Overcome Trastuzumab Resistance Research Articles

Feedback activation of STAT3 mediates trastuzumab resistance via upregulation of MUC1 and MUC4 expression.

Although HER2-targeting antibody trastuzumab confers a substantial benefit for patients with HER2-overexpressing breast and gastric cancer, overcoming trastuzumab resistance remains a large unmet need. In this study, we revealed a STAT3-centered positive feedback loop that mediates the resistance of trastuzumab. Mechanistically, chronic exposure of trastuzumab causes the upregulation of fibronection (FN), EGF and IL-6 in parental trastuzumab-sensitive breast and gastric cells and convergently leads to STAT3 hyperactivation. Activated STAT3 enhances the expression of FN, EGF and IL-6, thus constituting a positive feedback loop which amplifies and maintains the STAT3 signal; furthermore, hyperactivated STAT3 signal promotes the expression of MUC1 and MUC4, consequently mediating trastuzumab resistance via maintenance of persistent HER2 activation and masking of trastuzumab binding to HER2 respectively. Genetic or pharmacological inhibition of STAT3 disrupted STAT3-dependent positive feedback loop and recovered the trastuzumab sensitivity partially due to increased apoptosis induction. Combined trastuzumab with STAT3 inhibition synergistically suppressed the growth of the trastuzumab-resistant tumor xenografts in vivo. Taken together, our results suggest that feedback activation of STAT3 constitutes a key node mediating trastuzumab resistance. Combinatorial targeting on both HER2 and STAT3 may enhance the efficacy of trastuzumab or other HER2-targeting agents in HER2-positive breast and gastric cancer.

Src, a potential target for overcoming trastuzumab resistance in HER2-positive breast carcinoma

Background:Src is a non-receptor tyrosine kinase involved in signalling and crosstalk between growth-promoting pathways. We aim to investigate the relationship of active Src in response to trastuzumab of HER2-positive breast carcinomas.Methods:We selected 278 HER2-positive breast cancer patients with (n=154) and without (n=124) trastuzumab treatment. We performed immunohistochemistry on paraffin-embedded tissue microarrays of active Src and several proteins involved in the PI3K/Akt/mTOR pathway, PIK3CA mutational analysis and in vitro studies (SKBR3 and BT474 cancer cells). The results were correlated with clinicopathological factors and patients' outcome.Results:Increased pSrc-Y416 was demonstrated in trastuzumab-resistant cells and in 37.8% of tumours that correlated positively with tumour size, necrosis, mitosis, metastasis to the central nervous system, p53 overexpression and MAPK activation but inversely with EGFR and p27. Univariate analyses showed an association of increased active Src with shorter survival in patients at early stage with HER2/hormone receptor-negative tumours treated with trastuzumab.Conclusions:Src activation participates in trastuzumab mechanisms of resistance and indicates poor prognosis, mainly in HER2/hormone receptor-negative breast cancer. Therefore, blocking this axis may be beneficial in those patients.

Nuclear HER4 mediates acquired resistance to trastuzumab and is associated with poor outcome in HER2 positive breast cancer.

The role of HER4 in breast cancer is controversial and its role in relation to trastuzumab resistance remains unclear. We showed that trastuzumab treatment and its acquired resistance induced HER4 upregulation, cleavage and nuclear translocation. However, knockdown of HER4 by specific siRNAs increased trastuzumab sensitivity and reversed its resistance in HER2 positive breast cancer cells. Preventing HER4 cleavage by a γ-secretase inhibitor and inhibiting HER4 tyrosine kinase activity by neratinib decreased trastuzumab-induced HER4 nuclear translocation and enhanced trastuzumab response. There was also increased nuclear HER4 staining in the tumours from BT474 xenograft mice and human patients treated with trastuzumab. Furthermore, nuclear HER4 predicted poor clinical response to trastuzumab monotherapy in patients undergoing a window study and was shown to be an independent poor prognostic factor in HER2 positive breast cancer. Our data suggest that HER4 plays a key role in relation to trastuzumab resistance in HER2 positive breast cancer. Therefore, our study provides novel findings that HER4 activation, cleavage and nuclear translocation influence trastuzumab sensitivity and resistance in HER2 positive breast cancer. Nuclear HER4 could be a potential prognostic and predictive biomarker and understanding the role of HER4 may provide strategies to overcome trastuzumab resistance in HER2 positive breast cancer.

