Parental BT474 Cells Research Articles

Abstract 1690: JAK2 downmodulation leads to interferon gamma deficient response and resistance to immunotherapy in breast cancer

Abstract Introduction: Elimination of cancer cells by effector immune cells represents the culmination of a complex cascade of events, and disruption of any of those events may result in resistance. T cell-engaging therapies, such as T cell bispecific antibodies (TCBs) or chimeric antigen receptors (CARs), are raising extraordinary expectations as future treatments for virtually all cancers. Encouraging these expectations, TCBs and CARs have been recently approved to treat some hematologic malignancies. In contrast, TCBs and CARs against solid tumors tested to date, have failed to show clinical efficacy. This failure prompted intense research and the subsequent identification of mechanisms of primary and acquired resistance. Different strategies are being implemented to overcome these mechanisms of resistance. All these mechanisms impinge on the ability of T cells to reach cancer cells and/or on the inhibition of T cells. However, little is known about putative intrinsic mechanisms of resistance of cancer cells. That is, mechanisms deployed by tumor cells to resist killing by fully active and correctly engaged T cells. In this study, we attempted to identify novel intrinsic mechanisms of resistance. Methods: We have used TCBs and CARs targeting the cell surface receptor HER2 to identify a widespread mechanism of resistance to redirected T cells, using HER2-driven cell lines and Patient-Derived Xenografts (PDX). Results: We have generated a model of intrinsic resistance to a TCB targeting HER2 by treating during 6 months co-cultures of PBMCs and parental BT474 cells. These resistant cells, named as BT-R, are also resistant to a HER2-CAR, in vitro and in vivo. Using this model, we identified by RNA-seq, a downmodulation of the IFN-gamma signaling pathway. Interestingly, using gain and loss of function approaches, we demonstrated that JAK2 loss is the cause of IFN-gamma deficient response, and as a consequence, resistance to HER2-TCB and CAR-HER2. Conclusion: We have identified that the kinase JAK2, which transduces the signal initiated by interferon-gamma, is the component preferably disrupted to acquired resistance in all resistant models developed in vitro and in vivo. These results unveil a novel mechanism of resistance to T-cell based therapies, and imply the potential use of JAK2 and IFN-gamma response as a surrogate biomarker of response to immunotherapies. In addition, they open the avenue for the screening for therapies that can overcome deficient interferon-gamma response or restore JAK2 levels, which are promising potential candidates to increase the benefits of immunotherapies. Citation Format: Enrique Javier Arenas Lahuerta, Alex Martínez-Sabadell, Irene Rius Ruiz, Macarena Román Alonso, Marta Escorihuela, Antonio Luque, Carlos A. Fajardo, Alena Gros, Christian Klein, Joaquín Arribas. JAK2 downmodulation leads to interferon gamma deficient response and resistance to immunotherapy in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1690.

