 1) intraperitoneally with trastuzumab (1 mg/kg) alone, 2) subcutaneously with VEGF-Trap (2.5mg/kg) alone, 3) with a combination of trastuzumab and VEGF-Trap, 4) intraperitoneally with hIgG (10 mg/kg) or 5) subcutaneously with hFc (25 mg/kg). * denotes combination versus trastuzumab alone or VEGF-Trap alone (p= 0.02). § denotes combination versus control hIgG or control hFc (p= 0.008). (B), HE, CD31 and Ki67 staining in xenograft tumors. (C), Quantitation of CD31-positive cells in tumors. An asterisk denotes p = 0.01. ** denotes p = 0.005. (D), Quantitation of Ki67-positive cells in tumors. An asterisk denotes p < 0.05. ** denotes p = 0.008.

Inhibition of mTOR is required for optimal antitumor effect of HER2 inhibitors against HER2-overexpressing cancer cells

pmcid: 2787848

doi: 10.1158/1078-0432.CCR-09-1665

abstract: PurposeA significant fraction of HER2-overexpressing breast cancers exhibit resistance to the HER2 antibody trastuzumab. Hyperactivity of the phosphatidylinositol-3 kinase (PI3K)/AKT pathway confers trastuzumab resistance, and mTOR is a major downstream effector of PI3K/AKT. Therefore, we examined whether mTOR inhibitors synergize with trastuzumab.Experimental DesignImmunocompetent mice bearing HER2-positive mammary tumors were treated with trastuzumab, the mTOR inhibitor rapamycin, or the combination. Mice were imaged for tumor cell death using an optical Annexin-V probe and with [18F]FDG-PET. The signaling and growth effects of the mTOR inhibitor RAD001 on HER2+ cells treated with trastuzumab or lapatinib were evaluated.ResultsTreatment of mice with trastuzumab plus rapamycin was more effective than single-agent treatments, inducing complete regression of 26/26 tumors. The combination induced tumor cell death (Annexin-V binding) and inhibited FDG uptake. Rapamycin inhibited mTOR and tumor cell proliferation as determined by phospho-S6 and Ki67 immunohistochemistry, respectively. In culture, the combination of RAD001 plus trastuzumab inhibited cell growth more effectively than either drug alone. Trastuzumab partially decreased PI3K but not mTOR activity. Knockdown of TSC2 resulted in HER2-independent activation of mTOR and dampened the response to trastuzumab and lapatinib. Treatment with the HER2 inhibitor lapatinib decreased phospho-S6 and growth in TSC2-expressing but not in TSC2-knockdown cells.ConclusionsInhibition of PI3K and mTOR are required for the growth inhibitory effect of HER2 antagonists. These findings collectively support the combined use of trastuzumab and mTOR inhibitors for the treatment of HER2+ breast cancer.

pmcid: 2787848

Fig. 1 - Trastuzumab inhibits MMTV/HER2 tumor growth. Three groups of mice (A, B, and C) bearing MMTV/HER2 tumors derived from three transgenic donors were randomized to Tz or PBS when tumor volume reached ≥200 mm3. Average volume ± SEM is shown. *p<0.05 by Mann-Whitney U-test comparing Tz and PBS groups at each time point.

pmcid: 2787848

Fig. 2 - Rapamycin synergizes with trastuzumab to induce complete tumor regression. Two groups of MMTV/HER2 tumor-bearing mice (D and E) were randomized to Tz, rapamycin, or the combination once tumor volume reached ≥400 mm3 (A,B) or ≥200 mm3 (C,D). A,C) Average volume ± SEM is shown. *p<0.05, ♏p=0.052 by Mann-Whitney U-test comparing Tz and combination groups at each time point. #p<0.05 by all three Mann-Whitney U-tests comparing vehicle with each other treatment group. B,D) Tumor volume for each mouse after 2.5 weeks (B shows group D) and 2 weeks (D shows group E) of treatment is shown. Bars indicate average volumes. n0 = number of mice with tumors that completely regressed. Numbers of animals decreased slightly at later time points due to excessive tumor burden.

Comparative Impact of Trastuzumab and Cyclophosphamide on HER-2–Positive Human Breast Cancer Xenografts

pmcid: 2788792

doi: 10.1158/1078-0432.CCR-09-0931

abstract: PurposeMetronomic chemotherapy is a minimally toxic and frequently effective new treatment strategy that is beginning to show promising phase II clinical trial results, particularly for metastatic breast cancer when combined with various molecularly targeted antitumor agents. Here, we assessed a treatment strategy that uses trastuzumab plus daily oral metronomic cyclophosphamide on metastatic Her-2–positive human breast cancer models.Experimental DesignTreatments were initiated on orthotopic transplanted primary tumors as well as established visceral metastatic disease of two independent Her-2–positive breast cancer models, both independently derived from the human MDA-MB-231 breast cancer cell line. Outcome was assessed by noninvasive measurements of tumor cell–secreted human choriogonadotropin in the urine as a surrogate marker of relative tumor burden, or by whole body bioluminescent imaging, in addition to prolongation of survival.ResultsOrthotopic primary tumors responded to trastuzumab monotherapy with significant growth delays, whereas minimal antitumor effect was observed when mice with metastatic disease were treated. Nevertheless, trastuzumab showed a benefit in this latter setting when combined with metronomic low-dose cyclophosphamide as assessed by prolongation of survival. This benefit was similar to trastuzumab plus maximum tolerated dose cyclophosphamide, but was associated with lesser toxicity.ConclusionsTrastuzumab combined with metronomic cyclophosphamide may be an effective long-term maintenance strategy for the treatment of Her-2–positive metastatic breast cancer.

pmcid: 2788792

Fig. 1 - Impact of concurrent combination of trastuzumab-chemotherapy (CTX) therapy regimens on orthotopic primary H2N tumors in SCID mice. When tumors reached 250 mm3, mice were treated with saline (control, n = 3) or metronomic low-dose (Ld) CTX plus trastuzumab (Ld CTX + trastuzumab, n = 8), or with a MTD CTX plus trastuzumab (MTD CTX+ trastuzumab, where MTD CTX is 210 mg/kg every 21 d, n = 5). Top, tumor volume; bottom, mouse weights. Arrows, each MTD cycle (i.e., every 21 d). *, one mouse in each group had to be sacrificed at the indicated point. Trastuzumab was given twice weekly at 20 mg/kg i.p., and metronomic low-dose CTX was ~20 mg/kg/d.

pmcid: 2788792

Fig. 3 - A, effect on tumor volume (top) and hCG levels (bottom) of the different therapies on met2 orthotopically implanted primary tumors. When tumors reached 250 mm3, mice were treated with saline (control, n = 5), or trastuzumab alone (n = 6), or low-dose metronomic CTX alone (Ld CTX, n = 6) or the combination of trastuzumab plus low-dose metronomic CTX (Ld CTX+ trastuzumab, n = 6). The curves show that, similar to H2N tumors (see Fig. 1), the met2 line gave rise to primary tumors that are highly responsive to the low-dose metronomic CTX + trastuzumab combination therapy. The urine hCG values (corresponding to pooled urine hCG for each group, normalized to urine Creatinine levels) were found to be concordant with tumor volume measurements. B, treatment of met2 metastases and assessment of mouse weights. Orthotopically implanted met2 tumors were surgically removed, and 3 wk later, various therapies were initiated. Mice were treated with MTD CTX alone (MTD, n = 6) or low-dose CTX alone (n = 6) or control saline (n = 6), or trastuzumab alone (n = 6). In addition, other mice were given trastuzumab plus metronomic CTX (n = 12), or trastuzumab plus MTD CTX (n = 12; arrows, MTD dosing). Weight loss on day 115 for MTD therapies was significantly different (P < 0.05) versus the other regimens. Trastuzumab was given twice weekly at 20 mg/kg i.p., and low-dose metronomic CTX alone was ~20 mg/kg/d. C, top, hCG curves of met2 metastases treated with the regimens indicated. Note at the start of therapy (day 24), the low hCG levels made it difficult to accurately normalize the groups. As a consequence, some groups (e.g., low-dose metronomic CTX alone, see days 25–50) subsequently turned out to have relatively low hCG levels. Nonetheless, sequential hCG measurements showed controls and the monotherapies to rapidly increase after a lag phase (particularly long for the low-dose metronomic CTX group). In contrast, mice treated with the combination therapies did not show increases in hCG readings for 3 mo after treatment was initiated. The combination of MTD CTX + trastuzumab showed a decrease in hCG levels suggesting this approach to be the most effective therapy. Low-dose metronomic CTX + trastuzumab showed unchanged hCG levels for the first 3 mo of treatment, followed by an increase thereafter. Boxed graph, the same data with expanded Y-axis and only showing the hCG curves for the combination therapies for ease of comparison. Bottom, graph showing the same data on the full range of detected hCG values (i.e., 0–500), indicating that at the time of sacrifice monotherapies had 100-fold higher hCG burden than the ongoing combination therapies. Note that the data are of pooled urine hCG, normalized to creatinine levels. D, corresponding survival curve of therapy experiment for met2 metastases (see C) showing the impact of MTD CTX+ trastuzumab (P = 0.03) in this model, relative to the other regimens tested. MTD CTX is 210 mg/kg every 21 d. Trastuzumab was given twice weekly at 20 mg/kg i.p., and Ld CTX was ~20 mg/kg/d.

pmcid: 2788792

Fig. 3 - A, effect on tumor volume (top) and hCG levels (bottom) of the different therapies on met2 orthotopically implanted primary tumors. When tumors reached 250 mm3, mice were treated with saline (control, n = 5), or trastuzumab alone (n = 6), or low-dose metronomic CTX alone (Ld CTX, n = 6) or the combination of trastuzumab plus low-dose metronomic CTX (Ld CTX+ trastuzumab, n = 6). The curves show that, similar to H2N tumors (see Fig. 1), the met2 line gave rise to primary tumors that are highly responsive to the low-dose metronomic CTX + trastuzumab combination therapy. The urine hCG values (corresponding to pooled urine hCG for each group, normalized to urine Creatinine levels) were found to be concordant with tumor volume measurements. B, treatment of met2 metastases and assessment of mouse weights. Orthotopically implanted met2 tumors were surgically removed, and 3 wk later, various therapies were initiated. Mice were treated with MTD CTX alone (MTD, n = 6) or low-dose CTX alone (n = 6) or control saline (n = 6), or trastuzumab alone (n = 6). In addition, other mice were given trastuzumab plus metronomic CTX (n = 12), or trastuzumab plus MTD CTX (n = 12; arrows, MTD dosing). Weight loss on day 115 for MTD therapies was significantly different (P < 0.05) versus the other regimens. Trastuzumab was given twice weekly at 20 mg/kg i.p., and low-dose metronomic CTX alone was ~20 mg/kg/d. C, top, hCG curves of met2 metastases treated with the regimens indicated. Note at the start of therapy (day 24), the low hCG levels made it difficult to accurately normalize the groups. As a consequence, some groups (e.g., low-dose metronomic CTX alone, see days 25–50) subsequently turned out to have relatively low hCG levels. Nonetheless, sequential hCG measurements showed controls and the monotherapies to rapidly increase after a lag phase (particularly long for the low-dose metronomic CTX group). In contrast, mice treated with the combination therapies did not show increases in hCG readings for 3 mo after treatment was initiated. The combination of MTD CTX + trastuzumab showed a decrease in hCG levels suggesting this approach to be the most effective therapy. Low-dose metronomic CTX + trastuzumab showed unchanged hCG levels for the first 3 mo of treatment, followed by an increase thereafter. Boxed graph, the same data with expanded Y-axis and only showing the hCG curves for the combination therapies for ease of comparison. Bottom, graph showing the same data on the full range of detected hCG values (i.e., 0–500), indicating that at the time of sacrifice monotherapies had 100-fold higher hCG burden than the ongoing combination therapies. Note that the data are of pooled urine hCG, normalized to creatinine levels. D, corresponding survival curve of therapy experiment for met2 metastases (see C) showing the impact of MTD CTX+ trastuzumab (P = 0.03) in this model, relative to the other regimens tested. MTD CTX is 210 mg/kg every 21 d. Trastuzumab was given twice weekly at 20 mg/kg i.p., and Ld CTX was ~20 mg/kg/d.

Augmented inhibition of angiogenesis by combination of HER2 antibody chA21 and trastuzumab in human ovarian carcinoma xenograft

pmcid: 2939608

doi: 10.1186/1757-2215-3-20

abstract: BackgroundchA21 is a novel tumor-inhibitory antibody which recognized subdomain I of HER2 extracellular domain with an epitope distinct from other HER2 antibodies. Previously, we demonstrated that chA21 inhibits human ovarian carcinoma cell line SKOV-3 growth in vitro and in vivo study. In this study, we further investigated the anti-angiogenic efficacy combination of chA21 with trastuzumab in SKOV-3 xenograft model.MethodsNude mice were s.c. challenged with SKOV-3 cells and received treatment of chA21 alone, trastuzumab alone or both antibodies together twice a week for 21 days. Tumor volume and microvessel density (MVD) were evaluated. The effect of chA21 plus trastuzumab treament on vascular endothelial growth factor (VEGF) secretion, endothelial cells proliferation and migration, and the status of HER2 downstream pathway AKT/phosphorylated AKT (pAKT) were evaluated in vitro.ResultsIn vivo study combination of chA21 with trastuzumab resulted in reduce tumor growth and angiogenesis than each monotherapy. In vitro study, the combination of chA21 with trastuzumab inhibits VEGF secretion, endothelial cells proliferation and migration. Furthermore, the combination treatment inhibits pAKT expression.ConclusionOur findings suggested that the combination of chA21 with trastuzumab can cause augmented inhibition of angiogenesis in SKOV-3 xenograft model. Inhibition of agniogenesis may through suppression of AKT pathway. The therapeutic benefits of combination chA21 with trastuzumab warrant further study in an attempt to make the translation into the clinic.

pmcid: 2939608

Figure 1 - The tumor volume and the weight of SKOV-3 xenograft in the different treatment groups. (A) Either chA21 or trastuzumab treatment cause a marked growth inhibition in SKOV-3 xenograft compared with the control (P < 0.01), and the combination of chA21 with trastuzumab treatment induced a more efficient efficacy than the each antibody alone (P < 0.05). (B) When the experiment ended, all tumors were removed and weighted. Results are representative of the mean ± SD of 8 animals in each group. *, P < 0.01 compared with control. **, P < 0.01 compared with chA21 or trastuzumab alone.