Combining trastuzumab and cetuximab combats trastuzumab-resistant gastric cancer by effective inhibition of EGFR/ErbB2 heterodimerization and signaling.

The anti-ErbB2 antibody trastuzumab has currently been approved for ErbB2-positive gastric cancer. Despite the effectiveness of trastuzumab, resistance is common. Thus, there is an urgent need to overcome trastuzumab resistance. Here, we obtain a trastuzumab-resistant cell line, which is derived from the human gastric cancer NCI-N87 cell line, by modeling the development of acquired resistance in patients. Our data show that combining trastuzumab and cetuximab leads to a significant decrease in EGFR/ErbB2 heterodimers and signaling compared with either antibody alone, and the combination results in greater antitumor activity against the trastuzumab-resistant NCI-N87 cell line, both in vitro and in vivo, suggesting that a combined EGFR/ErbB2 inhibition may overcome trastuzumab resistance.

Dual HER2 inhibition in combination with anti-VEGF treatment is active in heavily pretreated HER2-positive breast cancer

BackgroundPreclinical data indicate that dual HER2 inhibition overcomes trastuzumab resistance and that use of an HER2 inhibitor with an anti-angiogenic agent may augment responses. Patients and methodsWe conducted a dose-escalation, phase I study of a combination of trastuzumab, lapatinib and bevacizumab. The subset of patients with metastatic breast cancer was analyzed for safety and response. ResultsTwenty-six patients with metastatic breast cancer (median = 7 prior systemic therapies) (all with prior trastuzumab; 23 with prior lapatinib; one with prior bevacizumab) received treatment on a range of dose levels. The most common treatment-related grade 2 or higher toxicities were diarrhea (n = 11, 42%) and skin rash (n = 2, 8%). The recommended phase 2 dose was determined to be the full Food and Drug Administration (FDA) approved doses for all the three agents (trastuzumab 8 mg/kg loading dose, 6 mg/kg maintenance dose, intravenously every 3 weeks; lapatinib 1250 mg daily, bevacizumab 15 mg/kg intravenously every 3 weeks). The overall rate of stable disease (SD) ≥6 months and partial or complete remission (PR/CR) was 50% (five patients with SD ≥6 months; seven PRs (including one unconfirmed); one CR). The rate of SD ≥6 months/PR/CR was not compromised in patients who had previously received study drugs, those with brain metastases, and patients treated at lower dose levels. ConclusionsThe combination of trastuzumab, lapatinib and bevacizumab was well-tolerated at maximally approved doses of each drug, and its activity in heavily pretreated patients with metastatic breast cancer suggests that it warrants further investigation. ClinTrials.gov IDNCT00543504.

Abstract B243: Targeting HER-2 positive breast cancer by inducing apoptosis and immune cell activation, overcoming trastuzumab resistance.