Abstract 2169: ACE1702: A potent and off-the-shelf oNK cell therapy product

Abstract Introduction Autologous or allogeneic natural killer (NK) cells possess efficient cytotoxicity against tumor cells without severe side effects such as CRS or graft-versus-host disease (GvHD). In addition to chimeric antigen receptor (CAR) strategy, antibody-cell conjugates (ACCTM) platform provides more efficient way to arm NK cells with binding specificity and enhanced potency against target cells. In this work, we develop a NK cell therapy product ACE1702, a novel NK cell line oNK conjugated with Trastuzumab, and assess its potency against HER2+ solid tumors. Methods oNK cells, a fluorescence-activated cell sorting (FACS) isolated CD16+ population sorted from NK-92 cells, was covalently conjugated with monoclonal antibody Trastuzumab after sublethal irradiation by our patented antibody-cell conjugates (ACCTM) platform to become our cryopreserved final product ACE1702 compliant with current good manufacturing practice (cGMP). Characterization of ACE1702 was analyzed by flow cytometry. Function of ACE1702 was validated by real-time xCELLigence analyzer in vitro. Efficacy of intraperitoneally (ip.) delivered ACE1702 was evaluated in tumor-bearing female immune compromised NSG mice. Tumorigenic potential of non-irradiated oNK and ACE1702 was evaluated in subcutaneous implantation female BALB/c nude mice model. Results We demonstrated that newly established CD16+ oNK cells harbor elevated basal cytotoxicity against target cells in the absence of antibodies without tumorigenic potential. The Trastuzumab-armed oNK cells, ACE1702, exerted human epidermal growth factor 2 (HER2) binding specificity and enhanced cytotoxicity against various types of cancer cells with grade 1 (MCF-7), grade 2 (OVCAR-3) and grade 3 (SK-OV-3) HER2 expression compared to control oNK cells in vitro. ACE1702 also displayed similar potency against Herceptin-resistant human breast cancer cell line BT-474 clone 5 and parental BT-474 cells in vitro. Furthermore, in vivo results in human ovarian cancer cell line SK-OV-3-bearing xenograft mouse model supported the in vitro observations. Compared to other immunotherapeutic strategies such as antibody-drug conjugate, allogeneic NK and chimeric antigen receptor (CAR) modification, ACCTM platform underscores its power in compatibility with diverse antibodies, ease for mass production compliant with good manufacturing practice and affordability to patients. Conclusion Here we introduced a novel Trastuzumab-modified oNK cell product with enhanced specificity against myriad types of HER2+ cancers even in the presence of Herceptin resistance, and demonstrated the non-tumorigenic potential of non-irradiated oNK cells and ACE1702. Citation Format: Hao-Kang H. Li, Tai-Sheng E. Wu, Ching-Wen S. Hsiao, Sen-Han S. Yang, Chia-Yun S. Lee, Yan-Liang J. Lin, Zih-Fei Z. Cheng, Yu-Bei Cheng, Yan-Da D. Lai, Hsiu-Ping C. Yang, Sai-Wen S. Tang, Wei-Lun S. Lo, Janet Pan, Shih-Chia S. Hsiao. ACE1702: A potent and off-the-shelf oNK cell therapy product [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2169.

Dynamic microRNA activity identifies therapeutic targets in trastuzumab-resistant HER2+ breast cancer.

MicroRNAs (miRNAs) are implicated in numerous physiologic and pathologic processes, such as the development of resistance to chemotherapy. Determining the role of miRNAs in these processes is often accomplished through measuring miRNA abundance by polymerase chain reaction, sequencing, or microarrays. We have developed a system for the large-scale monitoring of dynamic miRNA activity and have applied this system to identify the contribution miRNA activity to the development of trastuzumab resistance in a cell model of HER2+ breast cancer. MiRNA activity measurements identified significantly different activity levels between BT474 cells (HER2 + breast cancer) and BT474R cells (HER2 + breast cancer cells selected for resistance to trastuzumab). We created a library of 32 miRNA reporter constructs, which were delivered by lentiviral transduction into cells, and miRNA activity was quantified by bioluminescence imaging. Upon treatment with the bioimmune therapy, trastuzumab, the activity of 11 miRNAs were significantly altered in parental BT474 cells, and 20 miRNAs had significantly altered activity in the therapy-resistant BT474R cell line. A combination of statistical, network and classification analysis was applied to the dynamic data, which identified miR-21 as a controlling factor in trastuzumab response. Our data suggested downregulation of miR-21 activity was associated with resistance, which was confirmed in an additional HER2 + breast cancer cell line, SKBR3. Collectively, the dynamic miRNA activity measurements and analysis provided a system to identify new potential therapeutic targets in treatment-resistant cancers.

Abstract 903: Upregulation of DARPP32/PPP1R1B is a critical mediator of acquired lapatinib resistance in ErbB2-overexpressing breast cancer cells