Cyclin E amplification/overexpression is a mechanism of trastuzumab resistance in HER2+ breast cancer patients

pmcid: 3048107

doi: 10.1073/pnas.1014835108

abstract: Clinical benefits from trastuzumab and other anti-HER2 therapies in patients with HER2 amplified breast cancer remain limited by primary or acquired resistance. To identify potential mechanisms of resistance, we established trastuzumab-resistant HER2 amplified breast cancer cells by chronic exposure to trastuzumab treatment. Genomewide copy-number variation analyses of the resistant cells compared with parental cells revealed a focal amplification of genomic DNA containing the cyclin E gene. In a cohort of 34 HER2+ patients treated with trastuzumab-based therapy, we found that cyclin E amplification/overexpression was associated with a worse clinical benefit (33.3% compared with 87.5%, P < 0.02) and a lower progression-free survival (6 mo vs. 14 mo, P < 0.002) compared with nonoverexpressing cyclin E tumors. To dissect the potential role of cyclin E in trastuzumab resistance, we studied the effects of cyclin E overexpression and cyclin E suppression. Cyclin E overexpression resulted in resistance to trastuzumab both in vitro and in vivo. Inhibition of cyclin E activity in cyclin E-amplified trastuzumab resistant clones, either by knockdown of cyclin E expression or treatment with cyclin-dependent kinase 2 (CDK2) inhibitors, led to a dramatic decrease in proliferation and enhanced apoptosis. In vivo, CDK2 inhibition significantly reduced tumor growth of trastuzumab-resistant xenografts. Our findings point to a causative role for cyclin E overexpression and the consequent increase in CDK2 activity in trastuzumab resistance and suggest that treatment with CDK2 inhibitors may be a valid strategy in patients with breast tumors with HER2 and cyclin E coamplification/overexpression.

pmcid: 3048107

Fig. 4. - BT474 and BT474R sensitivity to CDK2 inhibition. (A) Quantification of crystal violet staining of BT474 or BT474R cells treated for 8 d with trastuzumab, CYC065, or the combination (T+C) at the indicated concentrations. Proliferation assays were performed in triplicate. (B) Western blot analysis of BT474 parental and resistant cell lines exposed to trastuzumab, CYC065, or the combination for 48 h at the indicated concentrations. Whole-cell extracts were analyzed with the indicated antibodies. Similar results were obtained comparing BT474 with BT474R2. (C) Cell death of BT474 or BT474R cells as delineated by sub-G1 population following treatment of trastuzumab, CYC065, or the combination for 48 h at the indicated concentrations. Similar results were obtained comparing BT474 with BT474R2. (D) Cell death of BT474 or BT474R cells as delineated by annexin V staining following treatment of trastuzumab, CYC065, or the combination for 48 h at the indicated concentrations. Similar results were obtained comparing BT474 with BT474R2. (E) Tumor growth inhibition in response to trastuzumab, CYC065, and the combination of the two agents. Student's t test was used to compare tumor sizes between the groups and data are expressed as mean ± SE *P = 0.0019 vs. trastuzumab; **P = 0.00085 vs. trastuzumab on day 12. The experiment was repeated two times with similar results.

Controlled Extracellular Matrix Degradation in Breast Cancer Tumors Improves Therapy by TrastuzumabStem Cell Gene Therapy of Breast Cancer

pmcid: 3048180

doi: 10.1038/mt.2010.256

abstract: Extracellular matrix (ECM) in solid tumors affects the effectiveness of therapeutics through blocking of intratumoral diffusion and/or physical masking of target receptors on malignant cells. In immunohistochemical studies of tumor sections from breast cancer patients and xenografts, we observed colocalization of ECM proteins and Her2/neu, a tumor-associated antigen that is the target for the widely used monoclonal antibody trastuzumab (Herceptin). We tested whether intratumoral expression of the peptide hormone relaxin (Rlx) would result in ECM degradation and the improvement of trastuzumab therapy. As viral gene delivery into epithelial tumors with extensive tumor ECM is inefficient, we used a hematopoietic stem cell (HSC)-based approach to deliver the Rlx gene to the tumor. In mouse models with syngeneic breast cancer tumors, HSC-mediated intratumoral Rlx expression resulted in a decrease of ECM proteins and enabled control of tumor growth. Moreover, in a model with Her2/neu-positive BT474-M1 tumors and more treatment-refractory tumors derived from HCC1954 cells, we observed a significant delay of tumor growth when trastuzumab therapy was combined with Rlx expression. Our results have implications for antibody therapy of cancer as well as for other anticancer treatment approaches that are based on T-cells or encapsulated chemotherapy drugs.

pmcid: 3048180

Figure 6 - Therapy studies in the BT474-M1 xenograft model. CB17-SCID-beige mice were transplanted with either mock- or Ins-SIN-LV-Rlx-transduced HSCs cells [Tx (Mock) and Tx (Rlx), respectively]. After engraftment of HSCs, BT474-M1 cells were implanted into the mammary fat pad. Intraperitoneal Dox or PBS injections started 7 days after tumor cell implantation. Trastuzumab injections started 21 days after BT474-M1 cell transplantation. (a) Tumor growth in Tx (Mock) and Tx (Rlx) mice without trastuzumab treatment. Shown is the relative increase of tumor volume. Tumor volumes at the day on which Dox/PBS injections were started (“b.t.”) were taken as 100%. b.t., before treatment; p.t., post-treatment. N = 7. Shown are the average tumor volumes and SD. (b) Tumor growth in Tx (Mock) and Tx (Rlx) mice with trastuzumab treatment. Note that the scale of the y-axis is different. (c,d) Representative tumor sections of mice without Rlx expression [Tx (Mock)] and with Dox-induced Rlx expression [Tx (Rlx)+Dox] stained with (c) H&E and for (d) Her2/neu. Tumors from trastuzumab-treated mice were either absent or too small and could therefore not be evaluated. (e,f) Representative sections stained for basement membrane using (e) Jones' periodic acid silver staining method and (f) collagen IV. Collagen staining in f appears in brown. Collagen IV stained sections were used for morphometry. Bar = 40 µm. Dox, doxycycline; GFP, green fluorescent protein; HSC, hematopoietic stem cell; LV, lentivirus; PBS, phosphate-buffered saline; Rlx, relaxin; SIN, self-inactivating.

Plant-Made Trastuzumab (Herceptin) Inhibits HER2/Neu+ Cell Proliferation and Retards Tumor GrowthPlant-Made Trastuzumab (Herceptin)

pmcid: 3048398

doi: 10.1371/journal.pone.0017541

abstract: BackgroundPlant biotechnology provides a valuable contribution to global health, in part because it can decrease the cost of pharmaceutical products. Breast cancer can now be successfully treated by a humanized monoclonal antibody (mAb), trastuzumab (Herceptin). A course of treatment, however, is expensive and requires repeated administrations of the mAb. Here we used an Agrobacterium-mediated transient expression system to produce trastuzumab in plant cells.Methodology/Principal FindingsWe describe the cloning and expression of gene constructs in Nicotiana benthamiana plants using intron-optimized Tobacco mosaic virus- and Potato virus X-based vectors encoding, respectively, the heavy and light chains of trastuzumab. Full-size antibodies extracted and purified from plant tissues were tested for functionality and specificity by (i) binding to HER2/neu on the surface of a human mammary gland adenocarcinoma cell line, SK-BR-3, in fluorescence-activated cell sorting assay and (ii) testing the in vitro and in vivo inhibition of HER-2-expressing cancer cell proliferation. We show that plant-made trastuzumab (PMT) bound to the Her2/neu oncoprotein of SK-BR-3 cells and efficiently inhibited SK-BR-3 cell proliferation. Furthermore, mouse intraperitoneal PMT administration retarded the growth of xenografted tumors derived from human ovarian cancer SKOV3 Her2+ cells.Conclusions/SignificanceWe conclude that PMT is active in suppression of cell proliferation and tumor growth.

pmcid: 3048398

Figure 6 - The treatment groups received their first doses (20 mg/kg) of PMT (n=7) and trastuzumab (n=10) in saline solution i.p. 6 days after SKOV3 implantation, and then for 16 days, they received 8 consecutive injections (10 mg/kg). The control group (n=34) received saline solution. Tumor volumes were recorded in intervals 10–14, 18–22 and 23–27 days after SKOV3 implantation using a caliper. Data are the mean ± standard deviations from two independent experiments. Asterisk shows P<0.05 by the unpaired two tailed Student's t-test for statistical significance of difference between the PMT and trastuzumab treatment and control.

Reduced Dose and Intermittent Treatment with Lapatinib and Trastuzumab for Potent Blockade of the HER Pathway in HER-2/neu Overexpressing Breast Tumor Xenografts

pmcid: 3060302

doi: 10.1158/1078-0432.CCR-10-1905

abstract: PurposeWe have shown that incomplete blockade of the Human Epidermal Growth Factor (HER) pathway is a mechanism of resistance to treatment with trastuzumab (T) in HER2-overexpressing tumor xenografts. We now investigate whether the addition of lapatinib (L), a dual HER1/2 kinase inhibitor, to T results in more potent inhibition of the pathway and therefore inhibition of tumor growth, and whether reduced dose and intermittent treatment with the combination is equally effective.Experimental DesignNude mice bearing HER2-overexpressing MCF7/HER2-18 or BT474 xenograft tumors were treated with L, T, alone or in various combinations with other HER inhibitors. L+T for short duration (14, 42 days), intermittent administration (14 days on/off), and reduced dosing (1/2 dose) was also investigated. Inhibition of tumor growth, downstream signaling, proliferation, and induction of apoptosis were assessed. All statistical tests were two-sided.ResultsL+T was the most effective regimen in both MCF7/HER2-18 and BT474 xenografts with complete tumor regression (CR) observed in all mice. Intermittent and reduced dose treatment (½ dose) resulted in high rates of CR and low rates of tumor recurrence that were comparable to full dose continuous treatment. L+T resulted in significantly reduced downstream signaling and proliferation, and increased apoptosis.ConclusionsL+T is a potent and effective combination even when given in reduced dose or intermittent schedule potentially resulting in lower toxicity and reduced cost if translated to patients. These findings warrant timely clinical testing.

pmcid: 3060302

Figure 1 - Growth of MCF7/HER2-18 xenograft tumors in athymic female mice treated with variable anti-HER single agents and combinations, with or without ER targeted therapy. A. E2 treatment alone or with lapatinib (E2+L), trastuzumab (E2+T), or their combination (E2+L+T). B. Tamoxifen treatment alone or lapatinib (Tam+L), trastuzumab (Tam+T), or their combination (Tam+L+T). C. Tamoxifen treatment in the presence of estrogen with the combination of lapatinib and (E2+Tam+L+T). D. Estrogen deprivation (ED) alone or along with lapatinib (ED+L), trastuzumab (ED+T), or their combination (ED+L+T). Results are presented as the mean tumor volume; error bars represent the standard error. In panels B, C, D, and E, for each group, the number of mice with complete tumor regression and the total number of mice are shown. E. Tamoxifen treatment with alternative combinations of HER family inhibitors—lapatinib and gefitinib (Tam+L+G), double dose lapatinib 200mg/kg/day (Tam+2L), and tamoxifen with lapatinib and pertuzumab (Tam+L+P). Complete regression was defined as complete tumor regression documented on 3 consecutive weekly measurements.

pmcid: 3060302

Figure 1 - Growth of MCF7/HER2-18 xenograft tumors in athymic female mice treated with variable anti-HER single agents and combinations, with or without ER targeted therapy. A. E2 treatment alone or with lapatinib (E2+L), trastuzumab (E2+T), or their combination (E2+L+T). B. Tamoxifen treatment alone or lapatinib (Tam+L), trastuzumab (Tam+T), or their combination (Tam+L+T). C. Tamoxifen treatment in the presence of estrogen with the combination of lapatinib and (E2+Tam+L+T). D. Estrogen deprivation (ED) alone or along with lapatinib (ED+L), trastuzumab (ED+T), or their combination (ED+L+T). Results are presented as the mean tumor volume; error bars represent the standard error. In panels B, C, D, and E, for each group, the number of mice with complete tumor regression and the total number of mice are shown. E. Tamoxifen treatment with alternative combinations of HER family inhibitors—lapatinib and gefitinib (Tam+L+G), double dose lapatinib 200mg/kg/day (Tam+2L), and tamoxifen with lapatinib and pertuzumab (Tam+L+P). Complete regression was defined as complete tumor regression documented on 3 consecutive weekly measurements.

pmcid: 3060302

Figure 2 - Growth of BT474 xenograft tumors in athymic mice treated with estrogen supplementation (E2) or estrogen deprivation (ED) alone or with HER blocking agents. A. Estrogen deprivation (ED) alone or along with lapatinib (ED+L), trastuzumab (ED+T), or their combination (ED+L+T). B. Continued Estrogen supplementation alone or with the combination of lapatinib and trastuzumab (E2+L+T). Results are presented as the mean tumor volume error bars represent the standard error. For each group, the number of mice with complete tumor regression and the total number of mice are shown. Complete regression was defined as complete disappearance of the tumor for 3 consecutive weeks.

pmcid: 3060302

Figure 3 - Growth of BT474 xenograft tumors in athymic mice treated with estrogen supplementation (E2), or estrogen deprivation (ED) alone and with Lapatinib (L), trastuzumab (T), or their combination (L+T) in various doses and schedules. A. Growth of tumors in animals treated with ED plus short durations of (L+T) for 14 days (ED+L+T 14) and 42 days (ED+L+T 42) compared to control groups treated with E2, ED, ED+L, and ED+T. B. Growth of tumors treated with ED with full continuous (L+T), reduced dose therapy (1/2L+1/2T), or intermittent therapy (ED+L+T 14 on/off). For each group, the number of mice with complete tumor regression and the total number of mice are shown. Complete regression was defined as complete disappearance of the tumor for 3 consecutive weeks.