Abstract Breast cancer is the most common cancer in women and the second leading cause of cancer-related deaths in females. Overexpression of HER-2 occurs in 20-30% of breast cancer patients and is associated with increased aggressiveness and significantly shortened disease-free and overall survival rates. Trastuzumab is the most commonly used adjuvant therapy for HER-2 positive breast cancer; however, therapeutic resistance often develops, likely due to tumor heterogeneity and a failure of effective immune response. Therefore, the overall survival rate of patients with HER-2 positive, metastatic breast cancer remains modest. In this study, we describe a novel targeted therapy for HER-2 positive breast cancer, which induces tumor cell apoptosis and activates immune surveillance against the tumor. Poly Inosine Poly Cytosine (pIC) is a known activator of apoptotic and inflammatory processes. We recently showed that targeted delivery of pIC into EGFR-overexpressing tumors induced tumor cell killing and the expression of pro-inflammatory cytokines both in vitro and in vivo. In the current study, we report on a novel chemical vector composed of linear polyethylenimine, polyethyleneglycol and a high affinity targeting moiety, HER-2 Affibody, (PPHAffibody), which selectively delivers pIC to HER-2 overexpressing breast cancer cells. We demonstrated that targeted delivery of pIC selectively induced apoptosis in HER-2 overexpressing cells, while cells with little to no HER-2 expression remained unaffected. Importantly, survival of trastuzumab resistant breast cancer significantly decreased upon pIC/PPHAffibody treatment. Confocal microscopy confirmed the selectivity of pIC/PPHAffibody binding and internalization into HER-2 overexpressing cells. To test the effect of pIC/PPHAffibody on immune cell recruitment and activation, ELISA and chemotaxis assays were utilized. Treatment of HER-2 overexpressing breast cancer cells with pIC/PPHAffibody induced the secretion of pro-inflammatory cytokines, such as IP-10 and RANTES, and promoted the chemotaxis of peripheral blood mononuclear cells (PBMC). In co-culture of HER-2 overexpressing tumor cells and PBMCs, pIC/PPHAffibody led to even more profound tumor cell killing than in the absence of PBMCs, indicating that the targeted pIC delivery indeed activates immune cells against the tumor. The in vivo efficacy of pIC/PPHAffibody treatment is currently under investigation. Furthermore, we will test whether pIC/PPHA synergizes with trastuzumab, both in vitro and in vivo. The data in this study indicate that targeted delivery of pIC restores immune surveillance and selectively induces breast cancer cell apoptosis, overcoming trastuzumab resistance. Thus, pIC/PPHA is a promising new addition to the available therapies for metastatic breast cancer patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B243. Citation Format: Maya Zigler, Salim Joubran, Alexei Shir, Noofar Edinger, Shoshana Klein, Alexander Levitzki. Targeting HER-2 positive breast cancer by inducing apoptosis and immune cell activation, overcoming trastuzumab resistance. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B243.

IS5–3 - HER2I Resistance

HER2 (ErbB2) is a member of the ErbB family of transmembrane receptor tyrosine kinases, which also includes the epidermal growth factor receptor (EGFR, ErbB1), HER3 (ErbB3) and HER4 (ErbB4). Given HER2 functions as an oncogene, gene amplification induces protein over-expression in cell membranes and regulates signal transduction in cellular processes, such as proliferation, differentiation, and cell survival. Aberrant HER2 expression or function has been associated in carcinogenesis of breast, gastric, ovarian, salivary gland, prostate and lung tumors. In particular HER2 overexpression or HER2 gene amplification has been reported in about 25% of breast cancers and is associated with aggressive disease. The use of anti-HER2 drugs, such as the monoclonal antibody trastuzumab and the TKI (tyrosin-kinase inhibitors) lapatinib, have changed the natural history of HER2-overexpressing breast cancer. Recently, trastuzumab efficacy has been demonstrated also in HER2-positive advanced gastric cancer. However, therapeutic resistance to trastuzumab or lapatinib, as either single-agents or in combination with chemotherapy in the metastatic setting, typically occurs within months of starting therapy. Several mechanisms of trastuzumab resistance have been reported including presence of truncated forms of HER2, as p95HER-2, phosphoinositide 3-kinase pathway activation or signaling pathway activation driven by HER3, epidermal growth factor receptor, and insulin-like growth factor 1 receptor. Alternative anti-HER2 strategies are currently in clinical development to overcome trastuzumab resistance. The use of antibody-drug conjugate is one of these strategies. Trastuzumab-DM1 (T-DM1) is a drug in which trastuzumab is linked to DM1, an antimicrotubule agent, enabling additional delivery of the cytotoxic to targeted cells. Pertuzumab is a recombinant humanized monoclonal antibody that binds to the dimerization epitope of HER-2, blocking its heterodimerization with EGFR and HER-3. Other strategies include: mTOR inhibitors, multi-HER TKIs as Neratinib, IGF-1R inhibitors, EGFR inhibitors and HSP-90 inhibitors.