Abstract ErbB2/Her2 overexpression is detected in approximately 30% of human breast cancers and is associated with poor prognosis. Lapatinib, a small molecule dual inhibitor targeting ErbB2 and EGFR, is FDA-approved for the treatment of ErbB2-positive (ErbB2+) advanced or metastatic breast cancer. However, lapatinib resistance (LR) is emerging as a critical issue in clinical oncology. To understand the molecular mechanisms of LR, we first developed a lapatinib-resistant cell line from ErbB2-overexpressing BT474 breast cancer cells by prolonged exposure of parental BT474 cells to gradually increasing concentrations of lapatinib (up to 8 µM). We performed microarray analyses on parental and LR cells to identify novel factors/pathways that contribute to LR. Our microarray data indicated that the gene expression profile of the BT474/LR cells is significantly different from the parental BT474 cells. We found that DARPP32/PPP1R1B is one of the most significantly upregulated genes in the BT474/LR cells. To investigate the role of DARPP32 in LR development, we examined the effect of DARPP32 knockdown via lentiviral shRNA on BT474/LR cell proliferation and apoptosis. Results from MTT, clonogenic, cell cycle, and cleaved caspase-3 and PARP analyses indicated that DARPP32 knockdown significantly sensitized BT474/LR cells to lapatinib. We also found that DARPP32 knockdown renders MDA-MB-361 breast cancer cells, which express high endogenous levels of DARPP32, more sensitive to lapatinib. These data demonstrate that DARPP32 overexpression contributes to LR. To understand the mechanism of DARPP32 overexpression-mediated LR, we focused on the regulation of transcription factor cAMP response element-binding protein (CREB) in this process. As such, DARPP32 overexpression was generally associated with CREB upregulation. We also found that CREB-mediated transcription was increased in BT474/LR cells, and DARPP32 knockdown in BT474/LR and MDA-MB-361 cells significantly downregulated CREB protein levels and CREB-mediated transcription, as indicated by luciferase reporter assays. CREB overexpression rendered BT474 cells resistant to lapatinib and reversed DARPP32 knockdown-induced sensitization in BT474/LR cells. In contrast, CREB knockdown sensitized the cells to lapatinib. Further analysis with MG132 blockage suggests that DARPP32 may regulate CREB protein levels through the proteasomal degradation pathway. Taken together, we have identified DARPP32 overexpression as a critical mediator of acquired lapatinib resistance. The underlying mechanisms involve the deregulation of CREB protein and activity levels. These novel findings advance our understanding of the molecular mechanisms of LR and provide fundamental support for testing the DARPP32-CREB axis in clinical settings for the management of LR breast cancers, which is of significant translational value. Citation Format: Zhikun Ma, Erin W. Howard, Amanda B. Parris, Xiaohe Yang. Upregulation of DARPP32/PPP1R1B is a critical mediator of acquired lapatinib resistance in ErbB2-overexpressing breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 903.

Abstract 477: Targeting of ErbB3 with functional cooperative miRNAs enhances efficacy of trastuzumab in ErbB2-overexpressing breast cancer cells

Abstract Introduction: ErbB3 serves as a critical co-receptor of erbB2 and plays a vital role in the development of erbB2-overexpressing (erbB2+) breast cancer. It is thought to be an important compensatory target for combinatorial strategy to improve the treatments for erbB2+ breast cancer patients. MiRNAs regulate gene expression by the sequence-specific targeting of mRNAs, leading to translational repression or mRNA degradation. ErbB3 is a direct target of both miR-125a and miR-205. Here, we study the potential activity of miRNA based erbB3-targeted therapy in erbB2+ breast cancer cells. Methods: Cell growth assays were used to determine cell viability. Western blot analyses were performed to assess the expression and activation of proteins. Flow cytometry analysis was carried out to examine cell cycle progression. Lentiviral vector containing one or two miRNAs was used to ectopically express miR-125a and/or miR-205. Results: Co-expression of miR-125a and miR-205 showed a potent activity to downregulate erbB3 while single miRNA had no or little effect on erbB3 when miR-125a or miR-205 was only increased below 10-fold in BT474 cells. Combination of the two miRNAs not only resulted in a dramatic reduction of phosphorylated erbB3 (P-erbB3) and the downstream signaling kinases Akt (P-Akt) and Src (P-Scr), it also inhibited cell proliferation and increased the cells at G1 phase. More importantly, concomitant expression of the two miRNAs significantly enhanced trastuzumab-mediated growth inhibition and cell cycle G1 arrest in BT474 cells. Interestingly, the expression levels of miR-125a, but not miR-205 in the trastuzumab-resistant BT474-HR20 cells were much lower than that in the parental BT474 cells. Ectopic expression of miR-125a alone profoundly inhibited proliferation of BT474-HR20 cells. These data suggested that reduced miR-125a might be a novel mechanism leading to trastuzumab resistance; and upregulation of miR-125a could be a useful tool to abrogate the resistance. Conclusions: Co-expression of miR-125a and miR-205 is a new approach to inhibit erbB3. Specific targeting of erbB3 via the functional cooperative miRNAs enhances efficacy of trastuzumab against erbB2+ breast cancer cells. Our data support further exploration of the possible role of miR-125a in the development of trastuzumab resistance. Keywords: ErbB3, ErbB2, miRNA, Trastuzumab, Breast Cancer Citation Format: Hui Lyu, Jingcao Huang, Bolun Wang, Amy Han, Bolin Liu. Targeting of ErbB3 with functional cooperative miRNAs enhances efficacy of trastuzumab in ErbB2-overexpressing breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 477.

