Trastuzumab-resistant BT474 Cells Research Articles

Abstract 2980: The role of USP7 and USP7 inhibitor in HER2+ breast cancer treatment

Abstract INTRODUCTION: Breast cancer is the most common invasive cancer and the second leading cause of cancer death in women. Approximately 20% of breast cancer tumors overexpress human epidermal growth factor receptor 2 (HER2), which is associated with an aggressive clinical phenotype and with a poorer prognosis. The development of trastuzumab, an immunoglobulin G monoclonal antibody directed against HER2, has changed the treatment paradigm for this disease. However, approximately 15-20% still does not respond to anti-HER2 therapy. Furthermore, despite advances in the management of metastatic HER2-positive breast cancer, response rate varies greatly, with most patients eventually succumbing to their disease. It is crucial to develop additional therapeutic strategies to improve outcomes for HER2 positive patients, especially those with tumors that do not respond to anti-HER2 therapy. METHODS and RESULTS: Using HER2+ breast cancer cell lines, we found that an USP7 inhibitor, P5091, dramatically sensitized cancer cells to HER2 inhibitor, trastuzumab treatment. To confirm it in vivo, we tested the drug combination in BT474 xenografts and found P5091 and trastuzumab synergistically suppressed tumor growth. Furthermore, using trastuzumab resistant HER2+ breast cancer cell line, we found that USP7 inhibitor, P5091, can dramatically re-sensitize trastuzumab-resistant BT474 cell line to trastuzumab treatment both in vitro and in vivo. More importantly, using patient derived xenograft (PDX) HER2+ models, we confirmed the synergistic combination effect of P5091 and trastuzumab. Mechanistically, USP7 inhibitor, P5091 led to significantly increased degradation of all four HER family members (HER1-4). When we knocked down USP7 by RNA interference or small hairpin RNA (shRNA), we observed similar substantial decrease in HER family member proteins. CONCLUSIONS: We have demonstrated the feasibility of using USP7 inhibitor P5091 to sensitize HER2+ breast cancers to trastuzumab treatment. These results provide a rationale for the use of combination therapy of USP7 inhibitors and HER2 inhibitors to treat HER2+ breast cancer. This combination therapy might also be effective in other cancers with upregulated HER signaling, such as GI cancer, lung cancer or ovarian cancer. Citation Format: Jia Yu, Bo Qin, Judy Boughey, Matthew Goetz, Liewei Wang. The role of USP7 and USP7 inhibitor in HER2+ breast cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2980.

DCZ3112, a novel Hsp90 inhibitor, exerts potent antitumor activity against HER2-positive breast cancer through disruption of Hsp90-Cdc37 interaction

Hsp90 regulates the stability of oncoproteins important in tumor development and progression, and represents a potential therapeutic target. However, all Hsp90 inhibitors currently in clinical trials target Hsp90 ATPase activity and exhibit low selectivity and high toxicity. In this study, we discovered a new Hsp90 inhibitor, DCZ3112, with a novel mechanism of action. DCZ3112 directly bound to the N-terminal domain of Hsp90 and inhibited Hsp90-Cdc37 interaction without inhibiting ATPase activity. DCZ3112 inhibited the proliferation predominantly in HER2-positive breast cancer cells, including those resistant to the classical Hsp90 inhibitor geldanamycin, which mainly targets ATPase. DCZ3112 produced synergistic in vitro activity in inhibiting cell proliferation, inducing G1-phase arrest and apoptosis, and reducing AKT and ERK phosphorylation. Consistent with this, DCZ3112 alone inhibited the growth of HER2-positive BT-474 xenografts, and exhibited enhanced antitumor activity when combined with the anti-HER2 antibody trastuzumab. Importantly, DCZ3112 also significantly inhibited the growth of trastuzumab-resistant BT-474 cells, and combined treatment retained synergistic antitumor activity. Thus, our findings show that disrupting Hsp90-Cdc37 interaction may represent a promising strategy against HER2-positive breast cancer, especially those with acquired resistance to trastuzumab.