Up-regulation of miR-21 Mediates Resistance to Trastuzumab Therapy for Breast Cancer*miR-21 Mediates Trastuzumab Resistance

pmcid: 3099726

doi: 10.1074/jbc.M110.216887

abstract: Trastuzumab resistance emerges to be a major issue in anti-human epidermal growth factor receptor 2 (HER2) therapy for breast cancers. Here, we demonstrated that miR-21 expression was up-regulated and its function was elevated in HER2+ BT474, SKBR3, and MDA-MB-453 breast cancer cells that are induced to acquire trastuzumab resistance by long-term exposure to the antibody, whereas protein expression of the PTEN gene, a miR-21 target, was reduced. Blocking the action of miR-21 with antisense oligonucleotides re-sensitized the resistant cells to the therapeutic activities of trastuzumab by inducing growth arrest, proliferation inhibition, and G1-S cell cycle checking in the presence of the antibody. Ectopic expression of miR-21 in HER2+ breast cancer cells confers resistance to trastuzumab. Rescuing PTEN expression with a p3XFLAG-PTEN-mut construct with deleted miR-21 targeting sequence at its 3′ UTR restored the growth inhibition of trastuzumab in the resistant cells by inducing PTEN activation and AKT inhibition. In vivo, administering miR-21 antisense oligonucleotides restored trastuzumab sensitivity in the resistant breast cancer xenografts by inducing PTEN expression, whereas injection of miR-21 mimics conferred trastuzumab resistant in the sensitive breast tumors via PTEN silence. Up-regulatin of miR-21 in tumor biopsies obtained from patients receiving pre-operative trastuzumab therapy was associated with poor trastuzumab response. Therefore, miR-21 overexpression contributes to trastuzumab resistance in HER2+ breast cancers and antagonizing miR-21 demonstrates therapeutic potential by sensitizing the malignancy to anti-HER2 treatment.

pmcid: 3099726

FIGURE 6. - miR-21 confers trastuzumab resistance on HER2+ breast cancer xenografts in vivo. A and B, tumor volume was measured in athymic nude mice xenografted in the mammary fat pads with trastuzumab-resistant (A) or parental (B) BT474 (1 × 107 cells) breast cancer cells. When the xenografts were palpable (around 150 mm3 in diameter), intratumor injection with Lipofectamine (5 μl) alone or in complex with 20 μg of miR-21 ASO or Lin4 ASO for the resistant BT474 xenografts (A), or with 15 μg of miR-21 or Lin4 mimics for the parental BT474 xenografts (B), followed by intravenous injection of trastuzumab or an isotype IgG 72 h later at 10 mg/kg, was performed biweekly for 3 consecutive weeks. *, p < 0.05; and **, p < 0.01 as compared with intratumor injection with Lipofectamine alone. ##, p < 0.01 versus xenografts without trastuzumab treatment. C and D, microscopic images of H&E staining and immunohistochemical staining for HER2, PCNA, and PTEN in the trastuzumab-resistant BT474 xenografts (C) treated as in A, and the parental BT474 xenografts (D) treated as in B.

Overcoming Trastuzumab Resistance in Breast Cancer by Targeting Dysregulated Glucose Metabolism

pmcid: 3129363

doi: 10.1158/0008-5472.CAN-11-0127

abstract: Trastuzumab shows remarkable efficacy in treatment of ErbB2-positive breast cancers when used alone or in combination with other chemotherapeutics. However, acquired resistance develops in most treated patients, necessitating alternate treatment strategies. Increased aerobic glycolysis is a hallmark of cancer and inhibition of glycolysis may offer a promising strategy to preferentially kill cancer cells. In this study, we investigated the antitumor effects of trastuzumab in combination with glycolysis inhibitors in ErbB2-positive breast cancer. We found that trastuzumab inhibits glycolysis via downregulation of heat shock factor 1 (HSF1) and lactate dehydrogenase A (LDH-A) in ErbB2-positive cancer cells, resulting in tumor growth inhibition. Moreover, increased glycolysis via HSF1 and LDH-A contributes to trastuzumab resistance. Importantly, we found that combining trastuzumab with glycolysis inhibition synergistically inhibited trastuzumab-sensitive and -resistant breast cancers in vitro and in vivo, due to more efficient inhibition of glycolysis. Taken together, our findings show how glycolysis inhibition can dramatically enhance the therapeutic efficacy of trastuzumab in ErbB2-positive breast cancers, potentially useful as a strategy to overcome trastuzumab resistance.

pmcid: 3129363

Figure 6 - The combination of trastuzumab and oxamate effectively inhibits tumor growth in vivo.

Glycoengineered Pichia produced anti-HER2 is comparable to trastuzumab in preclinical study

pmcid: 3149709

doi: 10.4161/mabs.3.3.15532

abstract: Mammalian cell culture systems are used predominantly for the production of therapeutic monoclonal antibody (mAb) products. A number of alternative platforms, such as Pichia engineered with a humanized N-linked glycosylation pathway, have recently been developed for the production of mAbs. The glycosylation profiles of mAbs produced in glycoengineered Pichia are similar to those of mAbs produced in mammalian systems. This report presents for the first time the comprehensive characterization of an anti-human epidermal growth factor receptor 2 (HER2) mAb produced in glycoengineered Pichia, and a study comparing the anti-HER2 from Pichia, which had an amino acid sequence identical to trastuzumab, with trastuzumab. The comparative study covered a full spectrum of preclinical evaluation, including bioanalytical characterization, in vitro biological functions, in vivo anti-tumor efficacy and pharmacokinetics in both mice and non-human primates. Cell signaling and proliferation assays showed that anti-HER2 from Pichia had antagonist activities comparable to trastuzumab. However, Pichia-produced material showed a 5-fold increase in binding affinity to FcγIIIA and significantly enhanced antibody dependent cell-mediated cytotoxicity (ADCC) activity, presumably due to the lack of fucose on N-glycans. In a breast cancer xenograft mouse model, anti-HER2 was comparable to trastuzumab in tumor growth inhibition. Furthermore, comparable pharmacokinetic profiles were observed for anti-HER2 and trastuzumab in both mice and cynomolgus monkeys. We conclude that glycoengineered Pichia provides an alternative production platform for therapeutic mAbs and may be of particular interest for production of antibodies for which ADCC is part of the clinical mechanism of action.

pmcid: 3149709

Figure 6 - Tumor growth inhibition by anti-HER2 mAb in comparison with trastuzumab in the BT474m1 breast cancer xenograft tumor model (each data point is calculated as mean from group of 10 mice). (A) Mean tumor volume of anti-HER2 mAb and trastuzumab treated BT474m1 xenografted mice at dosage of 0.003 mg/kg, 0.03 and 0.3 mg/kg. (B) Slope of mean tumor volume of all the treatment at all three different dosage.

The Potential Utility of Curcumin in the Treatment of HER-2-Overexpressed Breast Cancer: An In Vitro and In Vivo Comparison Study with Herceptin

pmcid: 3162976

doi: 10.1155/2012/486568

abstract: HER-2 is an important oncoprotein overexpressed in about 15–25% of breast cancers. We hypothesized that the ability of curcumin to downregulate HER-2 oncoprotein and inhibit the signal transduction pathway of PI3K/Akt, MAPK, and NF-κB activation may be important in the treatment of HER-2-overexpressed breast cancer. To examine the effect of curcumin on breast cancer cells, MCF-7, MDA-MB-231, MCF-10A, BT-474, and SK-BR-3-hr (a herceptin resistant strain from SK-BR-3) cells were used for in vitro analysis. The in vivo effect of curcumin on HER-2-overexpressed breast cancer was investigated with the HER-2-overexpressed BT-474 xenograft model. Cell growth, cell cycle change, the antimobility effect, signal transduction, and xenograft volume analysis between groups treated with herceptin and/or curcumin were tested. Curcumin decreased the cell growth of various breast cancer cell lines (MCF-7, MDA-MB-231, MCF-10A, BT-474, and SK-BR-3-hr). In Western blot analysis, the phosphorylation of Akt, MAPK, and expression of NF-κB were reduced in BT-474 cells, but not in SK-BR-3-hr cells, after treatment with herceptin. When treated with curcumin, the HER-2 oncoprotein, phosphorylation of Akt, MAPK and expression of NF-κB were decreased in both BT-474 and SK-BR-3-hr cells. In the BT-474 xenograft model, though not as much as herceptin, curcumin did effectively decrease the tumor size. The combination of curcumin with herceptin was not better than herceptin alone; however, the combination of taxol and curcumin had an antitumor effect comparable with taxol and herceptin. The results suggested that curcumin has potential as a treatment for HER-2-overexpressed breast cancer.

pmcid: 3162976

Figure 6 - In vivo effects of curcumin on the herceptin and/or taxol-treated HER-2-overexpressed breast cancer xenografts. The HER-2-overexpressed BT-474 cells were injected in 4–6-week-old, female, athymic, nude mice subcutaneously at 1 × 107 cells/tumor in the right flank region to form xenografts. Six mice per group were treated with different protocols with tumor volume monitored biweekly for consecutive 4 weeks. (a) The mean xenograft tumor volume change of control (0.1% DMSO), herceptin, curcumin, and combined curcumin and herceptin. (b) The mean xenograft tumor volume of taxol, taxol + herceptin, taxol + curcumin, and combined taxol + herceptin + curcumin. (c) The body weight change of these mice treated with herceptin and/or curcumin. DMSO, dimethyl sulfoxide.

The vitamin E analog, alpha-tocopheryloxyacetic acid enhances the anti-tumor activity of trastuzumab against HER2/neu-expressing breast cancer

pmcid: 3217981

doi: 10.1186/1471-2407-11-471

abstract: BackgroundHER2/neu is an oncogene that facilitates neoplastic transformation due to its ability to transduce growth signals in a ligand-independent manner, is over-expressed in 20-30% of human breast cancers correlating with aggressive disease and has been successfully targeted with trastuzumab (Herceptin®). Because trastuzumab alone achieves only a 15-30% response rate, it is now commonly combined with conventional chemotherapeutic drugs. While the combination of trastuzumab plus chemotherapy has greatly improved response rates and increased survival, these conventional chemotherapy drugs are frequently associated with gastrointestinal and cardiac toxicity, bone marrow and immune suppression. These drawbacks necessitate the development of new, less toxic drugs that can be combined with trastuzumab. Recently, we reported that orally administered alpha-tocopheryloxyacetic acid (α-TEA), a novel ether derivative of alpha-tocopherol, dramatically suppressed primary tumor growth and reduced the incidence of lung metastases both in a transplanted and a spontaneous mouse model of breast cancer without discernable toxicity.MethodsIn this study we examined the effect of α-TEA plus HER2/neu-specific antibody treatment on HER2/neu-expressing breast cancer cells in vitro and in a HER2/neu positive human xenograft tumor model in vivo.ResultsWe show in vitro that α-TEA plus anti-HER2/neu antibody has an increased cytotoxic effect against murine mammary tumor cells and human breast cancer cells and that the anti-tumor effect of α-TEA is independent of HER2/neu status. More importantly, in a human breast cancer xenograft model, the combination of α-TEA plus trastuzumab resulted in faster tumor regression and more tumor-free animals than trastuzumab alone.ConclusionDue to the cancer cell selectivity of α-TEA, and because α-TEA kills both HER2/neu positive and HER2/neu negative breast cancer cells, it has the potential to be effective and less toxic than existing chemotherapeutic drugs when used in combination with HER2/neu antibody.

pmcid: 3217981

Figure 4 - Tumor regression by α-TEA plus trastuzumab treatment. SCID mice (n = 9 to 10 mice per group) received a s.c. injection of MDA-MB-453 cells. After tumor establishment (day 15 post-tumor injection), mice received α-TEA in the diet (~2 mg/day/mouse) until day 63 (red bar) or 40 μg trastuzumab by i.p. injection, every 2 to 3 days for a total of 12 injections until day 40 (green bar). One mouse in the α-TEA + trastuzumab group died of unknown cause on day 35 post-tumor cell injection and was excluded from analysis. (A) Individual tumor volumes. (B) Tumor frequency.

pmcid: 3217981

Figure 5 - α-TEA inhibits tumor growth without modulating HER2/neu expression. SCID mice with established MDA-MB-453 tumors (day 15 post-tumor injection) received α-TEA in the diet (~2 mg/day/mouse) or 40 μg trastuzumab by i.p. injection, every 2 to 3 days for a total of 7 injections. On day 30 post-tumor injection, tumors were mechanically dissociated and a single cell suspension was prepared and analyzed for HER2/neu expression by flow cytometry. HER2/neu expression of MDA-MB-453 tumor cells used for transplantation is shown for comparison (pre-injection). Cells were gated on light scatter and non-viable cells were excluded from the analysis using viability stain.

Experimental α-particle radioimmunotherapy of breast cancer using 227Th-labeled p-benzyl-DOTA-trastuzumab

pmcid: 3250964

doi: 10.1186/2191-219X-1-18

abstract: BackgroundThe aim of the present study was to explore the biodistribution, normal tissue toxicity, and therapeutic efficacy of the internalizing low-dose rate alpha-particle-emitting radioimmunoconjugate 227Th-trastuzumab in mice with HER2-expressing breast cancer xenografts.MethodsBiodistribution of 227Th-trastuzumab and 227Th-rituximab in nude mice bearing SKBR-3 xenografts were determined at different time points after injection. Tumor growth was measured after administration of 227Th-trastuzumab, 227Th-rituximab, cold trastuzumab, and saline. The toxicity of 227Th-trastuzumab was evaluated by measurements of body weight, blood cell, and clinical chemistry parameters, as well as histological examination of tissue specimens.ResultsThe tumor uptake reached peak levels of 34% ID/g (4.6 kBq/g) 3 days after injection of 400 kBq/kg of 227Th-trastuzumab. The absorbed radiation dose to tumor was 2.9 Gy, while it was 2.4 Gy to femur due to uptake of the daughter nuclide 223Ra in bone; the latter already explored in clinical phases I and II trials without serious toxicity. A significant dose-dependent antitumor effect was observed for dosages of 200, 400, and 600 kBq/kg of 227Th-trastuzumab but no effect of 400 and 600 kBq/kg 227Th-rituximab (non-tumor binding). No serious delayed bone marrow or normal organ toxicity was observed, but there was a statistical significant reduction in blood cell parameters for the highest-dose group of 227Th-trastuzumab treatment.ConclusionInternalizing 227Th-trastuzumab therapy was well tolerated and resulted in a dose-dependent inhibition of breast cancer xenograft growth. These results warrant further preclinical studies aiming at a clinical trial in breast cancer patients with metastases to bone.

pmcid: 3250964

Figure 3 - Effects of 227Th-based RIT on growth of individual SKBR-3 tumor xenografts. Individual tumor growth after treatment with NaCl (a); 20, 100, and 250 μg cold trastuzumab (b); 227Th-rituximab at dosage of 400 and 600 kBq/kg (c); 200 kBq/kg (d); 400 kBq/kg (e) and 600 kBq/kg (f) of 227Th-trastuzumab. N = 9 to 19.