Bispecific Antibody to ErbB2 Overcomes Trastuzumab Resistance through Comprehensive Blockade of ErbB2 Heterodimerization

The anti-ErbB2 antibody trastuzumab has shown significant clinical benefits in metastatic breast cancer. However, resistance to trastuzumab is common. Heterodimerization between ErbB2 and other ErbBs may redundantly trigger cell proliferation signals and confer trastuzumab resistance. Here, we developed a bispecific anti-ErbB2 antibody using trastuzumab and pertuzumab, another ErbB2-specific humanized antibody that binds to a distinct epitope from trastuzumab. This bispecific antibody, denoted as TPL, retained the full binding activities of both parental antibodies and exhibited pharmacokinetic properties similar to those of a conventional immunoglobulin G molecule. Unexpectedly, TPL showed superior ErbB2 heterodimerization-blocking activity over the combination of both parental monoclonal antibodies, possibly through steric hindrance and/or inducing ErbB2 conformational change. Further data indicated that TPL potently abrogated ErbB2 signaling in trastuzumab-resistant breast cancer cell lines. In addition, we showed that TPL was far more effective than trastuzumab plus pertuzumab in inhibiting the growth of trastuzumab-resistant breast cancer cell lines, both in vitro and in vivo. Importantly, TPL treatment eradicated established trastuzumab-resistant tumors in tumor-bearing nude mice. Our results suggest that trastuzumab-resistant breast tumors remain dependent on ErbB2 signaling and that comprehensive blockade of ErbB2 heterodimerization may be an effective therapeutic avenue. The unique potential of TPL to overcome trastuzumab resistance warrants its consideration as a promising treatment in the clinic.

Helicobacter pylori: Gastric Cancer and Extragastric Malignancies – Clinical aspects

The best opportunity to reduce gastric cancer (GC)-related mortality remains prevention. Mass eradication of Helicobacter pylori infection in a Taiwanese population >30 years of age reduced GC incidence with an effectiveness of 25% (rate ratio 0.753, 95% CI 0.372-1.524). In the Shandong intervention trial conducted on a Chinese population aged 35-64 years, cancer incidence was reduced by 39% in subjects who received H. pylori treatment compared with the placebo group after 14.7 years of follow-up (absolute risk 3.0 vs 4.6%; odds ratio 0.61, 95% CI 0.38-0.96; p = .03). A high incidence of severe gastric atrophic changes and noninvasive gastric neoplasia has been reported in a Portuguese case-control study on first-degree relatives of patients with early-onset gastric carcinoma (i.e., diagnosed before 45 years), which emphasizes again the importance of GC screening in this population. For patients with advanced GC, new targeted therapies to improve survival are under scrutiny. Trastuzumab resistance may be present from early on, or develop during trastuzumab therapy in patients with GC, and an overexpression of the HER2/neu protein. New molecules to overcome trastuzumab resistance are also being evaluated. The association between H. pylori-induced gastritis and an increased risk of developing colonic neoplasms has been confirmed in a recent study, but the causality for this intriguing association has still to be clarified.

Pertuzumab: A New Targeted Therapy for HER2-Positive Metastatic Breast Cancer

Trastuzumab, a humanized monoclonal antibody, has become an important targeted therapy for patients with all stages of human epidermal growth factor receptor-2 (HER2)-positive breast cancer. However, primary and acquired resistance to trastuzumab remains a significant problem. Pertuzumab, a humanized monoclonal antibody that binds to a domain of the HER2 receptor separate from trastuzumab, may have the potential to overcome trastuzumab resistance. Clinical trials have shown that pertuzumab can be effectively combined with other biologic therapy or chemotherapy in patients with metastatic HER2-positive breast cancer. Pertuzumab is relatively well tolerated with minimal increases in hematologic and cardiac toxicity observed when added to trastuzumab and/or docetaxel. In addition to becoming the standard of care in combination with docetaxel and trastuzumab in patients with newly diagnosed HER2-positive metastatic breast cancer, clinical trials continue to evaluate pertuzumab in combination with other targeted therapy, chemotherapy, and in patients with early stage breast cancer. These trials will help to further determine the role of pertuzumab in the treatment of HER2-positive breast cancer.