ECM1 promotes the Warburg effect through EGF-mediated activation of PKM2

The Warburg effect is an oncogenic metabolic switch that allows cancer cells to take up more glucose than normal cells and favors anaerobic glycolysis. Extracellular matrix protein 1 (ECM1) is a secreted glycoprotein that is overexpressed in various types of carcinoma. Using two-dimensional digest-liquid chromatography–mass spectrometry (LC–MS)/MS, we showed that the expression of proteins associated with the Warburg effect was upregulated in trastuzumab-resistant BT-474 cells that overexpressed ECM1 compared to control cells. We further demonstrated that ECM1 induced the expression of genes that promote the Warburg effect, such as glucose transporter 1 (GLUT1), lactate dehydrogenase A (LDHA), and hypoxia-inducible factor 1 α (HIF-1α). The phosphorylation status of pyruvate kinase M2 (PKM-2) at Ser37, which is responsible for the expression of genes that promote the Warburg effect, was affected by the modulation of ECM1 expression. Moreover, EGF-dependent ERK activation that was regulated by ECM1 induced not only PKM2 phosphorylation but also gene expression of GLUT1 and LDHA. These findings provide evidence that ECM1 plays an important role in promoting the Warburg effect mediated by PKM2.

Abstract 1829: Regulation of Mixed Lineage Kinase-3 activity by Her2 and its implication in death or survival

Abstract Human Epidermal Growth Factor Receptor 2 (Her2) is a receptor tyrosine kinase that regulates cell growth and differentiation signaling pathways. The protein Her2 is significantly overexpressed in 20-30% of breast cancer. The available clinical data proves that Her2 amplification in breast cancer is associated with worst overall survival. Therefore, Her2 has become a very important candidate for drug targeting and currently Her2 positive patients are treated with humanized monoclonal antibody, Trastuzumab (Herceptin) and a small molecule, Lapatinib that blocks Her2 tyrosine kinase activity. It is reported that Her2 amplification leads to activation of multiple survival pathways, including PI3K-Akt. However, it is not clearly known how Trastuzumab promotes cell death and what pro-apoptotic pathways are activated downstream of Her2 blockage? It is also reported that patients on Trastuzumab develop subsequent resistance to the therapy. Previously we published that in breast cancer tumors, a pro-apoptotic kinase, Mixed Lineage Kinase-3 (MLK-3) was inhibited, specifically in estrogen receptor (ER) positive tumors. The kinase activity of MLK-3 was down regulated by estrogen and this prevented MLK-3-mediated cell death. Since Her2 serves as a drug target in breast cancer, therefore, we asked whether Her2 plays any role in regulating MLK-3 kinase activation and whether MLK-3 plays any role in Trastuzumab resistance. Our results showed that the endogenous MLK-3 kinase activity was up-regulated in SkBr3 cells upon treatment with Trastuzumab and Lapatinib. Whether the activation of MLK-3 upon Her2 blockage leads to cell death or survival is still not known and experiments are underway to investigate these aspects. We also observed that Her3 was the dimerization partner of Her2 in Trastuzumab- or Lapatinib- mediated MLK-3 activation. Not knowing any role of MLK-3 in Trastuzumab resistance, we used Trastuzumab sensitive and resistant BT474 cell model to investigate the role of MLK-3 in Trastuzumab resistance. Our data clearly showed that knockdown of MLK-3 caused spontaneous cell death in resistant cells. The Trastuzumab treatment further amplified cell death in MLK-3 knockdown resistant cells. The Akt1 activation in these cells was also diminished upon MLK-3 knockdown. Taken together, our data demonstrate that like ER, the Her2 also down regulates MLK-3 kinase activity and perhaps promote cell survival. Furthermore, paradoxically, the MLK-3 knockdown promoted cell death in Trastuzumab resistance BT474 cells, suggesting that MLK-3 could also serve as survival factor in resistant cells. Whether MLK-3 serves as a pro-apoptotic kinase or survival kinase in Trastuzumab mediated pathway needs to be extensively examined in near future. Citation Format: Subhasis Das, Gautam Sondarva, Navin Viswakarma, Rakesh Sathish Nair, Clodia Osipo, Basabi Rana, Ajay Rana. Regulation of Mixed Lineage Kinase-3 activity by Her2 and its implication in death or survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1829. doi:10.1158/1538-7445.AM2014-1829

Abstract 774: Role of t-Darpp in a shift to EGFR signaling in trastuzumab-resistant cells