In Pancreatic Carcinoma, Dual EGFR/HER2 Targeting with Cetuximab/Trastuzumab Is More Effective than Treatment with Trastuzumab/Erlotinib or Lapatinib Alone: Implication of Receptors' Down-regulation and Dimers' Disruption1

pmcid: 3306257

doi:

abstract: We previously demonstrated the synergistic therapeutic effect of the cetuximab (anti-epidermal growth factor receptor [EGFR] monoclonal antibody, mAb)-trastuzumab (anti-HER2 mAb) combination (2mAbs therapy) in HER2low human pancreatic carcinoma xenografts. Here, we compared the 2mAbs therapy, the erlotinib (EGFR tyrosine kinase inhibitor [TKI])-trastuzumab combination and lapatinib alone (dual HER2/EGFR TKI) and explored their possible mechanisms of action. The effects on tumor growth and animal survival of the three therapies were assessed in nude mice xenografted with the human pancreatic carcinoma cell lines Capan-1 and BxPC-3. After therapy, EGFR and HER2 expression and AKT phosphorylation in tumor cells were analyzed by Western blot analysis. EGFR/HER2 heterodimerization was quantified in BxPC-3 cells by time-resolved FRET. In K-ras-mutated Capan-1 xenografts, the 2mAbs therapy gave significantly higher inhibition of tumor growth than the erlotinib/trastuzumab combination, whereas in BxPC-3 (wild-type K-ras) xenografts, the erlotinib/trastuzumab combination showed similar growth inhibition but fewer tumor-free mice. Lapatinib showed no antitumor effect in both types of xenografts. The efficacy of the 2mAbs therapy was partly Fc-independent because F(ab′)2 fragments of the two mAbs significantly inhibited BxPC-3 growth, although with a time-limited therapeutic effect. The 2mAbs therapy was associated with a reduction of EGFR and HER2 expression and AKT phosphorylation. BxPC-3 cells preincubated with the two mAbs showed 50% less EGFR/HER2 heterodimers than controls. In pancreatic carcinoma xenografts, the 2mAbs therapy is more effective than treatments involving dual EGFR/HER2 TKIs. The mechanism of action may involve decreased AKT phosphorylation and/or disruption of EGFR/HER2 heterodimerization.

pmcid: 3306257

Figure 1 - Comparison of the antitumor activity of the 2mAbs therapy (cetuximab/trastuzumab) and of the erlotinib/trastuzumab combination in nude mice xenografted subcutaneously with Capan-1 and BxPC-3 human pancreatic carcinoma cells. The weight of the animals was followed up during treatment in the Capan-1 (A) and BxPC-3 (B) xenograft models. At days 14 (Capan-1 cells) (C) and 26 (BxPC-3 cells) (D) after graft, groups of 10 mice were treated with the two mAbs (ratio 1:1; 2 mg/kg of each mAb, twice a week), erlotinib (100 mg/kg; daily) plus trastuzumab (2 mg/kg, twice a week), or sterile PBS (daily). Results are presented as the mean tumor volume for each group. Bars, SD of the mean. C + T indicates cetuximab/trastuzumab; E + T, erlotinib/trastuzumab.

pmcid: 3306257

Figure 2 - Comparison of the effect of lapatinib (dual HER2/EGFR TKI) and of the 2mAbs therapy on tumor growth in nude mice xenografted subcutaneously with BxPC-3 (A), Capan-1 (B), or SKOV-3 (C) cells and randomized in different groups (n = 10 per group). At days 14 (Capan-1), 20 (BxPC-3), and 11 (SKOV-3 cells) after graft, mice were treated with lapatinib (100, 200, or 300 mg/kg, daily), the 2mAbs therapy (ratio 1:1; 2 mg/kg of each mAb, twice per week) or sterile PBS (daily). Results are presented as the mean tumor volume of each group. Bars, SD of the mean. C indicates cetuximab; Lap, lapatinib; T, trastuzumab.

pmcid: 3306257

Figure 3 - Comparison of the antitumor effect, in vitro and in vivo, of the 2mAbs therapy (cetuximab and trastuzumab) using intact mAbs or the F(ab′)2 fragments. (A) ADCC was assessed in vitro by incubating 51Cr-labeled BxPC-3 target cells with peripheral blood mononuclear cells (effector cells) at different concentrations in the presence of cetuximab and/or trastuzumab (whole mAbs) or their F(ab′)2 fragments. ADCC was determined by measuring the 51Cr released in the supernatant. (B) SCID/Beige mice bearing BxPC-3 pancreatic carcinoma xenografts were treated at day 20 after graft by daily coinjection of F(ab′)2 fragments of the two mAbs or twice per week with the intact mAbs. Results are presented as the mean tumor volume of each treated group. Bars, SD of the mean. C indicates cetuximab; double-head arrow, period of treatment; T, trastuzumab.

Different mechanisms for resistance to trastuzumab versus lapatinib in HER2- positive breast cancers -- role of estrogen receptor and HER2 reactivation

pmcid: 3326563

doi: 10.1186/bcr3067

abstract: IntroductionThe human epidermal growth factor receptor 2 (HER2)-targeted therapies trastuzumab (T) and lapatinib (L) show high efficacy in patients with HER2-positive breast cancer, but resistance is prevalent. Here we investigate resistance mechanisms to each drug alone, or to their combination using a large panel of HER2-positive cell lines made resistant to these drugs.MethodsResponse to L + T treatment was characterized in a panel of 13 HER2-positive cell lines to identify lines that were de novo resistant. Acquired resistant lines were then established by long-term exposure to increasing drug concentrations. Levels and activity of HER2 and estrogen receptor (ER) pathways were determined by qRT-PCR, immunohistochemistry, and immunoblotting assays. Cell growth, proliferation, and apoptosis in parental cells and resistant derivatives were assessed in response to inhibition of HER or ER pathways, either pharmacologically (L, T, L + T, or fulvestrant) or by using siRNAs. Efficacy of combined endocrine and anti-HER2 therapies was studied in vivo using UACC-812 xenografts.ResultsER or its downstream products increased in four out of the five ER+/HER2+ lines, and was evident in one of the two intrinsically resistant lines. In UACC-812 and BT474 parental and resistant derivatives, HER2 inhibition by T reactivated HER network activity to promote resistance. T-resistant lines remained sensitive to HER2 inhibition by either L or HER2 siRNA. With more complete HER2 blockade, resistance to L-containing regimens required the activation of a redundant survival pathway, ER, which was up-regulated and promoted survival via various Bcl2 family members. These L- and L + T-resistant lines were responsive to fulvestrant and to ER siRNA. However, after prolonged treatment with L, but not L + T, BT474 cells switched from depending on ER as a survival pathway, to relying again on the HER network (increased HER2, HER3, and receptor ligands) to overcome L's effects. The combination of endocrine and L + T HER2-targeted therapies achieved complete tumor regression and prevented development of resistance in UACC-812 xenografts.ConclusionsCombined L + T treatment provides a more complete and stable inhibition of the HER network. With sustained HER2 inhibition, ER functions as a key escape/survival pathway in ER-positive/HER2-positive cells. Complete blockade of the HER network, together with ER inhibition, may provide optimal therapy in selected patients.

pmcid: 3326563

Figure 6 - Growth of UACC-812 xenografts treated with various anti-HER2 treatments, with or without estrogen deprivation. (A) Treatment in the presence of estrogen supplementation, representing no endocrine therapy. Treatments included: Estrogen alone (E2) or with lapatinib (E2 + L), trastuzumab (E2 + T), or their combination (E2 + L + T). (B) Treatments in the presence of endocrine therapy in the form of estrogen deprivation. Treatments included: Estrogen (E2), estrogen deprivation (ED) alone, or along with lapatinib (ED + L), trastuzumab (ED + T), or their combination (ED + L + T). Results are presented as the mean tumor volume; error bars represent the standard error.

Polymalic Acid–Based Nanobiopolymer Provides Efficient Systemic Breast Cancer Treatment by Inhibiting both HER2/neu Receptor Synthesis and Activity

pmcid: 3428373

doi: 10.1158/0008-5472.CAN-10-3093

abstract: Biodegradable nanopolymers are believed to offer great potential in cancer therapy. Here, we report the characterization of a novel, targeted, nanobiopolymeric conjugate based on biodegradable, nontoxic, and nonimmunogenic PMLA [poly(β-l-malic acid)]. The PMLA nanoplatform was synthesized for repetitive systemic treatments of HER2/neu-positive human breast tumors in a xenogeneic mouse model. Various moieties were covalently attached to PMLA, including a combination of morpholino antisense oligonucleotides (AON) directed against HER2/neu mRNA, to block new HER2/neu receptor synthesis; anti-HER2/neu antibody trastuzumab (Herceptin), to target breast cancer cells and inhibit receptor activity simultaneously; and transferrin receptor antibody, to target the tumor vasculature and mediate delivery of the nanobiopolymer through the host endothelial system. The results of the study showed that the lead drug tested significantly inhibited the growth of HER2/neu-positive breast cancer cells in vitro and in vivo by enhanced apoptosis and inhibition of HER2/neu receptor signaling with suppression of Akt phosphorylation. In vivo imaging analysis and confocal microscopy demonstrated selective accumulation of the nanodrug in tumor cells via an active delivery mechanism. Systemic treatment of human breast tumor-bearing nude mice resulted in more than 90% inhibition of tumor growth and tumor regression, as compared with partial (50%) tumor growth inhibition in mice treated with trastuzumab or AON, either free or attached to PMLA. Our findings offer a preclinical proof of concept for use of the PMLA nanoplatform for combination cancer therapy.

pmcid: 3428373

Figure 6 - Mouse tumor inhibition, pathology, signaling, and apoptosis marker expression. A, 2 representative animals for each group and histopathologic analysis of respective tumors (H&E staining) are shown after treatment with different drug variants. Variable amounts of dead tissue are present in all nanobiopolymer-treated groups. In accordance with tumor size reduction data, the lead drug P/mPEG/LOEt/AON/Herceptin/TfRM) caused pronounced disappearance of tumor cells with mostly necrotic areas present. B, tumor growth inhibition inmice. Animals treated with Herceptin, P/mPEG/LOEt/Herceptin, or P/mPEG/LOEt/AON/TfRH/M) showed significant inhibition compared with PBS control (P < 0. 03). P/mPEG/LOEt/AON/Herceptin/TfRM) treatment produced the highest inhibition of tumor growth resulting in 80% to 95% tumor regression during the follow-up period compared with other treatment groups (P < 0. 02 vs. Herceptin and other drugs; P < 0. 001 vs. PBS). Error bars denote SEM. C, Expression of select markers after treatment of HER2/neu-positive tumors in vivo. Western blot analysis revealed the decrease in HER2/neu and p-Akt (but not total Akt) expression in Herceptin-, P/mPEG/LOEt/Herceptin-, or P/mPEG/LOEt/AON/TfRH/M)-treated mice compared with PBS-treated ones. P/mPEG/LOEt/AON/Herceptin/TfRM) further inhibited HER2/neu expression, with near disappearance of p-Akt band. PARP cleavage as a measure of apoptosis was also the most pronounced in P/mPEG/LOEt/AON/Herceptin/TfRM)-treated mice compared with other groups. GAPDH was an internal control to normalize gel loading.

Regulation of ERBB2 Receptor by t-DARPP Mediates Trastuzumab Resistance in Human Esophageal Adenocarcinoma

pmcid: 3432752

doi: 10.1158/0008-5472.CAN-12-1119

abstract: Esophageal adenocarcinoma (EAC) is an aggressive malignancy with a poor outcome. Although targeting ERBB2 with trastuzumab is evaluated in clinical trials, the molecular mechanisms of trastuzumab resistance remain uncharacterized in EAC. The dopamine and cyclic AMP-regulated phosphoprotein of Mr 32,000 (DARPP-32), also known as PPP1R1B, is located together with ERBB2 at the 17q12-q21 amplicon. We evaluated the expression of a transcript variant of DARPP-32 (t-DARPP) and ERBB2 in 141 primary tumors and investigated the role of t-DARPP in trastuzumab resistance using OE19 and OE33 EAC cell models. Overexpression of t-DARPP mRNA was detected in two-thirds of tumors with a correlation between ERBB2 and t-DARPP overexpression levels (r=0.58, p=0.003). Cell viability and clonogenic survival assays showed that t-DARPP increased survival by 40% in response to trastuzumab (p<0.01). The Annexin-V staining and Western blot analysis indicated that t-DARPP effectively abrogated trastuzumab-induced apoptosis, inhibited cleavage of caspase-3 and blocked trastuzumab-induced dephosphorylation of ERBB2 and AKT proteins. The knockdown of endogenous t-DARPP reversed these effects and sensitized cells to trastuzumab (p<0.01). The cycloheximide-based protein degradation analysis indicated that t-DARPP extended the half-life of ERBB2 explaining the increase in the basal levels of ERBB2, p-ERBB2(Y1248), and p-AKT(S473). Coimmunoprecipitation and Western blot analysis demonstrated that t-DARPP associated with ERBB2 in a protein complex, and interfered with trastuzumab binding to the ERBB2 receptor. Using EAC-xenografted mouse model, t-DARPP enhanced tumor growth and rendered tumors unresponsive to trastuzumab. This study establishes t-DARPP as a mediator of trastuzumab resistance and underscores its potential importance in clinical trials of EAC.

pmcid: 3432752

Figure 7 - OE19 cells stably expressing t-DARPP or pcDNA3 control vector were injected subcutaneously (4 × 106 cells per site) into nude mice. When Tumor volume reached 200 mm3, the mice were treated with 20 mg/kg trastuzumab twice weekly for 20 days. A) A representative sacrificed control mouse with xenograft tumors (left panel), and resected xenograft tumors (right panel) at the end of experiment. B) A representative sacrificed t-DARPP mouse with xenograft tumors (left panel), and resected xenograft tumors (right panel) at the end of experiment. C) Tumor growth curve from OE19 cells stably expressing t-DARPP or control vector before the treatment (left panel) and after the treatment (right panel). Each data point represents the mean ± standard deviation. The data show that t-DARPP significantly enhanced tumor growth rate (p<0.01), and inhibited response to treatment with trastuzumab (p<0.001) as compared to control. D) The H&E staining at the end of trastuzumab treatment shows effectively diminished control tumors leaving necrotic and fibrotic lesions (left panel) whereas t-DARPP tumors were unaffected (right panel).