Neratinib overcomes trastuzumab resistance in HER2 amplified breast cancer

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.

Optimal Targeting of HER2–PI3K Signaling in Breast Cancer: Mechanistic Insights and Clinical Implications

The combination of a PI3K inhibitor with trastuzumab has been shown to be effective at overcoming trastuzumab resistance in models of HER2(+) breast cancer by inhibiting HER2-PI3K-FOXO-survivin signaling. In this review the potential clinical implications of these findings are discussed.

Novel Targeted Therapies to Overcome Trastuzumab Resistance in HER2- Overexpressing Metastatic Breast Cancer

Overexpression of the human epidermal growth factor receptor 2 (HER2) is identified in approximately 25- 30% of breast cancers and indicates a poor prognosis. Trastuzumab, the anti-HER2 monoclonal antibody (mAb), has shown significant clinical effects selectively in HER2-overexpressing metastatic breast cancer (MBC) with improved overall survival and reduced recurrent risk. However, there is an urgent need to develop new strategies to overcome innate and acquired trastuzumab resistance, which has increasingly occurred. Recently, an increased understanding of mechanisms of trastuzumab resistance significantly promotes the development of novel targeted therapies for trastuzumab refractory disease. It is believed that aberrant activations of several signaling pathways involving the human epidermal growth factor receptor (EGFR/HER) family, phosphoinositide 3 kinase/Akt (PI3K/Akt) pathway, and vascular endothelial growth factor (VEGF) family, contribute to the development of trastuzumab resistance. Novel agents that target these relevant signal pathways provide some potential solutions, such as tyrosine kinase inhibitors (TKIs) and mAbs. HER2 is also recognized as an immunotherapeutic target. The failure to induce immune-mediated antitumor response is another important reason for trastuzumab resistance. Strategies to boost T cell-mediated immune responses specific to HER2 including HER2 vaccines and bispecific antibodies (bsAbs) could be developed as a promising way to prevent relapse or combat trastuzumab resistance. In this review, the emerging data from preclinical and clinical studies related to these novel therapies will be discussed.

Abstract LB-215: Concomitant targeting of tumor cells and induction of T cell response synergizes to effectively inhibit trastuzumab-resistant breast cancer.

Abstract Introduction: Trastuzumab is an iconic example of rationally designed targeted therapy for HER2-positive breast cancers; however, trastuzumab resistance is a imposing clinical challenge that calls for novel approaches to better benefit patients. The purpose of this study was to find alternative combinatorial targeted therapies to overcome trastuzumab resistance. Methods: We used two distinct PTEN-loss mediated trastuzumab resistant mammary tumor mouse models. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and Akt inhibitor triciribine (TCN) were applied alone or in combination with 7.16.4 antibody, the mouse equivalent of the trastuzumab, for the treatment of tumor-bearing mice. Immunoblotting, immunohistochemistry, in vivo cytotoxicity assay and quantitative real time PCR were employed to dissect the in vivo effects of the treatments and molecular changes. Results: Concomitant targeting of tumor cells with Akt inhibitor TCN plus trastuzumab, and activation of T cells with anti-CTLA-4 antibody in the tumor microenvironment results in a synergistic inhibitory effect on tumor growth and overcomes trastuzumab resistance in both mammary tumor mouse models. In vivo combinatorial treatment with the 7.16.4 HER2/Neu antibody and TCN effectively inhibited tumor growth in both models via inhibiting PI3K/AKT and MAPK signaling accompanied by increased T cell infiltration in the tumor microenvironment. We demonstrated that both CD8+ and CD4+ T cells were essential to the optimal antitumor effect of the combination treatment in an IFN-γ-dependent manner. Importantly, the antitumor activities of HER2/Neu antibody and TCN combination treatment were further improved when we blocked co-inhibitory receptor cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) to enhance the T cell response. Conclusion: Our data indicate that multi-targeted combinatorial therapies targeting tumor cells and concomitantly enhancing T-cell response in the tumor microenvironment cooperated to exert maximal therapeutic activity. We propose that concomitant targeting tumor cells and tumor microenvironment is a promising clinical strategy for treating trastuzumab-resistant breast cancers and other advanced malignancies. Citation Format: Qingfei Wang, Shau-Hsuan Li, Hai Wang, Yi Xiao, Ozgur Sahin, Samuel W. Brady, Ping Li, Elizabeth M. Jaffee, Gabriel N. Hortobagyi, Dihua Yu. Concomitant targeting of tumor cells and induction of T cell response synergizes to effectively inhibit trastuzumab-resistant breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-215. doi:10.1158/1538-7445.AM2013-LB-215