Abstract Elucidating the molecular mechanism of both primary and acquired resistance to trastuzumab is vital to gaining insight into ways to improve therapeutic outcomes in breast cancer patients. We have previously shown that levels of the 32kDa dopamine and cAMP-regulated phosphoprotein Darpp-32 (Dp32) and its amino-truncated isoform t-Darpp (tDp) are altered in trastuzumab-resistant BT474 cells. Specifically, tDp is upregulated in models of trastuzumab resistance and over-expression of the protein is sufficient to confer resistance. We have further demonstrated that trastuzumab-resistant BT474 cells are more sensitive to the EGFR tyrosine kinase inhibitor (TKI), AG1478, in the presence of trastuzumab than in its absence (1), suggesting that these cells gain a dependence on EGFR when HER2 signaling is shut down. We have now examined the mechanism by which tDp confers resistance in HER2+ breast cancer cells. We used SK-Br-3 cells stably expressing an empty vector, tDp or flag-tDp, as well as two trastuzumab-resistant BT474 clones, BT1.0C and BT1.0E, that endogenously over-express moderate and very high levels of tDp, respectively. Cells with elevated tDp levels had a higher basal level of p-EGFR (Y1068) and we observed an increase in p-EGFR upon trastuzumab treatment in all of the HER2+ cells. To further investigate if EGFR signaling plays a role in tDp's resistance mechanism, we performed SRB assays to test the sensitivity of tDp over-expressing cells to EGFR TKIs, AG1478 and Gefitinib. We found that cells expressing high tDp are 2.5- to 7.5-fold more sensitive to EGFR inhibition compared to controls (p<0.01). Furthermore, the combination treatment of AG1478 and trastuzumab was synergistic in inhibiting the trastuzumab-resistant BT1.0E cell line that highly over-expresses tDp at an ED50, ED75 and ED90 CI of 0.61, 0.46, and 0.35, respectively. The parental BT474 cells and BT1.0C clone, which expresses lower levels of tDp, showed antagonism at the ED50 but synergism at the ED75 and ED90. Using siRNA knock-down of tDp, we will show whether tDp is required in causing an increased dependence on EGFR signaling. We will also report on similar combination experiments using tDp-transfected SK-Br-3 cells, to determine if tDp expression is sufficient for conferring synergism, and with a HER2 TKI in combination with EGFR TKI. Taken together, our results suggest that tDp is a novel molecular target that can mediate resistance to trastuzumab by inducing a compensatory shift to EGFR signaling when HER2 is inhibited. 1. Chan CT, Metz MZ, & Kane SE (2005) Differential sensitivities of trastuzumab (Herceptin)-resistant human breast cancer cells to phosphoinositide-3 kinase (PI-3K) and epidermal growth factor receptor (EGFR) kinase inhibitors. Breast Cancer Res Treat 91(2):187-201. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 774. doi:1538-7445.AM2012-774

Plasma microRNA 210 levels correlate with sensitivity to trastuzumab and tumor presence in breast cancer patients

Trastuzumab is part of the standard treatment for patients with human epidermal growth factor receptor 2 (HER-2)-positive breast cancer, but not all patients respond to trastuzumab. Altered microRNA (miR) expression levels in cancer cells have been correlated with prognosis and response to chemotherapy. The authors of this report hypothesized that altered miR expression levels in plasma are associated with sensitivity to trastuzumab in patients with HER-2 positive breast cancer. Quantitative reverse transcriptase-polymerase chain reaction was used to analyze plasma samples, including samples from patients with breast cancer who were enrolled in a clinical trial of neoadjuvant trastuzumab-based chemotherapy. Expression levels of miR-210, miR-21, miR-29a, and miR-126 were analyzed according to the type of response (pathologic complete response [n = 18] vs residual disease [n = 11]). MicroRNA expression levels also were compared in trastuzumab-sensitive and trastuzumab-resistant breast cancer cells derived from BT474 cells and in an independent set of preoperative plasma samples (n = 39) and postoperative plasma samples (n = 30) from 43 breast cancer patients who did not receive any treatment. At baseline before patients received neoadjuvant chemotherapy combined with trastuzumab, circulating miR-210 levels were significantly higher in those who had residual disease than in those who achieved a pathologic complete response (P = .0359). The mean expression ratio for miR-210 was significantly higher in trastuzumab-resistant BT474 cells, and miR-210 expression was significantly higher before surgery than after surgery (P = .0297) and in patients whose cancer metastasized to the lymph nodes (P = .0030). Circulating miR-210 levels were associated with trastuzumab sensitivity, tumor presence, and lymph node metastases. These results suggest that plasma miR-210 may be used to predict and perhaps monitor response to therapies that contain trastuzumab.

Targeting both Notch and ErbB-2 signalling pathways is required for prevention of ErbB-2-positive breast tumour recurrence.