Dual inhibition of IGF1R and ER enhances response to trastuzumab in HER2 positive breast cancer cells.

Although HER2 targeted therapies have improved prognosis for HER2 positive breast cancer, HER2 positive cancers which co-express ER have poorer response rates to standard HER2 targeted therapies, combined with chemotherapy, than HER2 positive/ER negative breast cancer. Administration of hormone therapy concurrently with chemotherapy and HER2 targeted therapy is generally not recommended. Using publically available gene expression datasets we found that high expression of IGF1R is associated with shorter disease-free survival in patients whose tumors are ER positive and HER2 positive. IGF1R is frequently expressed in HER2 positive breast cancer and there is significant evidence for crosstalk between IGF1R and both HER2 and ER. Therefore, we evaluated the therapeutic potential of targeting ER and IGF1R in cell line models of HER2/ER/IGF1R positive breast cancer, using tamoxifen and two IGF1R targeted tyrosine kinase inhibitors (NVP-AEW541 and BMS-536924). Dual inhibition of ER and IGF1R enhanced growth inhibition in the four HER2 positive cell lines tested and caused an increase in cell cycle arrest in G1 in BT474 cells. In addition, combined treatment with trastuzumab, tamoxifen and either of the IGF1R TKIs enhanced response compared to dual targeting strategies in three of the four HER2 positive breast cancer cell lines tested. Furthermore, in a cell line model of trastuzumab-resistant HER2 positive breast cancer (BT474/Tr), tamoxifen combined with an IGF1R TKI produced a similar enhanced response as observed in the parental BT474 cells suggesting that this combination may overcome acquired trastuzumab resistance in this model. Combining ER and IGF1R targeting with HER2 targeted therapies may be an alternative to HER2 targeted therapy and chemotherapy for patients with HER2/ER/IGF1R positive breast cancer.

Abstract 319: Role of GPR110 on tumorigenesis and metastasis in HER2+ breast cancer in the context of anti-HER2 drug resistance

Abstract Drug resistance is a major limitation of treatment regimens targeting the human epidermal growth factor receptor-2 (HER2) in breast cancers that overexpress this protein. Therefore, identification of novel drug targets to overcome anti-HER2 drug resistance is an unmet clinical need. Our previous studies have suggested that GPR110 may be a potential target for anti-HER2 drug resistance and tumorigenicity. Furthermore, GPR110 is an orphan receptor in the adhesion G-protein coupled receptor family, members of which have shown a role in cancer metastasis. In this study, our objectives were (1) to assess the gene expression of GPR110 using the Taqman RT-PCR assay in anti-HER2 drug resistant and in cancer stem-like cell population of various HER2+ breast cancer cell line models and (2) to investigate the effects of GPR110 gene knockdown on tumorigenesis and metastasis using various cell-based assays in HER2+ breast cancer cell lines and their anti-HER2-resistant derivatives. Cells transfected with non-targeting and 3 independent GPR110-targeting siRNAs were used to assess anchorage-independent cell growth, mammosphere formation, and cell invasion and migration. We found that GPR110 gene expression was significantly higher (>/ = 2-fold) in lapatinib-resistant (LR), trastuzumab-resistant (TR), and lapatinib + trastuzumab-resistant (LTR) derivatives of BT474, SKBR3 and UACC812 cell lines compared to the parental cells. GPR110 gene expression was also significantly higher (>/ = 1.9-fold) in ALDH+ versus ALDH- cell populations of BT474, SKBR3, MDA-MB-361 and HCC1569 cell lines. In addition, GPR110 gene knockdown led to a 55-85% (p<0.05) decrease in anchorage-independent cell growth in BT474 and SKBR3 LTR derivatives but not in parental cells. A significant reduction in mammosphere count upon GPR110 knockdown was also seen in LTR derivatives (50-70%, p<0.05) and not in parental BT474 cells. Interestingly, GPR110 knockdown in SKBR3 parental cells resulted in a significant reduction in cell invasion (40-50%, p<0.05) and migration (40-50%, p<0.05) suggesting a potential role of GPR110 in tumor cell dissemination. Overall, these results demonstrate that GPR110 is overexpressed in multiple anti-HER resistant derivatives as well as breast cancer stem-like cell population of various HER2+ breast cancer cell line models. Our studies also suggest a role of GPR110 in tumorigenesis specifically in the context of anti-HER2 drug resistance and in metastatic processes. Future in vivo studies will assess GPR110 as a novel drug target to overcome anti-HER2 drug resistance and combat metastasis in HER2+ breast cancer. Citation Format: Debashish Sahay, Raksha Bhat, Motthafar Rimawi, C. Kent Osborne, Rachel Schiff, Meghana V. Trivedi. Role of GPR110 on tumorigenesis and metastasis in HER2+ breast cancer in the context of anti-HER2 drug resistance. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 319.