Abstract 3736: IL6 feedback loop regulates breast cancer stem cells which mediate trastuzumab resistance.

Abstract The development of HER2 targeting agents such as trastuzumab and lapatinib represents one of the greatest achievements in clinical oncology demonstrating the effectiveness of molecularly targeted therapeutics (2). Despite the clinical benefits of trastuzumab in improving progression free and overall survival, majority of patients demonstrate “intrinsic” resistance or develop “acquired” resistance within one-to-two years. Although inactivation of the PTEN gene has been implicated in the development of resistance to the HER2 targeting antibody trastuzumab, the mechanisms mediating this resistance remain to be investigated. We generated trastuzumab resistant cells by knocking down PTEN expression in HER2 overexpressing breast cancer cell lines and demonstrated that development of trastuzumab resistance in these cells is mediated by activation of an IL6 inflammatory feedback loop leading to expansion of breast cancer stem cell (CSC) population. We and others previously demonstrated that cytokines including IL6 and IL8 regulates breast cancer stem cell self-renewal. Furthermore, elevated serum levels of these cytokines in breast cancer patients strongly correlated with metastatic disease and therapeutic resistance. We therefore hypothesized that activation of IL6 feedback loop may mediate trastuzumab resistance. Utilizing these models, we demonstrated that long term trastuzumab treatment generates highly enriched CSCs which display an EMT phenotype secreting over 100-fold more IL6 than parental cells. Recombinant IL6 or conditioned medium from high-IL6 secreting cells resulted in trastuzumab resistance in parental PTEN wild type cells expanding CSCs with EMT phenotype characterized by the CD44+/CD24- expression and EMT gene signature. An IL6 receptor antibody interrupted this inflammatory feedback loop reducing the cancer stem cell population resulting in decreased tumor growth and metastasis in mouse xenographs. These studies have important clinical implications demonstrating that trastuzumab resistance may be mediated by an IL-6 inflammatory loop and suggest that blocking this loop represents a novel strategy to overcome trastuzumab resistance. . Citation Format: April L. Davis, Hasan Korkaya, Maria Ouzounova, Fayaz Malik, Gwangil Kim, Ahmed A. Quraishi, Suling Liu, Shawn G. Clouthier, Max S. Wicha. IL6 feedback loop regulates breast cancer stem cells which mediate trastuzumab resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3736. doi:10.1158/1538-7445.AM2013-3736

Heat Shock Factor 1 (HSF1) Controls Chemoresistance and Autophagy through Transcriptional Regulation of Autophagy-related Protein 7 (ATG7)

Heat shock factor 1 (HSF1), a master regulator of heat shock responses, plays an important role in tumorigenesis. In this study we demonstrated that HSF1 is required for chemotherapeutic agent-induced cytoprotective autophagy through transcriptional up-regulation of autophagy-related gene ATG7. Interestingly, this is independent of the HSF1 heat shock response function. Treatment of cancer cells with the FDA-approved chemotherapeutic agent carboplatin induced autophagy and growth inhibition, which were significantly increased upon knockdown of HSF1. Mechanistic studies revealed that HSF1 regulates autophagy by directly binding to ATG7 promoter and transcriptionally up-regulating its expression. Significantly, breast cancer patient sample study revealed that a higher ATG7 expression level is associated with poor patient survival. This novel finding was further confirmed by analysis of two independent patient databases, demonstrating a prognostic value of ATG7. Furthermore, a strong positive correlation was observed between levels of HSF1 and ATG7 in triple-negative breast cancer patient samples, thus validating our in vitro findings. This is the first study identifying a critical role for HSF1 in controlling cytoprotective autophagy through regulation of ATG7, which is distinct from the HSF1 function in the heat shock response. This is also the first study demonstrating a prognostic value of ATG7 in breast cancer patients. These findings strongly argue that combining chemotherapeutic agents with autophagy inhibition by repressing HSF1/ATG7 axis represents a promising strategy for future cancer treatment.