Background:We reported that Notch-1, a potent breast oncogene, is activated in response to trastuzumab and contributes to trastuzumab resistance in vitro. We sought to determine the preclinical benefit of combining a Notch inhibitor (γ-secretase inhibitor (GSI)) and trastuzumab in both trastuzumab-sensitive and trastuzumab-resistant, ErbB-2-positive, BT474 breast tumours in vivo. We also studied if the combination therapy of lapatinib plus GSI can induce tumour regression of ErbB-2-positive breast cancer.Methods:We generated orthotopic breast tumour xenografts from trastuzumab- or lapatinib-sensitive and trastuzumab-resistant BT474 cells. We investigated the antitumour activities of two distinct GSIs, LY 411 575 and MRK-003, in vivo.Results:Our findings showed that combining trastuzumab plus a GSI completely prevented (MRK-003 GSI) or significantly reduced (LY 411 575 GSI) breast tumour recurrence post-trastuzumab treatment in sensitive tumours. Moreover, combining lapatinib plus MRK-003 GSI showed significant reduction of tumour growth. Furthermore, a GSI partially reversed trastuzumab resistance in resistant tumours.Conclusion:Our data suggest that a combined inhibition of Notch and ErbB-2 signalling pathways could decrease recurrence rates for ErbB-2-positive breast tumours and may be beneficial in the treatment of recurrent trastuzumab-resistant disease.

Analysis of truncated HER2 expression and activation in breast cancer.

1099 Background: De novo and acquired resistance to trastuzumab-based therapy is an active area of research. One of the mechanisms of resistance is expression of p95HER2 which is clinically associated with aggressive disease, poor prognosis, and lack of response to trastuzumab. Clinical studies on p95HER2 are limited due to lack of highly sensitive and specific assays to accurately measure its expression and activation. Methods: A novel technology capable of specifically detecting expression and phosphorylation of receptor tyrosine kinases (RTKs) (Prometheus proprietary COPIA assay) was used to differentially detect p95HER2 from full length HER2. Using this assay, we analyzed the expression and activation of p95HER2 as well as other major oncogenic pathways including HER1, HER2, HER3, IGF1-R, c-MET, PI3K, Shc, VEGFR, and CK in 229 frozen breast cancer tissues (stage II to IV). Expression and activation of HER2, p95HER2, and PI3K were also measured in trastuzumab-resistant and -sensitive BT474 cells. Results: Here, we report the successful profiling of HER2 and p95HER2 expression and activation in frozen primary breast cancer tissues as determined by IHC and tested in COPIA. Approximately 50% of IHC 3+ samples had activated p95HER2. The level of expression and activation of p95HER2 in the IHC 2+, 1+ and negative subsets was dramatically lower although some tissues have significant levels. All 10 markers tested revealed diverse activation of signal pathways. In our preclinical studies, trastuzumab-resistant BT474 cells showed significantly higher activation of p95HER2, and PI3K compared to sensitive BT474 cells upon trastuzumab treatment. Conclusions: The highly sensitive, specific, and quantitative p95HER2 COPIA assay allows accurate detection of HER2 and total and activated p95HER2 in small tumor samples such as fine needle aspirates or core biopsies and could select patients who are most likely to respond to tyrosine kinase inhibitors. Our COPIA assay enables detection of multiple kinases simultaneously in the same samples, which sheds some light on the mechanism of p95HER2 associated trastuzumab resistance. Future studies are warranted to measure p95HER2 expression and activation in clinical responders and non-responders to trastuzumab treatment. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Prometheus Labs Prometheus Labs

Notch-1 Confers Resistance to ErbB-2 Targeted Inhibition by Regulating PTEN, p27, and IGF-1R.