Autophagy Protects from Trastuzumab-Induced Cytotoxicity in HER2 Overexpressing Breast Tumor Spheroids.

Multicellular tumor spheroids represent a 3D in vitro model that mimics solid tumor essential properties including assembly and development of extracellular matrix and nutrient, oxygen and proliferation gradients. In the present study, we analyze the impact of 3D spatial organization of HER2-overexpressing breast cancer cells on the response to Trastuzumab. We cultured human mammary adenocarcinoma cell lines as spheroids with the hanging drop method and we observed a gradient of proliferating, quiescent, hypoxic, apoptotic and autophagic cells towards the inner core. This 3D organization decreased Trastuzumab sensitivity of HER2 over-expressing cells compared to monolayer cell cultures. We did not observe apoptosis induced by Trastuzumab but found cell arrest in G0/G1 phase. Moreover, the treatment downregulated the basal apoptosis only found in tumor spheroids, by eliciting protective autophagy. We were able to increase sensitivity to Trastuzumab by autophagy inhibition, thus exposing the interaction between apoptosis and autophagy. We confirmed this result by developing a resistant cell line that was more sensitive to autophagy inhibition than the parental BT474 cells. In summary, the development of Trastuzumab resistance relies on the balance between death and survival mechanisms, characteristic of 3D cell organization. We propose the use of spheroids to further improve the understanding of Trastuzumab antitumor activity and overcome resistance.

Abstract 3573: Novel G protein-coupled receptor targets in HER2+ breast cancer

Abstract Background: G protein-coupled receptors (GPCRs) are excellent drug targets. However, not many GPCRs have been identified as potential drug targets in breast cancer (BC). This may be due to two factors: (1) The primary mode of GPCR regulation is at the level of signaling rather than gene expression, which is the common method for large-scale target identification. (2) GPCR expression in BC stem cells or drug-resistant cells could have been overlooked because these cells exist in small fractions. In this study, we aimed to identify novel GPCR targets based on large panel functional screening and differential gene expression in HER2+ BC stem cells and drug resistant cells. Methods: (I) For the functional screening, each of the 370 GPCR genes was knocked down using targeted Silencer® Select siRNA library comprising of 3 individual siRNAs per target in BT474 and SKBR3 cell lines (N = 3). Candidate GPCRs were selected based on at least 20% alteration in cell growth by at least 2 out of 3 siRNAs. (II) For the expression screening, gene expression of 343 GPCRs was measured in ALDH+ BC stem cells vs. ALDH- cells as well as in anti-HER2 drug-resistant derivatives vs. parental cells of BT474 cells using Taqman RT-PCR arrays (N = 2-4). Melatonin MT1 receptor (MTNR1A) gene knockdown and pharmacological modulation were employed to functionally validate its role in anchorage-dependent (MTT assay) and -independent (soft agar colony formation assay) cell growth. Results: Gene knockdown of 11 GPCRs (ADORA, ADRA1D, CCR8, GNRHR, GPR26, GPR113, GPR120, LPAR3, MC5R, MTNR1A, NTSR2) altered cell growth of HER2+ BC cells by at least 20%. The MT1 receptor was selected as a candidate GPCR for this study because (1) its knockdown resulted in at least 20% inhibition of cell growth in both BT474 and SKBR3 cells, and (2) its expression was increased in ALDH+ vs. ALDH- as well as in drug-resistant cells vs. parental BT474 cells. MTNR1A knockdown resulted in 25-35% inhibition in the anchorage-dependent cell growth of both BT474 and SKBR3 cells. Furthermore, MTNR1A knockdown caused a marked (3-fold) decrease in the number of colonies in soft agar assay. MT1 receptor antagonist luzindole reduced the anchorage-dependent cell growth of BT474 cells in a concentration-dependent manner (IC50 = 20-55 nM). However, the efficacy of luzindole was only ∼50% of lapatinib efficacy in reducing cell growth. Luzindole (1μM) also significantly reduced (by 2-fold) anchorage-independent cell growth of BT474 cells. Neither MTNR1A knockdown nor luzindole significantly improved the efficacy of lapatinib (1nM) in inhibiting cell growth of BT474 cells. Conclusion: In summary, functional knockdown screening and differential gene expression of GPCRs is a powerful tool for identifying GPCRs as candidate targets in BC. Using the combination of these two approaches, we have discovered that melatonin MT1 receptor as a potential drug target in HER2+ BC cells. The place in therapy for MT1 receptor antagonists, however, still remains to be determined. Citation Format: Raksha Bhat, Puja Yadav, Pavel Christiny, Rachel Schiff, Meghana V. Trivedi. Novel G protein-coupled receptor targets in HER2+ breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3573. doi:10.1158/1538-7445.AM2015-3573