Trastuzumab anti-tumor efficacy in patient-derived esophageal squamous cell carcinoma xenograft (PDECX) mouse models

pmcid: 3485623

doi: 10.1186/1479-5876-10-180

abstract: BackgroundTrastuzumab is currently approved for the clinical treatment of breast and gastric cancer patients with HER-2 positive tumors, but not yet for the treatment of esophageal carcinoma patients, whose tumors typically show 5 ~ 35% HER-2 gene amplification and 0 ~ 56% HER-2 protein expression. This study aimed to investigate the therapeutic efficacy of Trastuzumab in patient-derived esophageal squamous cell carcinoma xenograft (PDECX) mouse models.MethodsPDECX models were established by implanting patient esophageal squamous cell carcinoma (ESCC) tissues into immunodeficient (SCID/nude) mice. HER-2 gene copy number (GCN) and protein expression were determined in xenograft tissues and corresponding patient EC samples by FISH and IHC analysis. Trastuzumab anti-tumor efficacy was evaluated within these PDECX models (n = 8 animals/group). Furthermore, hotspot mutations of EGFR, K-ras, B-raf and PIK3CA genes were screened for in the PDECX models and their corresponding patient’s ESCC tissues. Similarity between the PDECX models and their corresponding patient’s ESCC tissue was confirmed by histology, morphology, HER-2 GCN and mutation.ResultsNone of the PDECX models (or their corresponding patient’s ESCC tissues) harbored HER-2 gene amplification. IHC staining showed HER-2 positivity (IHC 2+) in 2 PDECX models and negativity in 3 PDECX models. Significant tumor regression was observed in the Trastuzumab-treated EC044 HER-2 positive model (IHC 2+). A second HER-2 positive (IHC 2+) model, EC039, harbored a known PIK3CA mutation and showed strong activation of the AKT signaling pathway and was insensitive to Trastuzumab treatment, but could be resensitised using a combination of Trastuzumab and AKT inhibitor AZD5363. In summary, we established 5 PDECX mouse models and demonstrated tumor regression in response to Trastuzumab treatment in a HER-2 IHC 2+ model, but resistance in a HER-2 IHC 2+/PIK3CA mutated model.ConclusionsThis study demonstrates Trastuzumab-induced tumor regressions in HER-2 positive tumors, and highlights PIK3CA mutation as a potential resistance mechanism to Trastuzumab treatment in pre-clinical patient-derived EC xenograft models.

pmcid: 3485623

Figure 3 - Anti-tumor efficacy of Trastuzumab in the 5 PDECX mouse models. Tumor-bearing mice were treated starting from tumor volume 100~200mm3. The subcutaneous tumor volume was measured by caliper and calculated as mean±SEM. Y-axis represented the volume of the tumor (mean±SEM), and X-axis represents the number of days following treatment start day.

Combined targeting of HER2 and VEGFR2 for effective treatment of HER2-amplified breast cancer brain metastasesTreatment of breast cancer brain metastases

pmcid: 3494882

doi: 10.1073/pnas.1216078109

abstract: Brain metastases are a serious obstacle in the treatment of patients with human epidermal growth factor receptor-2 (HER2)–amplified breast cancer. Although extracranial disease is controlled with HER2 inhibitors in the majority of patients, brain metastases often develop. Because these brain metastases do not respond to therapy, they are frequently the reason for treatment failure. We developed a mouse model of HER2-amplified breast cancer brain metastasis using an orthotopic xenograft of BT474 cells. As seen in patients, the HER2 inhibitors trastuzumab and lapatinib controlled tumor progression in the breast but failed to contain tumor growth in the brain. We observed that the combination of a HER2 inhibitor with an anti–VEGF receptor-2 (VEGFR2) antibody significantly slows tumor growth in the brain, resulting in a striking survival benefit. This benefit appears largely due to an enhanced antiangiogenic effect: Combination therapy reduced both the total and functional microvascular density in the brain xenografts. In addition, the combination therapy led to a marked increase in necrosis of the brain lesions. Moreover, we observed even better antitumor activity after combining both trastuzumab and lapatinib with the anti-VEGFR2 antibody. This triple-drug combination prolonged the median overall survival fivefold compared with the control-treated group and twofold compared with either two-drug regimen. These findings support the clinical development of this three-drug regimen for the treatment of HER2-amplified breast cancer brain metastases.

pmcid: 3494882

Fig. 1. - Imaging of the breast cancer brain metastasis model and the effect of anti-HER2 therapies on tumor growth. (A) Imaging of an established BT474-Gluc tumor after direct injection into the brain parenchyma. The cranial window was used for intravital microscopy. (Left) Representative bioluminescence image of the tumor symbolizes its approximate size at treatment initiation. (Scale bar: 5 mm.) Intravital multiphoton microscopy images illustrate the brain metastatic lesion boundary (Center) and the abnormality of the tumor vasculature (Right). (Scale bars: 100 μm.) Tumor cells expressing CFP are green, and blood vessels (red) are contrast-enhanced by i.v. injection of tetramethylrhodamine dextran (2,000,000 molecular weight). (B) Effect of trastuzumab (red, 5 mg/kg twice a week) or lapatinib (green, 100 mg/kg daily) on the growth of BT474-Gluc breast cancer cells when growing in the mammary fat pad (Left) or brain parenchyma (Right). Tumor growth curves for each treatment group are shown. Data are expressed as the mean ± SEM. (Left) BT474-Gluc mammary fat pad tumors were allowed to reach ~75 mm3 in volume before treatment initiation; the y axis is the fold-change of tumor volume (n = 6–8 mice). (Right) BT474-Gluc brain metastatic tumors were allowed to reach a blood Gluc activity of roughly 10 RLU/s, corresponding to a volume of ~10 mm3, before treatment initiation; the y axis is the fold-change of blood Gluc activity (n = 8–13 mice). (Treatment was initiated for BT474-Gluc brain metastatic tumors at ~10 mm3 in all subsequent experiments. Data are expressed as the mean ± SEM in all subsequent figures.)

pmcid: 3494882

Fig. 2. - Effects of anti-HER2 and anti-VEGFR2 therapies, and their combination on breast cancer growth in the brain parenchyma and mouse survival. Trastuzumab and lapatinib were dosed as previously mentioned, and DC101 was dosed at 40 mg/kg via i.p. injection twice a week. Tumor size was monitored twice a week via blood Gluc activity (Left), and animal survival was ascertained (Right). (A) Tumor growth plot (Left) and Kaplan–Meier survival plot (Right) of brain metastatic tumor-bearing mice treated with control (black), trastuzumab (red), DC101 (blue), or trastuzumab and DC101 (magenta) (n = 8–10 mice; except in the case of trastuzumab treatment, where n = 5 mice). **P < 0.01. (B) Tumor growth plot (Left) and Kaplan–Meier survival plot (Right) of brain metastatic tumor-bearing mice treated with control (black), lapatinib (green), DC101 (blue), or lapatinib and DC101 (orange) (n = 8–10 mice). ***P < 0.001. (Final tumor growth points occur when at least 3 mice are still alive.)

pmcid: 3494882

Fig. 3. - Imaging of metastatic breast cancer growth in the brain parenchyma and response to the combination of anti-HER2 and anti-VEGFR2 therapy. (A) MRI analysis of control or trastuzumab and DC101 treatment on BT474-Gluc metastatic tumors in the brain. MRI tumor volume was measured on days 0, 5, and 14 of treatment. (Left) Representative MRI of control- and combination-treated tumors is shown. (Right) Tumor volume, calculated using MRI, is plotted for each individual mouse (# corresponds to the tumors illustrated by the MRI scans in A). (B) Bioluminescence imaging analysis of control or trastuzumab and DC101 treatment on metastatic BT474-Gluc tumors in the brain. (Left) Images of control- and combination-treated tumors 14 d after treatment initiation are shown. (Right) Average bioluminescence signal for the three control- and three combination-treated tumors is depicted. *P < 0.05.

pmcid: 3494882

Fig. 6. - Effects of dual HER2 targeting with and without anti-VEGFR2 therapy on BT474-Gluc brain metastatic tumors. (A) Tumor growth plot (Left) and Kaplan–Meier survival plot (Right) of tumor-bearing mice treated with control (black), trastuzumab (red), lapatinib (green), or trastuzumab and lapatinib (brown) (n = 8–20 mice). ***P < 0.001. (B) Tumor growth plot (Left) and Kaplan–Meier survival plot (Right) of tumor-bearing mice treated with control (black); trastuzumab and DC101 (magenta); lapatinib and DC101 (orange); trastuzumab and lapatinib (brown); and the triple combination of trastuzumab, lapatinib, and DC101 (cyan) (n = 6–9 mice). ***P < 0.001. (Final tumor growth points occur when at least three mice are still alive.)

pmcid: 3494882

Fig. P1. - Effect of various combinations of anti-HER2 and anti-VEGFR2 treatments on the growth of established breast cancer brain metastases. Breast cancers were allowed to grow to roughly 10 mm3 in size in the brain parenchyma before the initiation of treatment, which consisted of either a dual or triple combination of anti-HER2 agents (trastuzumab and lapatinib) and an anti-VEGFR2 antibody (DC101). (A) MRI scans of control and the combination treatment of trastuzumab and DC101 at days 0, 5, and 14. Arrows indicate tumor location. (B) Bioluminescence images of control- and trastuzumab plus DC101-treated mice at day 14. ph, photons; sr, steradian. (C) Tumor growth plot (Upper, mean ± SEM) and Kaplan–Meier survival plot (Lower) of tumor-bearing mice treated with control (black); trastuzumab plus DC101 (magenta); lapatinib plus DC101 (orange); trastuzumab plus lapatinib (brown); and the triple combination of trastuzumab, lapatinib, and DC101 (cyan) (n = 6–9 mice). RLU/s, Relative Light Units per second.

Ultrasound-mediated blood-brain/blood-tumor barrier disruption improves outcomes with trastuzumab in a breast cancer brain metastasis model

pmcid: 3502612

doi: 10.1016/j.jconrel.2012.09.007

abstract: Trastuzumab has shown positive results in many patients with metastatic HER2-positive breast cancer, but it is less effective for controlling metastases in the CNS, which remains a site of relapse. The poor prognosis for patients with brain metastases is thought to be largely due to the presence of the blood-brain barrier (BBB) that prevents delivery of most drugs to the CNS and to the heterogeneous and limited permeability of the blood-tumor barrier (BTB). Focused ultrasound (FUS) bursts combined with circulating microbubbles can temporarily permeabilize both the BBB and the BTB. This technique has been investigated as a potential noninvasive method for targeted drug delivery in the brain. Here, we investigated whether BBB/BTB permeabilization in the tumor and surrounding brain tissue induced by FUS and microbubbles can slow tumor growth and improve survival in a breast cancer brain metastases model. HER2/neu-positive human breast cancer cells (BT474) were inoculated in the brains of 41 nude (nu/nu) rats. Animals in the treatment group received six weekly treatments of BTB/BBB permeabilization under MRI guidance combined with IV administration of trastuzumab (2 mg/kg). Tumor growth and survival rates were monitored via MRI for seven weeks after sonication. Starting at week seven and continuing through the end of the study, the mean tumor volume of the FUS+trastuzumab group was significantly (P<0.05) less than those of the three control groups (no treatment, FUS alone, trastuzumab alone). Furthermore, in four out of 10 rats treated with FUS+trastuzumab, the tumor appeared to be completely resolved in MRI, an outcome which was not observed in any of the 31 rats in three control groups. Trastuzumab improved median survival by 13% compared to the no treatment group, a difference which was significant (P=0.044). Treatment with FUS+trastuzumab produced the most significant benefit compared to the no-treatment controls (P=0.0084). More than half (6/10) animals survived at the study endpoint, leading to a median survival time greater than 83 days (at least 32% longer than the untreated control group). Overall, this work suggests that BBB/BTB permeabilization induced by FUS and microbubbles can improve outcomes in breast cancer brain metastases.

pmcid: 3502612

Fig. 4 - Tumor volume measurements for each group over 13 weeks. The measurements were obtained in MRI acquired before each of the six treatments and weekly afterwards. (a) Tumor volume vs. time; (b) Measurements normalized to the volume measured at week 1; (c) Individual measurements for all tumors at week 7 (the last week where all animals survived). The mean tumor growth with FUS+trastuzumab was substantially reduced compared to the control groups. Tumor volume was significantly less (P<0.05) than each of the three control groups at week 7. This improvement was largely driven by four animals where the tumor appeared in MRI to be completely resolved; tumor growth for the six “non-responders” was similar to the trastuzumab-only group. At the study endpoint (week 13), only the four “responders” survived. The tumor volume at week 1 was approximately 2 mm3 (mean ± standard error shown).