Trastuzumab emtansine for the treatment of HER2-positive metastatic breast cancer

Trastuzumab emtansine is an antibody-drug conjugate comprised of the receptor tyrosine-protein kinase erbB-2 (HER2) antibody trastuzumab, and a derivative of the cytostatic agent maytansinoid DM1, covalently linked by a thiol linker. The drug was developed in an attempt to overcome trastuzumab resistance in patients with HER2-positive breast carcinoma, but it is also of potential use in other HER2-positive cancers. The preclinical antitumor activity of trastuzumab emtansine was established in HER2-positive breast cancer cell lines and murine xenograft models. Preclinically, trastuzumab emtansine was efficacious in HER2-positive cells that were resistant to trastuzumab or lapatinib. Clinically, the drug is well tolerated in most patients, with a predictable pharmacokinetic profile and minimal systemic exposure to free cytotoxic DM1. Unlike with trastuzumab, cardiac toxicity has not been seen in patients receiving trastuzumab emtansine and less adverse events have been reported than with other chemotherapy regimens. Results from a number of phase II studies and early results from a phase III investigation (EMILIA) demonstrated response rates of 25-35% in patients with breast cancer who had previously received trastuzumab. Several phase II and III studies are under way investigating trastuzumab emtansine in combination with other regimens in patients with HER2-positive cancers.

The HER2 Receptor in Breast Cancer: Pathophysiology, Clinical Use, and New Advances in Therapy

Human epidermal growth factor receptor 2 (HER2) is overexpressed in around 20–30% of breast cancer tumors. It is associated with a more aggressive disease, higher recurrence rate, and increased mortality. Trastuzumab is a HER2 receptor blocker that has become the standard of care for the treatment of HER2 positive breast cancer. The effectiveness of Trastuzumab has been well validated in research as well as in clinical practice. The addition of Trastuzumab to standard of care chemotherapy in clinical trials has been shown to improve outcomes for early stage as well as metastatic HER2 positive breast cancer. The most clinically significant side effect of Trastuzumab is the risk of cardiac myocyte injury, leading to the development of congestive heart failure. The emergence of patterns of resistance to Trastuzumab has led to the discovery of new monoclonal antibodies and other targeted agents aimed at overcoming Trastuzumab resistance and improving survival in patients diagnosed with HER2 positive breast cancers.

Abstract P4-08-01: PI3K/mTOR inhibition overcomes in vitro and in vivo trastuzumab resistance independent of feedback activation of pAKT