Abstract 1971: Inhibition of CXCR4 pathway augments trastuzumab sensitivity in HER2 positive breast cancer cells with intrinsic and acquired trastuzumab resistance

Abstract The clinical benefit from trastuzumab (T) in HER2-positive breast cancers is limited by de novo or acquired resistance. By serial exposure of HER2+ breast cancer cell lines BT474 and SKBr3 to T at 200 µg/ml for 1 year and clonogenic selection, we have developed T-resistant cell lines and identified differentially expressing genes including consistent overexpression of CXCR4 at the mRNA and protein levels. CXCR4 is involved in breast cancer metastasis and proliferation and its overexpression in human breast cancer specimens is linked to poor prognosis. We sought to functionally validate CXCR4 as a resistance mechanism to T. CXCR4 mRNA was quantified using qRT-PCR, and CXCR4 protein expression was quantified by Western blotting. Stable clones overexpressing CXCR4 were selected by transfecting CXCR4 full length sequence-containing lentivirus into parental trastuzumab sensitive BT474 and SKBR3 cells. Stable CXCR4 knockdown clones were selected by transfecting three CXCR4 sequence specific shRNA containing lentivirus along with scrambled sequence as a control, into acquired T-resistant cell lines termed BT474R1 and SKBR3R1 and intrinsically T-resistant HCC-1419 cell line. Stable clones of BT474R1 with tetracycline controlled (tet-on) CXCR4 expression, were created using the pSLIK-hygro lentiviral system. CXCR4 expression was assessed by Western blot assay and by flow cytometry. MTT cell viability analysis was performed on these transfected cell lines to characterize T dose responsiveness with parental sensitive and T-resistant BT474R1 cells as control. BT474R cells demonstrated a 100-fold increase in IC50 to T treatment when compared to parental BT474 cells and also exhibited a 4-fold increase in CXCR4 mRNA and a corresponding increase in CXCR4 protein. Transfection of CXCR4 into sensitive BT474 and SkBR3 cell lines showed a greater than 100 fold increase in the IC50 value by MTT assay after treatment with T as compared to the parental cell lines. Semi-quantitative analysis by Western-blotting confirmed a 7 to 8 fold increase in CXCR4 expression in CXCR4-transfected cells as compared to parental cells. shRNA-mediated knockdown of CXCR4 in BT474R1 as well as primarily resistant HCC-1419 cell line showed a significantly improved responsiveness to trastuzumab with a 100 fold decrease in the IC50 value whereas a moderate response was seen in the SKBR3R1 cells. CXCR4 overexpression and activation of the CXCR4/CXCL-12 pathway contributes to trastuzumab resistance in human breast cancer cell lines. shRNA-mediated inhibition of the CXCR4 pathway in resistant cells significantly augments trastuzumab sensitivity. CXCR4 targeting may represent a therapeutic strategy to reverse de novo and acquired resistance and to improve the efficacy of trastuzumab in HER2-positive breast cancer. Citation Format: Arjun Mehta, Gloria Yang-Kolodji, Debu Tripathy. Inhibition of CXCR4 pathway augments trastuzumab sensitivity in HER2 positive breast cancer cells with intrinsic and acquired trastuzumab resistance. [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 1971. doi:10.1158/1538-7445.AM2014-1971

Abstract P5-08-04: Overexpression of CXCR-4 confers trastuzumab resistance in human breast cancer cell lines