Anti-HER3 Domain 1 and 3 Antibodies Reduce Tumor Growth by Hindering HER2/HER3 Dimerization and AKT-Induced MDM2, XIAP, and FoxO1 Phosphorylation12

pmcid: 3593156

doi:

abstract: Blockade of the human epidermal growth factor receptor 3 (HER3) and of the downstream phosphatidylinositide 3-kinase (PI3K)/AKT pathway is a prerequisite for overcoming drug resistance and to develop novel treatments for cancers that are not eligible for the currently approved targeted therapies. To this end, we generated specific antibodies (Abs) against domain 1 (D1) and domain 3 (D3) of HER3 that recognize epitopes that do not overlap with the neuregulin-binding site. The fully human H4B-121 Ab and the mouse monoclonal Abs 16D3-C1 and 9F7-F11 inhibited tumor growth in nude mice xenografted with epidermoid, pancreatic, or triple-negative breast cancer cells. The combination of one anti-HER3 Ab and trastuzumab improved tumor growth inhibition in mice xenografted with HER2low cancer cell lines, for which trastuzumab alone shows no or moderate efficiency. Ab-induced disruption of tumor growth was associated with G1 cell cycle arrest, proliferation inhibition, and apoptosis of cancer cells. Anti-HER3 Abs blocked HER2/HER3 heterodimerization and HER3 phosphorylation at the cell membrane, leading to inhibition of phosphorylation of the downstream AKT targets murine double minute 2, X-linked inhibitor of apoptosis, and forkhead box O1. This study demonstrates that anti-HER3 D1 and D3 Abs could represent a new option for immunotherapy of pancreatic and triple-negative breast cancers.

pmcid: 3593156

Figure 5 - Dual treatment with the anti-HER3 D1 Ab 16D3-C1 and trastuzumab (●) improves tumor growth inhibition in nude mice xenografted with HER2low A431 epidermoid (A) or A549 lung (B) carcinoma cells in comparison to mice treated with vehicle (NaCl; ), 16D3-C1 () or trastuzumab (○) alone. Tumor growth data are presented as the mean tumor volume ± SEM for each group of eight nude mice.

A single proteolytic cleavage within the lower hinge of trastuzumab reduces immune effector function and in vivo efficacy

pmcid: 3680949

doi: 10.1186/bcr3240

abstract: IntroductionRecent studies reported that human IgG antibodies are susceptible to specific proteolytic cleavage in their lower hinge region, and the hinge cleavage results in a loss of Fc-mediated effector functions. Trastuzumab is a humanized IgG1 therapeutic monoclonal antibody for the treatment of HER2-overexpressing breast cancers, and its mechanisms of action consist of inhibition of HER2 signaling and Fc-mediated antibody-dependent cellular cytotoxicity (ADCC). The objective of this study is to investigate the potential effect of proteinase hinge cleavage on the efficacy of trastuzumab using both a breast cancer cell culture method and an in vivo mouse xenograft tumor model.MethodsTrastuzumab antibody was incubated with a panel of human matrix metalloproteinases, and proteolytic cleavage in the lower hinge region was detected using both western blotting and mass spectrometry. Single hinge cleaved trastuzumab (scIgG-T) was purified and evaluated for its ability to mediate ADCC and inhibition of breast cancer cell proliferation in vitro as well as anti-tumor efficacy in the mouse xenograft tumor model. Infiltrated immune cells were detected in tumor tissues by immunohistochemistry.ResultsscIgG-T retains HER2 antigen binding activity and inhibits HER2-mediated downstream signaling and cell proliferation in vitro when compared with the intact trastuzumab. However, scIgG-T lost Fc-mediated ADCC activity in vitro, and had significantly reduced anti-tumor efficacy in a mouse xenograft tumor model. Immunohistochemistry showed reduced immune cell infiltration in tumor tissues treated with scIgG-T when compared with those treated with the intact trastuzumab, which is consistent with the decreased ADCC mediated by scIgG-T in vitro.ConclusionTrastuzumab can be cleaved by matrix metalloproteinases within the lower hinge. scIgG-T exhibited a significantly reduced anti-tumor efficacy in vivo due to the weakened immune effector function such as ADCC. The results suggest that the lower hinge cleavage of trastuzumab can occur in the tumor microenvironment where matrix metalloproteinases often have high levels of expression and scIgG-T might compromise its anti-tumor efficacy in the clinic. However, further studies are needed to validate these hypotheses in the clinical setting.

pmcid: 3680949

Figure 5 - Trastuzumab and single hinge cleaved trastuzumab tumor inhibition efficacy in breast cancer xenograft mouse model. (A) BALB/c nu/nu mice (n = 5) were subcutaneously inoculated with 5×106 BT474 human breast cancer cells. Mice were treated with the antibodies at 5 mg/kg weekly for a total of five doses when tumors reached an average size of 100 mm3. Tumor sizes were measured and compared among the treatment groups. *P < 0.05 and **P < 0.005. (B) Tumor lysates were prepared from the frozen xenograft tumor tissues. Total HER2 expression, pHER2 (Y1248), pAKT (S473), and pErk1/2 were determined by western blotting. scIgG-T, single hinge cleaved trastuzumab.

Autophagy-related gene 12 (ATG12) is a novel determinant of primary resistance to

HER2-targeted therapies: Utility of transcriptome analysis of the autophagy interactome to guide breast cancer treatment pmcid: 3681498

doi:

abstract: The autophagic process, which can facilitate breast cancer resistance to endocrine, cytotoxic, and molecularly targeted agents, is mainly regulated at the post-translational level. Although recent studies have suggested a possible transcriptome regulation of the autophagic genes, little is known about either the analysis tools that can be applied or the functional importance of putative candidate genes emerging from autophagy-dedicated transcriptome studies. In this context, we evaluated whether the constitutive activation of the autophagy machinery, as revealed by a transcriptome analysis using an autophagy-focused polymerase chain reaction (PCR) array, might allow for the identification of novel autophagy-specific biomarkers for intrinsic (primary) resistance to HER2-targeted therapies. Quantitative real-time PCR (qRT-PCR)-based profiling of 84 genes involved in autophagy revealed that, when compared to trastuzumab-sensitive SKBR3 cells, the positive regulator of autophagic vesicle formation ATG12 (autophagy-related gene 12) was the most differentially up-regulated gene in JIMT1 cells, a model of intrinsic cross-resistance to trastuzumab and other HER1/2-targeting drugs. An analysis of the transcriptional status of ATG12 in > 50 breast cancer cell lines suggested that the ATG12 transcript is commonly upregulated in trastuzumab-unresponsive HER2-overexpressing breast cancer cells. A lentiviral-delivered small hairpin RNA stable knockdown of the ATG12 gene fully suppressed the refractoriness of JIMT1 cells to trastuzumab, erlotinib, gefitinib, and lapatinib in vitro. ATG12 silencing significantly reduced JIMT1 tumor growth induced by subcutaneous injection in nude mice. Remarkably, the outgrowth of trastuzumab-unresponsive tumors was prevented completely when trastuzumab treatment was administered in an ATG12-silenced genetic background. We demonstrate for the first time the usefulness of low-density, autophagy-dedicated qRT-PCR-based platforms for monitoring primary resistance to HER2-targeted therapies by transcriptionally screening the autophagy interactome. The degree of predictive accuracy warrants further investigation in the clinical situation.

pmcid: 3681498

Figure 5 - Shown are the mean tumor volumes (±SD) in JIMT1 and ATG12 shRNA-JIMT1 xenograft-bearing nude mice following injection with trastuzumab (5 mg/kg/week) for nine weeks. Data from control shRNA-JIMT1 cells were superimposable with those obtained in JIMT1 parental cells and have been omitted for simplicity.

The anti-malarial chloroquine overcomes Primary resistance and restores sensitivity to Trastuzumab in HER2-positive breast cancer

pmcid: 3749547

doi: 10.1038/srep02469

abstract: Autophagy may control the de novo refractoriness of HER2 gene-amplified breast carcinomas to the monoclonal antibody trastuzumab (Herceptin). Tumor cells originally obtained from a patient who rapidly progressed on trastuzumab ab initio display increased cellular levels of the LC3-II protein—a finding that correlates with increased numbers of autophagosomes—and decreased levels of the autophagy receptor p62/SQSTM1, a protein selectively degraded by autophagy. Trastuzumab-refractory cells are in a state of “autophagy addiction” because genetic ablation of autophagy-specific genes (ATG8, ATG5, ATG12) notably reduces intrinsic refractoriness to trastuzumab. When the anti-malarial lysosomotropic drug chloroquine impedes autophagic resolution of the accumulation of autophagolysosomes formed in the presence of trastuzumab, cells commit to die by apoptosis. Accordingly, combination treatment with trastuzumab and chloroquine radically suppresses tumor growth by > 90% in a tumor xenograft completely refractory to trastuzumab. Adding chloroquine to trastuzumab-based regimens may therefore improve outcomes among women with autophagy-addicted HER2-positive breast cancer.

pmcid: 3749547

Figure 5 - Left. Shown are the mean tumor volumes (±SD) in JIMT-1 xenograft-bearing nude mice following i.p. injection with trastuzumab (5 mg/kg/week) and/or chloroquine (20 ng/kg/daily) for nine weeks. Note that tumor volumes in the trastuzumab + chloroquine group were drastically reduced in a synergistic manner compared to the untreated control group and with the single agent treatments. Changes in Bax:Bcl-2 ratios in JIMT-1 xenografts treated with trastuzumab in the absence or presence of chloroquine are also shown. The results are presented as the means (columns) and 95% confidence intervals (bars) of two independent western blotting experiments following densitometric analyses of Bax and Bcl-2 proteins (calculations were based on the arbitrary values of the bands to show the Bax:Bcl-2 ratio in each tumor). Statistically significant difference (one-factor ANOVA analysis) between experimental condition groups and control groups are shown. Right. HER2 gene-amplified breast cancer cells with inherent (primary) resistance to trastuzumab are addicted to autophagy as the need for complementary bioenergetic resources in response to HER1/2-targeting drugs, which significantly affect cancer cells' addiction to certain fuel sources and metabolic pathways (“metabolic reprogramming”), is satisfied by the autophagy's ability to promote large-scale recycling of cytoplasmic macromolecules and organelles including mitochondria to regenerate energy and building blocks. In addition, protective autophagy in trastuzumab-refractory breast cancer cells appears to involve a rapid targeting of HER2 protein aggregates for degradation in the autophagolysosome. In this scenario, the concurrent combination of trastuzumab with pharmacologically (i.e., chloroquine)-induced autophagy dysfunction, with blockade of autophagosome and lysosome function, and accumulation of autophagosomes and autophagy substrates, can lead to an aberrant accumulation of unmetabolized substrate with deleterious consequences (i.e., apoptotic cell death) in autophagy-addicted, trastuzumab-refractory breast cancer cells.

Single-Antibody, Targeted Nanoparticle Delivery of Camptothecin

pmcid: 3795804

doi: 10.1021/mp300702x

abstract: We have developed a new method for assembling targeted nanoparticles that utilizes the complexation between targeting agents that contain boronic acids and polymer-drug conjugates that possess diols. Here, we report the first in vivo, antitumor results of a nanoparticle formed via this new assembly methodology. A nanoparticle consisting of a mucic acid polymer conjugate of camptothecin (CPT), MAP-CPT; and containing on average one Herceptin antibody is investigated in nude mice bearing HER2 overexpressing BT-474 human breast cancer tumors. Nontargeted MAP-CPT and antibody-containing MAP-CPT nanoparticles of ca. 30–40 nm diameter and slightly negative zeta potential show prolonged in vivo circulation and similar biodistributions after intravenous tail vein injections in mice. The maximum tolerated dose (MTD) of the nontargeted and Herceptin-containing MAP-CPT nanoparticles are found to be 10 and 8 mg CPT/kg, respectively, in mice. Mice bearing BT-474 human breast tumors treated with nontargeted MAP-CPT nanoparticles at 8 mg CPT/kg show significant tumor growth inhibition (mean tumor volume of 63 mm3) when compared to Irinotecan at 80 mg/kg (mean tumor volume of 575 mm3) and CPT at 8 mg/kg (mean tumor volume of 808 mm3) at the end of the study. Herceptin antibody treatment at 5.9 mg/kg results in complete tumor regressions in 5 out of 8 mice, with a mean tumor volume of 60 mm3 at the end of the study. Mice treated with MAP-CPT nanoparticles at 1 mg CPT/kg do not show tumor inhibition. However, all mice receiving administrations of MAP-CPT nanoparticles (1 mg CPT/kg) that contain on average a single Herceptin molecule per nanoparticle (5.9 mg Herceptin equivalent/kg) show complete tumor regression by the end of the study. These results demonstrate that the antitumor efficacy of nanoparticles carrying anticancer drugs can be enhanced by incorporating on average a single antibody.

pmcid: 3795804

Figure 4 - Antitumor efficacy studies in female NCr nude mice bearing BT-474 xenograft tumors. Mean tumor volumes as functions of time. Groups containing Herceptin received 2 weekly doses, all other groups received 3 weekly doses.

Optical metabolic imaging identifies glycolytic levels, sub-types and early treatment response in breast cancer

pmcid: 3801432

doi: 10.1158/0008-5472.CAN-13-0527

abstract: Abnormal cellular metabolism is a hallmark of cancer, yet there is an absence of quantitative methods to dynamically image this powerful cellular function. Optical metabolic imaging (OMI) is a non-invasive, high-resolution, quantitative tool for monitoring cellular metabolism. OMI probes the fluorescence intensities and lifetimes of the autofluorescent metabolic co-enzymes reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD). We confirm that OMI correlates with cellular glycolytic levels across a panel of human breast cell lines, using standard assays of cellular rates of glucose uptake and lactate secretion (p<0.05, r=0.89). Additionally, OMI resolves differences in the basal metabolic activity of untransformed from malignant breast cells (p<0.05), and between breast cancer sub-types (p<0.05), defined by estrogen receptor (ER) and/or HER2 expression or absence. In vivo OMI is sensitive to metabolic changes induced by inhibition of HER2 with the antibody trastuzumab (Herceptin) in HER2-overexpressing human breast cancer xenografts in mice. This response was confirmed with tumor growth curves and stains for Ki67 and cleaved caspase-3. OMI resolved trastuzumab-induced changes in cellular metabolism in vivo as early as 48 hours post-treatment (p<0.05), while FDG-PET did not resolve any changes with trastuzumab up to 12-days post-treatment (p>0.05). In addition, OMI resolved cellular sub-populations of differing response in vivo that are critical for investigating drug resistance mechanisms. Importantly, OMI endpoints remained unchanged with trastuzumab-treatment in trastuzumab-resistant xenografts (p>0.05). OMI has significant implications for rapid cellular-level assessment of metabolic response to molecular expression and drug action, which would greatly accelerate drug development studies.

pmcid: 3801432

Figure 6 - (a) HR6 tumor size (mean +/− SE) treated with trastuzumab (10 mg/kg, 2 × weekly) compared to control IgG treated tumors. (b) Ki67 staining of HR6 control IgG and trastuzumab-treated tumors. (c) Cleaved Caspase 3 staining of HR6 control IgG and trastuzumab-treated tumors. (d) Representative FDG-PET image (T=tumor). (e) FDG uptake increases in control tumors at day 12 compared to control tumors at day 2, * P<0.05. No significant difference in FDG uptake between trastuzumab-treated and control tumors is observed at any time. n = 10. (f) Representative OMI images, scale bar is 50 μm.. (g) Redox ratio, (h) NADH τm, and (i) FAD τm of control and trastuzumab-treated tumors. * P<0.05; n = 6 tumors.