Abstract Background: Amplification, over-expression, and/or hyperactivity of ErbB-2 occur in 30% of breast tumors. Metastatic breast tumors that overexpress ErbB-2 are difficult to treat and generally resistant to trastuzumab or lapatinib. We have recently published that overexpression of ErbB-2 suppresses Notch-1 activity and this is reversed by inhibitors of ErbB-2, trastuzumab or a tyrosine kinase inhibitor (Osipo et al., 2008. Oncogene). Furthermore, we demonstrated that trastuzumab resistance is reversed when Notch-1 is downregulated by siRNA or when cells are treated with a gamma-secretase inhibitor. Overexpression of IGF-1R and/or downregulation of PTEN, and p27Kip1 have been shown to be very sensitive markers of trastuzumab resistance. Based on our results and what is known from the literature, we asked by what mechanism Notch-1 contributes to trastuzumab resistance and can a combination therapy prevent or reverse the resistant phenotype by using a pre-clinical in vivo model.Methods: Using a pcDNA3.1-NIC construct expressing activated Notch-1 and a Notch-1 siRNA, we either increased NIC levels in trastuzumab sensitive or downregulated Notch-1 in trastuzumab resistant BT474 cells, respectively and measured protein and mRNA expressions of PTEN, p27Kip1, and the IGF-1R. In addition, BT474 breast tumor xenografts were generated in athymic mice and randomized to vehicle, MRK-003 or Ly411,575 gamma-secretase inhibitor, trastuzumab, or trastuzumab plus a gamma-secretase inhibitor. Tumor size was measured up to 14 weeks with treatments and up to 12 weeks after stopping treatments.Results: The results showed that intracellular, active Notch-1 (ICN) increased mRNA and protein expression of the IGF-1R, while simultaneously decreased protein expressions of PTEN and p27Kip1 in ErbB-2 positive breast cancer cells sensitive to trastuzumab. Conversely, knockdown of Notch-1 by siRNA in resistant cells decreased IGF-1R, while increasing PTEN and p27Kip1. The xenograft studies demonstrated that combining a GSI with trastuzumab either completely prevented tumor recurrence (MRK-003 GSI) or significantly delayed tumor recurrence (Ly 411,575 GSI).Conclusions: Taken together, these results suggest that Notch-1 potentially contributes to trastuzumab resistance by upregulating the IGF-1R and downregulating PTEN and p27Kip1 in vitro. The benefit of inhibiting Notch activity using a GSI in combination with trastuzumab is prevention of resistance and tumor recurrence. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3123.

ErbB-2 inhibition activates Notch-1 and sensitizes breast cancer cells to a γ-secretase inhibitor

ErbB-2 overexpression in breast tumors is associated with poor survival. Expression of Notch-1 and its ligand, Jagged-1, is associated with the poorest survival, including ErbB-2-positive tumors. Trastuzumab plus chemotherapy is the standard of care for ErbB-2-positive breast cancer. A proportion of tumors are initially resistant to trastuzumab and acquired resistance to trastuzumab occurs in metastatic breast cancer and is associated with poor prognosis. Thus, we investigated whether Notch-1 contributes to trastuzumab resistance. ErbB-2-positive cells have low Notch transcriptional activity compared to non-overexpressing cells. Trastuzumab or a dual epidermal growth factor receptor (EGFR)/ErbB-2 tyrosine kinase inhibitor (TKI) increased Notch activity by 2- to 6-fold in SKBr3, BT474 and MCF-7/HER2-18 cells. The increase in activity was abrogated by a Notch inhibitor, gamma-secretase inhibitor (GSI) or Notch-1 small-interfering RNA (siRNA). Trastuzumab decreased Notch-1trade mark precursor, increased amount and nuclear accumulation of active Notch-1(IC) and increased expression of targets, Hey1 and Deltex1 mRNAs, and Hes5, Hey1, Hes1 proteins. Importantly, trastuzumab-resistant BT474 cells treated with trastuzumab for 6 months expressed twofold higher Notch-1, twofold higher Hey1, ninefold higher Deltex1 mRNAs and threefold higher Notch-1 and Hes5 proteins, compared to trastuzumab-sensitive BT474 cells. The increase in Hey1 and Deltex1 mRNAs in resistant cells was abrogated by a Notch-1 siRNA. Cell proliferation was inhibited more effectively by trastuzumab or TKI plus a GSI than either agent alone. Decreased Notch-1 by siRNA increased efficacy of trastuzumab in BT474 sensitive cells and restored sensitivity in resistant cells. Trastuzumab plus a GSI increased apoptosis in sensitive cells by 20-30%. A GSI alone was sufficient to increase apoptosis in trastuzumab-resistant BT474 cells by 20%, which increased to 30% with trastuzumab. Notch-1 siRNA alone decreased cell growth by 30% in sensitive and more than 50% in resistant BT474 cells. Furthermore, growth of both trastuzumab sensitive and resistant cells was completely inhibited by combining trastuzumab plus Notch-1 siRNA. More importantly, Notch-1 siRNA or a GSI resensitized trastuzumab-resistant BT474 cells to trastuzumab. These results demonstrate that ErbB-2 overexpression suppresses Notch-1 activity, which can be reversed by trastuzumab or TKI. These results suggest that Notch-1 might play a novel role in resistance to trastuzumab, which could be prevented or reversed by inhibiting Notch-1.