Abstract Background: Trastuzumab significantly improves survival in patients with HER2 positive breast cancer; however, de novo or acquired resistance ensues in the advanced setting. We have identified differentially expression genes in acquired trastuzumab resistance cell line models following long-term trastuzumab exposure and have demonstrated consistent overexpression of CXCR-4 at the mRNA and protein levels. The interaction between the cognate ligand CXCL-12 and its receptor CXCR-4 is known to promote malignant cell proliferation and angiogenesis via activation of Akt and MAPK pathways. CXCR-4 shows no to low expression in normal breast epithelium while its expression sequentially increases from precancerous lesions to carcinomas. CXCR-4 overexpression in human breast cancer is also associated with a worse outcome in small studies. We sought to explore the role of CXCR-4 expression on trastuzumab drug responsiveness. Methods: Trastuzumab-resistant cell lines were generated by serial passaging of HER+ cells lines BT-474 and SKBR-3 for 1 year with continual exposure to 200 μg/ml of trastuzumab. For these resistant cell lines, termed BtRT and SkRT, CXCR-4 mRNA was quantified using qRT-PCR, and CXCR-4 protein expression was quantified by Western blot. Stable clones overexpressing CXCR-4 receptor were selected by transfecting CXCR-4 sequence-containing lentivirus into sensitive parental sensitive BT-474 and SKBR3 cells. Expression of CXCR-4 receptor was assessed by Western blot assay and by flow cytometry. MTT cell viability analysis was performed on these transfected cell lines to characterize trastuzumab dose responsiveness with parental cells as control. Results: Trastuzumab-resistant cell line BtRT demonstrated a 100-fold increase in IC50 to trastuzumab treatment when compared to parental BT-474 cells and also exhibited a 3-fold further increase in CXCR-4 mRNA and a corresponding increase in CXCR-4 protein. CXCR-4-transfected sensitive BT474 cell lines showed a greater than 100 fold increase in the IC50 value on MTT assay after treatment with trastuzumab as compared to the parental cell line. Semi-quantitative analysis by Western-blot confirmed a 5-fold increase in CXCR-4 expression in CXCR-4-transfected cells as compared to parental cells. CXCR-4 expression in HER2+ human breast tumors and responsiveness in patients treated with neoadjuvant trastuzumab-based therapy will be presented. Conclusion: CXCR-4 overexpression and activation of the CXCR-4/CXCL-12 pathway contributes to trastuzumab resistance in human breast cancer cell lines. CXCR-4 targeting may represent a therapeutic strategy to improve the efficacy of trastuzumab in HER2-positive breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-08-04.

Abstract P4-06-02: Identification of novel G-protein coupled receptor targets in HER2-positive breast cancer.

Abstract Background: HER2 is a member of human epidermal growth factor receptor (HER) superfamily and HER2-overexpressing (HER2+) breast cancer (BC) is an aggressive tumor. Despite the clinical success of anti-HER2 therapies, de novo and acquired drug resistance occur in many patients. Identification of novel drug targets to overcome anti-HER2 therapy resistance is an unmet need. Since G-protein coupled receptors (GPCRs) are known to cross-talk with the HER superfamily, it is possible that some GPCRs may signal to modulate the HER2 pathway. GPCRs are considered excellent drug targets due to their plasma membrane localization, unique ligand-binding pocket, and availability of high throughput assays for drug screening. The expression and function of the majority of GPCRs are largely unknown in HER2+ BC. The goal of this study was to identify novel GPCR targets in HER2+ BC, in the context of anti-HER2 therapy resistance. Methods: We examined the differential GPCRs expression in BC stem cells, suggested to be involved in resistance, as well as in anti-HER2 treatment-resistant BT474 cell line model of HER2+ BC. BC stem cells were identified as aldehyde dehydrogenase-positive (ALDH+) cells using the Aldefluor assay. Brightly fluorescent ALDH+ cells were separated from ALDH- cells using FACS Aria II cell sorter. Anti-HER2 resistant derivatives of BT474 cells were established by long-term exposure to increasing drug concentration of trastuzumab (T), lapatinib (L), or their combination (T+L). RNA was isolated and subjected to profiling using TaqMan real time RT-PCR GPCR 384-well microarray to quantify the expression of mRNA encoding 343 GPCRs from 50 different subfamilies. Only overexpressed GPCRs were considered of interest as drug targets, and the overexpression of GPCRs was verified by RT-PCR and western blotting for the selected targets. Publically available TCGA dataset for mRNA expression was also interrogated to determine differential expression of selected GPCRs in HER2+ vs. other subtypes of BC. Results: Nine GPCRs [BAI3, EDNRA, GPR110, GPR116, GPR124, MTNR1A, EDG2, EMR2, GCGR] were upregulated in ALDH+ compared to ALDH- BT474 cells. In addition, 11 GPCRs [CCBP2, CCR9, F2RL1, GALR2, GPR1, GPR24, GPR87, GPR110, GPR183, LGR4, OXER1] were over-expressed in the resistant derivatives (T, L, and T+L) compared to the parental BT474 cells. Out of these, 13 belong to Class A and 6 to Class B, designated by The International Union of Basic and Clinical Pharmacology (IUPHAR). GPR110 was the only GPCR common to BC stem cells as well as resistant derivatives of BT474 cells. In TCGA dataset, GPR110 expression was significantly higher in HER2+ and basal subtypes of BC compared to ER+ luminal A and B subtypes. GPR110 as well as other differentially expressed GPCRs are currently being investigated as potential targets by determining the effects of their downregulation or exogenous over-expression on the growth and drug-sensitivity of these BC cells. Conclusions: We report for the first time the differential expression of a large panel of GPCRs in the BC stem cell population and in anti-HER2 resistant derivatives of the BT474 cell line model of HER2+ BC. Results of the functional studies will guide novel strategies to improve the treatment for HER2+ BC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-06-02.