The anti-erbB3 antibody MM-121/SAR256212 in combination with trastuzumab exerts potent antitumor activity against trastuzumab-resistant breast cancer cells

pmcid: 3829386

doi: 10.1186/1476-4598-12-134

abstract: BackgroundElevated expression of erbB3 receptor has been reported to induce resistance to therapeutic agents, including trastuzumab in erbB2-overexpressing breast cancer. Our recent studies indicate that erbB3 interacts with both erbB2 and IGF-1 receptor to form a heterotrimeric complex in trastuzumab-resistant breast cancer cells. Herein, we investigate the antitumor activity of MM-121/SAR256212, a fully human anti-erbB3 antibody (Ab), against two erbB2-overexpressing breast cancer cell lines resistant to trastuzumab.MethodsMTS-based proliferation assays were used to determine cell viability upon treatment of trastuzumab and/or MM-121/SAR256212. Cell cycle progression was examined by flow cytometric analysis. Western blot analyses were performed to determine the expression and activation of proteins. Tumor xenografts were established by inoculation of the trastuzumab-resistant BT474-HR20 cells into nude mice. The tumor-bearing mice were treated with trastuzumab and/or MM-121/SAR256212 via i.p injection to determine the Abs’ antitumor activity. Immunohistochemical analyses were carried out to study the Abs’ inhibitory effects on tumor cell proliferation and induction of apoptosis in vivo.ResultsMM-121 significantly enhanced trastuzumab-induced growth inhibition in two sensitive and two resistant breast cancer cell lines. MM-121 in combination with trastuzumab resulted in a dramatic reduction of phosphorylated erbB3 (P-erbB3) and Akt (P-Akt) in the in vitro studies. MM-121 combined with trastuzumab did not induce apoptosis in the trastuzumab-resistant cell lines under our cell culture condition, rather induced cell cycle G1 arrest mainly associated with the upregulation of p27kip1. Interestingly, in the tumor xenograft model established from the trastuzumab-resistant cells, MM-121 in combination with trastuzumab as compared to either agent alone dramatically inhibited tumor growth correlated with a significant reduction of Ki67 staining and increase of cleaved caspase-3 in the tumor tissues.ConclusionsThe combination of MM-121 and trastuzumab not only inhibits erbB2-overexpressing breast cancer cell proliferation, but also promotes the otherwise trastuzumab-resistant cells undergoing apoptosis in an in vivo xenografts model. Thus, MM-121 exhibits potent antitumor activity when combined with trastuzumab under the studied conditions. Our data suggest that further studies regarding the suitability of MM-121 for treatment of breast cancer patients whose tumors overexpress erbB2 and become resistant to trastuzumab may be warranted.

pmcid: 3829386

Figure 5 - MM-121 in combination with trastuzumab significantly inhibits in vivo growth of tumor xenografts established from BT474-HR20 trastuzumab-resistant breast cancer cells. BT474-HR20 cells were s.c. injected into nude mice to establish tumor xenografts. The tumor-bearing mice (n=5) received i.p. injections of PBS, trastuzumab, MM-121, or trastuzumab plus MM-121 as described in the Materials and Methods. After 6 treatments, the mice were euthanized at day 36 post injection of tumor cells, and all tumors were excised for histology and IHC analysis. A, The graphs show the tumor growth curves. Bars, SD. The combinations of MM-121 and trastuzumab significantly inhibited tumor growth as compared to control or single Ab treatment. B, Data show the representative tumors with hematoxylin and eosin (H&E) staining and IHC analysis of erbB2 and erbB3. The residual tumor cells obtained from combinatorial treatments retained similar expression levels of erbB2/erbB3 receptors on the cell membrane.

Combined blockade of HER2 and VEGF exerts greater growth inhibition of HER2-overexpressing gastric cancer xenografts than individual blockadeHER2 and VEGF dual blockade in gastric cancer

pmcid: 3849567

doi: 10.1038/emm.2013.111

abstract: Gastric cancer overexpressing the human epidermal growth factor 2 (HER2) protein has a poor outcome, although a combination of chemotherapy and the anti-HER2 antibody trastuzumab has been approved for the treatment of advanced gastric cancer. Vascular endothelial growth factor (VEGF) expression in gastric cancer is correlated with recurrence and poor prognosis; however, the anti-VEGF antibody bevacizumab has shown limited efficacy against gastric cancer in clinical trials. In this study, we evaluated the antitumor effects of trastuzumab; VEGF-Trap binding to VEGF-A, VEGF-B and placental growth factor (PlGF); and a combination of trastuzumab and VEGF-Trap in a gastric cancer xenograft model. Although trastuzumab and VEGF-Trap each moderately inhibited tumor growth, the combination of these agents exerted greater inhibition compared with either agent alone. Immunohistochemical analyses indicated that the reduction in tumor growth was associated with decreased proliferation and increased apoptosis of tumor cells and decreased tumor vascular density. The combined treatment resulted in fewer proliferating tumor cells, more apoptotic cells and reduced tumor vascular density compared with treatment with trastuzumab or VEGF-Trap alone, indicating that trastuzumab and VEGF-Trap had additive inhibitory effects on the tumor growth and angiogenesis of the gastric cancer xenografts. These data suggest that trastuzumab in combination with VEGF-Trap may represent an effective approach to treating HER2-overexpressing gastric cancer.

pmcid: 3849567

Figure 4 - The combined inhibition of human epidermal growth factor 2 (HER2) and vascular endothelial growth factor (VEGF) reduces tumor growth more efficiently than single-agent inhibition. (a) Mice bearing NCI-N87Luc+ tumors were divided into four groups and treated with an isotype-control antibody, VEGF-Trap (VT), trastuzumab (Tr) or a combination of VEGF-Trap and trastuzumab (VT+Tr) for 28 days. (b) Changes in body weight. No significant change in body weight was observed among the treatment groups. (c) Dissected tumor weight after 28 days (n=7; *P<0.05 versus isotype control; #P<0.05 versus VEGF-Trap; $P<0.05 versus trastuzumab). IgG, immunoglobulin G.

Neratinib overcomes trastuzumab resistance in HER2 amplified breast cancer

pmcid: 3858548

doi:

abstract: Trastuzumab has been shown to improve the survival outcomes of HER2 positive breast cancer patients. However, a significant proportion of HER2-positive patients are either inherently resistant or develop resistance to trastuzumab. We assessed the effects of neratinib, an irreversible panHER inhibitor, in a panel of 36 breast cancer cell lines. We further assessed its effects with or without trastuzumab in several sensitive and resistant breast cancer cells as well as a BT474 xenograft model. We confirmed that neratinib was significantly more active in HER2-amplified than HER2 non-amplified cell lines. Neratinib decreased the activation of the 4 HER receptors and inhibited downstream pathways. However, HER3 and Akt were reactivated at 24 hours, which was prevented by the combination of trastuzumab and neratinib. Neratinib also decreased pHER2 and pHER3 in acquired trastuzumab resistant cells. Neratinib in combination with trastuzumab had a greater growth inhibitory effect than either drug alone in 4 HER2 positive cell lines. Furthermore, trastuzumab in combination with neratinib was growth inhibitory in SKBR3 and BT474 cells which had acquired resistance to trastuzumab as well as in a BT474 xenograft model. Innately trastuzumab resistant cell lines showed sensitivity to neratinib, but the combination did not enhance response compared to neratinib alone. Levels of HER2 and phospho-HER2 showed a direct correlation with sensitivity to neratinib. Our data indicate that neratinib is an effective anti-HER2 therapy and counteracted both innate and acquired trastuzumab resistance in HER2 positive breast cancer. Our results suggest that combined treatment with trastuzumab and neratinib is likely to be more effective than either treatment alone for both trastuzumab-sensitive breast cancer as well as HER2-positive tumors with acquired resistance to trastuzumab.

pmcid: 3858548

Figure 6 - A) Left, Mice bearing BT474 xenograft tumors were treated with either control (vehicle), neratinib, trastuzumab or their combination for 16 days. During this time, tumor size was measured in each mouse and the tumor volume calculated. B) At the end of the experiment from A, tumor samples were collected and embedded in paraffin. The slides were cut and analysed for the percentage of connective tissue per section. The differences in means of the percentage of connective tissue between the groups were analyzed by Anova with Bonferroni's multiple comparison test and statistically significant changes were indicated in the figure. C-F), Sections were cut from paraffin-embedded xenograft samples and stained for HER2, phosphorylated HER2 (pHER2), phosphorylated Akt (pAkt) and phosphorylated ERK (pERK). Sections were then scored for the intensity and percentage of staining. The IRS scoring from each condition was shown in the figures.

Combating trastuzumab resistance by targeting SRC, a common node downstream of multiple resistance pathways

pmcid: 3877934

doi: 10.1038/nm.2309

abstract: Trastuzumab is a successful rationally designed ERBB2-targeted therapy. However, about half of individuals with ERBB2-overexpressing breast cancer do not respond to trastuzumab-based therapies, owing to various resistance mechanisms. Clinically applicable regimens for overcoming trastuzumab resistance of different mechanisms are not yet available. We show that the nonreceptor tyrosine kinase c-SRC (SRC) is a key modulator of trastuzumab response and a common node downstream of multiple trastuzumab resistance pathways. We find that SRC is activated in both acquired and de novo trastuzumab-resistant cells and uncover a novel mechanism of SRC regulation involving dephosphorylation by PTEN. Increased SRC activation conferred considerable trastuzumab resistance in breast cancer cells and correlated with trastuzumab resistance in patients. Targeting SRC in combination with trastuzumab sensitized multiple lines of trastuzumab-resistant cells to trastuzumab and eliminated trastuzumab-resistant tumors in vivo, suggesting the potential clinical application of this strategy to overcome trastuzumab resistance.

pmcid: 3877934

Figure 1 - SRC hyperactivation is a key signaling alteration in acquired trastuzumab-resistant cells. (a) MTS assay comparing cell proliferation of indicated parental breast cancer cell lines and their corresponding acquired TtzmR sublines upon treatment with freshly added trastuzumab (Ttzm, 2 µg ml−1) for 4 d. (b) Tumor volume of mammary fat pad xenografts derived from either BT474 parental (P) or TtzmR subline upon treatment of IgG control or Ttzm (10 mg per kg body weight, intraperitoneally) weekly. Tumor volume at various times of treatment is presented as percentage of original tumor size at day 0 of treatment. (c) Representative histograms from flow cytometric analysis of EGFR and ERBB2 abundance in BT474 parental and TtzmR cells. (d) Relative amounts of EGFR, ERBB2, HER3 and IGF-1R in the indicated parental and TtzmR cells analyzed by flow cytometry. (e) Immunoblots comparing major cell signaling changes between the indicated parental and TtzmR sublines. P indicates phosphorylation; for example, P-EGFR-Y1068 is EFGR phosphorylated at Tyr1068 (f) Immunoblots assessing the impact of overexpression of EGFR and IGF-1R in BT474 parental (BT474.P) cells on signaling. (g) MTS assay evaluating trastuzumab resistance of BT474 cells overexpressing EGFR or IGF-1R, treated as in a. (h) Immunoblots of EGFR and phosphorylated SRC after shRNA-mediated EGFR knockdown in BT474.TtzmR cells. (i) MTS assay evaluating sensitivity of TtzmR to trastuzumab after EGFR knockdown. Cells were treated as in a. (j) Left, immunoblots of knock-down of SRC in BT474.TtzmR cells by SRC shRNA. Right, MTS assay assessing trastuzumab sensitivity of TtzmR cells after SRC knockdown. All error bars, s.e.m. All quantitative data were generated from a minimum of three replicates.

pmcid: 3877934

Figure 3 - SRC is a key modulator of trastuzumab response. (a) Immunoblots comparing SRC phosphorylation status in the indicated cells expressing a constitutively active SRC mutant (Y527F) or a kinase-dead SRC mutant (K295R). (b) MTS assay assessing trastuzumab sensitivity of cells transfected with SRC Y527F or SRC K295R mutant. Cells were treated as in Figure 1a. Error bars, s.e.m. (c) 3D tumor spheroid assay comparing response to trastuzumab treatment of BT474.GFP and BT474.SRC Y527F. 3D tumor spheroid assay was carried out as described in Online Methods. Scale bar, 100 µm. (d) Top, representative BT474 orthotopic xenograft tumors. Scale bar, 1 cm. Bottom, volume of mammary fat pad xenograft tumors derived from either GFP-labeled BT474 parental (GFP) or SRC Y527F–expressing cells upon treatment with IgG or Ttzm. Tumor volume at various times of treatment is percentage of original tumor size at day zero of treatment. Error bars, s.e.m. Ttzm-treated GFP group versus SRC Y527F group (ANOVA, P < 0.001, two-sided). (e) Correlation between clinical response rate and amount of tumor phospho-SRC-Y416 (pSRC) in patients who received first-line trastuzumab-based therapy. Complete response (CR), partial response (PR) and stable disease (SD) were grouped together and compared with SD. Patient response was compared by Fisher’s exact test (P = 0.011, two-sided). (f) Low versus high tumor phospho-SRC-Y416 abundance and overall survival of patients who received first-line trastuzumab-based therapy. Difference of overall survival was analyzed by Kaplan-Meier survival model with log-rank test (P = 0.044, two-sided).

pmcid: 3877934

Figure 6 - Trastuzumab plus saracatinib combinatorial treatment overcomes trastuzumab resistance in vivo. (a) Top, representative immunofluorescence images of SRC knockdown in PTEN.shRNA xenografts using intratumoral injection of SRC.shRNA-containing virus. Scale bar, 100 µm. Bottom, volume of trastuzumab-resistant PTEN-deficient tumors with or without SRC knockdown upon treatment with IgG or Ttzm. Tumor volume at various times of treatment is presented as percentage of original tumor size at day zero of treatment. (b) Top, representative immunohistochemistry (IHC) images of in vivo inhibition of SRC-Y416 phosphorylation by saracatinib or AKT-S473 phosphorylation by triciribine in BT474.TtzmR xenograft tumors. Scale bar, 100 µm. Bottom, TtzmR xenograft tumor volume in response to different treatments. (c) Left, representative in vivo luciferase images of mice at day 0 and 21 days after indicated treatment. Left side of animal, BT474 control.shRNA tumors; right side, BT474 PTEN.shRNA tumors. Right, tumor volume in response to different treatments. (d) Left, representative IHC staining of AKT-S473 phosphorylation after different treatments (vehicle or trastuzumab plus saracatinib) in BT474.PTEN.shRNA xenograft tumors. Scale bar, 50 µm. Right, overall AKT-S473 phosphorylation IHC staining intensity between trastuzumab-alone group and combination-treatment group. Phospho-AKT (pAKT) staining was compared between each group by Fisher’s exact test (P = 0.049, two-sided). (e) Top, representative tumor sections with TUNEL staining. Scale bar, 50 µm. Bottom, in situ TUNEL staining of apoptotic cells in tumors treated as indicated. All error bars, s.e.m. All in vivo data were generated from a minimum of five replicates. (f) Model of SRC as a common node downstream of multiple resistance pathways and conquering trastuzumab resistance by targeting SRC.