Abstract Background: Aberrant activity of the PI3K/mTOR pathway has been implicated in resistance to trastuzumab and anti-hormonal therapy for HER2-amplified and ER-positive breast cancer, respectively. Previous studies in our laboratory and others have shown that increased PI3K/mTOR signaling, either through PTEN loss or activating PIK3CA mutations, can confer resistance to trastuzumab therapy. In this study, we assessed the potential of targeting the PI3K/mTOR pathway in overcoming both de novo and acquired trastuzumab resistance. Materials and Methods: The in vitro activity of the pan-PI3K inhibitor BKM120, the mTORC1 inhibitor RAD001 and the dual PI3K/mTORC1/2 inhibitor BEZ235 were evaluated in a panel of 49 human breast cancer and immortalized cell lines. The in vivo activity of these molecules was assessed in six cell line xenografts models representing, ER+/HER2− (KPL-1, ZR75-1), ER+/HER2+ (UACC812, MDA361), ER−/HER2+ (SUM190), PIK3CA mutant (SUM190, MDA361), PTEN-null (ZR75-1) and trastuzumab resistant (BT-TR) breast cancer. Finally, the effect of PI3K/mTOR inhibition on feedback activation of PI3K signaling and compensatory pathways was measured by Western blot, immunohistochemistry (IHC) and reverse phase protein analysis (RPPA) of control and treated cell lysates/tumors. Results: Using a sensitivity cut-off of an IC50 of < 1 µmol/L, 16 of the 18 HER2-amplified breast cancer cell lines and 8 of 10 cell lines with activating mutations in PIK3CA were sensitive to the pan-PI3K inhibitor BKM120. BEZ235 showed the most potent efficacy across the panel with IC50s < 100 nmol/L for each of the 49 cell lines tested. The HER2-amplified/PIK3CA mutant cell lines were also unexpectedly sensitive to the mTORC1 inhibitor RAD001, this was despite the silencing of mTORC1 signaling being followed by a feedback increase in phospho-AKT signaling. Furthermore, each of these molecules showed remarkable in vivo activity across the panel of xenografts models. BKM120, RAD001 and BEZ235 induced both tumor stabilization and regression independent of PTEN, PI3K, ER and HER2 status. Pharmacodynamic analysis of tumor tissue revealed that BEZ235 and RAD001 both inhibited mTORC1 signaling as indicated by a reduction in the levels of phosphorylated ribosomal protein S6 (pS6). However, in contrast to BKM120 and BEZ235, RAD001 did not induce a reduction in the levels of pAKT (S473 or TH308) yet showed comparable in vivo efficacy to each of these molecules. Finally, combined targeting of HER2 and PI3K/mTOR in vitro increased the anti-proliferative activity of the molecules and led to an increased induction of apoptosis. The efficacy of these molecules (alone or in combination with trastuzumab) was assessed in a model of in vivo trastuzumab resistance generated through long-term treatment of the trastuzumab sensitive BT474 cells (BT-TR). All PI3Ki induced complete inhibition of tumor proliferation in monotherapy, while the combination of trastuzumab and each of these molecules induced tumor regression in the trastuzumab resistant tumors. Discussion: These pre-clinical data indicate that targeting the PI3K/mTOR pathway either alone or in combination with trastuzumab is effective strategy for overcoming trastuzumab resistance. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-08-01.

Photochemical internalization (PCI) of HER2-targeted toxins: Synergy is dependent on the treatment sequence

BackgroundPhotochemical internalization (PCI) is a modality for cytosolic release of drugs trapped in endocytic vesicles. The method is based upon photosensitizers localized in the membranes of endocytic vesicles which create membrane rupture upon light exposure by generating reactive oxygen species (ROS), predominantly singlet oxygen (1O2). MethodsThe human epidermal growth factor receptor 2 (HER2)-targeted immunotoxin (IT), trastuzumab–saporin, was evaluated in combination with PCI using TPCS2a (Amphinex®), a new photosensitizer approved for clinical use. ResultsPCI synergistically enhanced the cytotoxicity of trastuzumab–saporin on trastuzumab-resistant HER2+ Zr-75-1 cells. The PCI effect was only observed when the IT was administered prior to the photochemical treatment (“light after” strategy), while administration of a non-targeted drug may equally well be performed after light exposure. Mechanistic studies showed reduced ligand-induced HER2 phosphorylation and receptor-mediated endocytosis after TPCS2a-PDT. Photochemical disruption of the cytoplasmic domain of HER2 was found to be induced by 1O2 generated both by photosensitizer located in the endocytic vesicles and in the outer leaflet of the plasma membrane. ConclusionsAdministration of the HER2-targeted toxin prior to light exposure is a prerequisite for successful PCI-mediated delivery of HER2-targeted toxins. General significancePCI of HER2-targeted toxins is demonstrated as a highly effective treatment modality which may overcome trastuzumab resistance. The mechanistic studies of the lack of PCI effect of the “light first” procedure is of outermost importance when designing a clinical PCI treatment protocol for delivery of HER2-targeted therapies.