Epigenetic silencing of miR-375 induces trastuzumab resistance in HER2-positive breast cancer by targeting IGF1R

pmcid: 3974046

doi: 10.1186/1471-2407-14-134

abstract: BackgroundResistance to humanized monoclonal erbB2/HER2 antibody, trastuzumab (Herceptin), has become a pivotal obstacle for targeted therapy of HER2-positive breast cancers. The activation of alternative growth factor receptors, in particular, the insulin-like growth factor 1 receptor (IGF1R), represents a common feature of trastuzumab-refractory cells; however, the underlying mechanism remains elusive.MethodsTrastuzumab-resistant breast cancer SKBr-3 cells were generated by long-term in vitro culture of SKBr-3 cells in the presence of trastuzumab. Among the differentially expressed microRNAs (miRNAs) screened by microarray analysis, candidate miRNA(s) predicted to target IGF1R was studied for its role in conferring trastuzumab resistance. The mechanism underlying decreased expression of IGF1R-targeted miRNA in refractory cells was also addressed.ResultsmiR-375, which was downregulated and predicted to target IGF1R in trastuzumab-resistant HER2-positive breast cancer cells, could indeed inhibit the cellular luciferase activity in a reporter construct containing the 3′-UTR of IGF1R. Overexpression of miR-375 restored the sensitivity of cells to trastuzumab, while inhibition of miR-375 conferred trastuzumab resistance on HER2-positive breast cancer cells. Blockade of DNA methylation and histone deacetylation restored the expression of miR-375 in trastuzumab-resistant cells. A reverse correlation between the levels of miR-375 and IGF1R was validated in clinical breast cancers.ConclusionsEpigenetic silencing of miR-375 causes the upregulation of IGF1R, which at least partially underlies trastuzumab resistance of breast cancer cells. Our study has implications for miR-375 as a potential target in combination with trastuzumab for treating HER2-positive breast cancers.

pmcid: 3974046

Figure 4 - miR-375 modulates trastuzumab resistance of HER2-positive breast cancer xenografts. Nude mice were inoculated in the mammary fat pad with trastuzumab-resistant SKBr-3 cells overexpressing pre-miR-375 or control pre-miRNA to allow tumor development. Mice were intravenously injected with 10 mg/kg trastuzumab twice a week. A. Tumor volume in the trastuzumab-treated mice. Data are represented as the mean±SD of six mice. *P <0.05. B. Tumor weight at the end of the treatment period (45 days after first trastuzumab injection). The bars represent the mean±SD of six mice. C. Kaplan-Meier survival curves of the trastuzumab-treated mice (n=6).

Engagement of immune effector cells by trastuzumab induces HER2/ERBB2 downregulation in cancer cells through STAT1 activation

pmcid: 4053225

doi: 10.1186/bcr3637

abstract: IntroductionTrastuzumab has been widely used for the treatment of human epidermal growth factor receptor 2 (HER2) overexpressing breast cancer for more than a decade. However, reports on the involvement of HER2 downregulation in trastuzumab’s mechanism of action are inconsistent. The aim of this study is to investigate if the dependence of trastuzumab-mediated cancer cell HER2 downregulation on immune effector cells represents a novel mechanism of action for trastuzumab.MethodsHER2 expression was evaluated by Western blotting, flow cytometry, and real-time polymerase chain reaction (PCR) in cell lysates from co-cultures of multiple cancer cell lines with peripheral blood mononuclear cells (PBMCs) in the presence or absence of trastuzumab. The engagement of immune cells by trastuzumab through Fc gamma receptors (FcγRs) was tested using three trastuzumab variants with compromised or no Fc (fragment crystallizable) functions and FcγRs blocking experiments. The engagement of immune cells by trastuzumab in HER2 downregulation was also evaluated in in vivo mouse xenograft tumor models.ResultsHER2 downregulation of cancer cells by trastuzumab occurred only when trastuzumab was actively engaged with immune cells and cancer cells, as demonstrated consistently in co-cultures of cancer cell lines with PBMCs and in vivo mouse xenograft tumor models. We further demonstrated that HER2 downregulation in cancer cells by immune-cell-engaged trastuzumab was at the transcriptional level, not through the HER2 degradation pathway. Activation of signal transducer and activator of transcription 1 (STAT1) in cancer cells by the increased interferon gamma (IFN-γ) production in immune cells played an important role in downregulating HER2 in cancer cells upon engagement of immune cells by trastuzumab. Furthermore, HER2 downregulation in cancer cells induced by trastuzumab engagement of immune cells was correlated with the antibody’s antitumor efficacy in vivo.ConclusionsThis study reveals that engagement of immune effector cells by trastuzumab induces HER2 downregulation in HER2-expressing cancer cells, which represents a new function of immune cells in trastuzumab-mediated antitumor efficacy and serves as a novel mechanism of action for trastuzumab. Our results imply that HER2 downregulation in cancer cells treated by trastuzumab may predict active engagement of immune effector cells in tumor microenvironment.

pmcid: 4053225

Figure 5 - HER2 downregulation and tumor inhibition by trastuzumab in mouse xenograft tumor models. Tumors from different treatment groups (n=3) were collected one day after last administration of the antibody. (A) Formaldehyde-fixed BT474 tumor tissues were used for IHC detection of HER2. (B) The same amounts of tumor lysates were loaded on SDS-PAGE and HER2 was detected by WB. (C) Tumor inhibition after four weekly treatments with trastuzumab (tras) or the variant scIgG-T. The left bar graph is from the BT474 tumor model and the right graph is from the MCF7/HER2 tumor model. Percentage of tumor inhibition was calculated, **P <0.01. HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; scIgG-T, single hinge cleaved trastuzumab; WB, Western blotting.

In vivo hyperspectral imaging of microvessel response to trastuzumab treatment in breast cancer xenografts

pmcid: 4102362

doi: 10.1364/BOE.5.002247

abstract: HER2-amplified (HER2 + ) breast cancers are treated with the anti-HER2 monoclonal antibody trastuzumab. Although trastuzumab reduces production of the angiogenic factor VEGF in HER2 + tumors, the acute and sustained effects of trastuzumab on the tumor vasculature are not understood fully, particularly in trastuzumab-resistant tumors. We used mouse models of trastuzumab sensitive and trastuzumab-resistant HER2 + breast cancers to measure dynamic changes in tumor microvessel density and hemoglobin oxygenation (sO2) in vivo using quantitative hyperspectral imaging at 2, 5, 9, and 14 days after antibody treatment. Further analysis quantified the distribution of microvessels into low and high oxygenation groups, and monitored changes in these distributions with trastuzumab treatment. Gold standard immunohistochemistry was performed to validate complementary markers of tumor cell and vascular response to treatment. Trastuzumab treatment in both responsive and resistant tumors resulted in decreased sO2 5 days after initial treatment when compared to IgG-treated controls (p<0.05). Importantly, responsive tumors showed significantly higher vessel density and significantly lower sO2 than all other groups at 5 days post-treatment (p<0.05). Distribution analysis of vessel sO2 showed a significant (p<0.05) shift of highly oxygenated vessels towards lower oxygenation over the time-course in both trastuzumab-treated responsive and resistant tumors. This study suggests that longitudinal hyperspectral imaging of microvessel sO2 and density could distinguish trastuzumab-responsive from trastuzumab-resistant tumors, a finding that could be exploited in the post-neoadjuvant setting to guide post-surgical treatment decisions.

pmcid: 4102362

Fig. 1 - Tumor response in trastuzumab-responsive (a-c) and –resistant (d-f) xenografts treated with control IgG and trastuzumab. BT474 (trastuzumab-responsive) tumor growth curves (a). Percent of cells positive for Ki67 (% Ki67 + ), in BT474 tumors (b), and percent of cells positive for CC-3 (% cleaved caspase-3 + ), in BT474 tumors (c) at days 2, 5, and 14 post treatment. HR6 (trastuzumab-resistant) tumor growth curves (d). Ki67 immunohistochemistry in HR6 tumors (e), and CC-3 immunohistochemistry in HR6 tumors (f) at days 2, 5, and 14 post treatment. Green arrows in (a) and (d) indicate treatment timepoints (days 0, 3, 7, 10). Asterisks (*) indicate p<0.05 between control IgG and trastuzumab treated groups. Bar indicates mean with SEM error bars.(modified from Walsh et al, 2013 [31]).

Co-Treatment with Panitumumab and Trastuzumab Augments Response to the MEK Inhibitor Trametinib in a Patient-Derived Xenograft Model of Pancreatic Cancer1

pmcid: 4198828

doi: 10.1016/j.neo.2014.06.004

abstract: Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations and epidermal growth factor receptor (EGFR) family signaling are drivers of tumorigenesis in pancreatic ductal adenocarcinoma (PDAC). Previous studies have demonstrated that combinatorial treatment of PDAC xenografts with the mitogen-activated protein kinase–extracellular-signal-regulated kinase (ERK) kinase1/2 (MEK1/2) inhibitor trametinib and the dual EGFR/human epidermal growth factor receptor 2 (HER2) inhibitor lapatinib provided more effective inhibition than either treatment alone. In this study, we have used the therapeutic antibodies, panitumumab (specific for EGFR) and trastuzumab (specific for HER2), to probe the role of EGFR and HER2 signaling in the proliferation of patient-derived xenograft (PDX) tumors. We show that dual anti-EGFR and anti-HER2 therapy significantly augmented the growth inhibitory effects of the MEK1/2 inhibitor trametinib in three different PDX tumors. While significant growth inhibition was observed in both KRAS mutant xenograft groups receiving trametinib and dual antibody therapy (tumors 366 and 608), tumor regression was observed in the KRAS wild-type xenografts (tumor 738) treated in the same manner. Dual antibody therapy in conjunction with trametinib was equally or more effective at inhibiting tumor growth and with lower apparent toxicity than trametinib plus lapatinib. Together, these studies provide further support for a role for EGFR and HER2 in pancreatic cancer proliferation and underscore the importance of therapeutic intervention in both the KRAS–rapidly accelerated fibrosarcoma kinase (RAF)–MEK–ERK and EGFR-HER2 pathways to achieve maximal therapeutic efficacy in patients.

pmcid: 4198828

Figure 3 - In vivo response of PDX tumors to trametinib and antibody treatment. (A, C, and E) In vivo response of three different established PDAC tumors (100-500 mm3 before starting therapy) to treatment with vehicle control (black bar), trametinib (Tra), panitumumab (P), or trametinib plus panitumumab (Tra + P) (treated mice, hatched bars). The number of mice in each treatment group is indicated in parentheses. An MRI was obtained just before the start of treatment to establish an index tumor volume for each mouse. Subsequent interval MRI studies were used to assess the change in tumor volume while on treatment (see Figure W2). To calculate the relative change in tumor volume (fold change) for each tumor, the interval tumor volumes were divided by the index tumor volume and linear regression was used to model a line of best fit for the tumor fold change data plotted relative to time. The slope of that line of best fit demonstrates the tumor growth rate for each treatment group expressed as the fold change per week on treatment. Mean fold change per week plus the standard error of the mean are displayed for each treatment group as bar graphs. Significance is denoted as *P < .05. Following the initial treatment period, a group of mice treated with trametinib was maintained on trametinib. Mice in the panitumumab (P) group were switched to panitumumab plus trastuzumab (P + T) and mice in the trametinib plus panitumumab (Tra + P) group were switched to combined treatment with panitumumab, trastuzumab, and trametinib (Tra + P + T) for an additional 2 to 4 weeks (open bars, Figure W2). (B, D, and F) Representative MRI images of control and triple therapy–treated (Tra + P + T) tumors obtained just before the onset of treatment (Start Rx) and at the conclusion of treatment just before sacrifice (End Rx). The tumors are outlined and these images depict the axial slice with the largest cross-sectional area in each tumor.

pmcid: 4198828

Figure 4 - Comparison of in vivo response of PDX tumors to trametinib plus lapatinib versus trametinib plus antibody treatment. (A and B) In vivo response of two different PDAC tumors to treatment with vehicle control, trametinib plus lapatinib (Tra + L), trametinib plus pertuzumab (Tra + PZ), or trametinib plus panitumumab plus trastuzumab (Tra + P + T). Tumors were allowed to grow to a starting volume of 100 to 500 mm3 before the onset of treatment. Mice were treated with vehicle control or drug therapy for 4 weeks (the number of mice in each treatment group is indicated in parentheses). Initial tumor volume was assessed by MRI before the start of dosing and subsequent weekly MRI assessments were carried out to calculate the change in relative tumor volume (see also Figure W3). The mean relative tumor volume ± the standard error of the mean over time is plotted in line graph form for each treatment group. Linear regression was used to model a line of best fit for the tumor fold change data plotted relative to time. The slope of that line of best fit that served as an estimate of the tumor growth rate for each treatment group expressed as fold change per week on treatment was determined as described in the Materials and Methods section. The mean fold change per week ± the standard error of the mean are displayed for each treatment group as bar graphs. Significance is denoted as *P < .05.