Abstract 3812: Deguelin as a novel therapeutic agent for Herceptin resistant breast cancers

Abstract Human breast cancers overexpressing HER-2 are generally highly aggressive. Current therapeutic strategies to target HER-2 signaling pathway with antibodies or small molecule tyrosine kinase inhibitors have shown impressive success in the management of these tumor types, although most women exhibit short term response to these therapies. Acquired resistance to HER-2 target drugs, especially Herceptin is a major challenge to clinicians. Patients receiving Herceptin therapy invariably recur and most often with highly aggressive tumors. Thus identifying new drugs which target multiple molecular pathways is desired to treat Herceptin resistant breast cancer. Using paired HER-2 overexpressing parental Herceptin responsive (BT-474) and its Herceptin resistant variant cells (BT-474HR) we showed that acquired resistance to Herceptin is associated with enhanced expression of HER-2, EGFR, CyclinD1, P-AKT (Ser-473), PI-3K, JAB1 proteins and decreased expression of nuclear pten and p27 proteins. These results are similar to those observed in recurrent breast tumors in patients following Herceptin treatment. Deguelin, a natural product derivative of rotenoids and has been shown to inhibit growth of diverse cancer types. Deguelin is a known AKT inhibitor, however its efficacy is never tested in breast cancers resistant to Herceptin which express high levels of p-AKT. We examined effect of Deguelin on HER-2 overexpressing BT-474 BT-474-HRcells. Deguelin inhibited growth of both, BT-474 and BT-474HR cells in concentration (0-250nM) and time dependent (24-72h) manner. There was a 40-50%- inhibition of both BT474and BT-474HR cells at 25nM Deguelin treatment at 72h. Deguelin decreased cell surface expression of HER-2 in both these cell lines. In parental BT-474 cells both Herceptin 5-10ug/ml and Deguelin down regulated HER-2 expression, but only Deguelin reduced HER-2 (125-250nM) over expression in both BT-474 and BT-474HR cells. Down regulation of HER-2 following Deguelin treatment was associated with simultaneous down regulation of nuclear accumulation of cyclin D1, and JAB1, and p-AKT (Ser-473) proteins and up regulation of nuclear p27 and p53in both BT-474 and BT474 HR cells. These results suggest that Deguelin inhibits growth of both HER-2 overexpressing and those resistant to Herceptin therapy breast cancer cells. Effect of Deguelin appears to be mediated through down regulation of HER-2, PI3K AKT, JAB1, Cyclin D1, and p27 pathway. Our preliminary results warrant further investigation into Deguelin as a potential therapeutic agent for Herceptin resistant breast cancers. (This work was supported by NCI CA140321.) 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 3812. doi:1538-7445.AM2012-3812

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

P2-01-14: High Level of CD44 Expression and Soluble CD44 Secretion Mediate Trastuzumab Resistance in HER2 Positive Breast Cancer.

Abstract Background: Overexpression of HER2 occurs in 15–25% of invasive breast cancer. Trastuzumab, a humanized monoclonal antibody targeting the extracellular domain of HER2, markedly improves disease-free and overall survival in patients with HER2−positive breast cancer. However, resistance to trastuzumab develops in most patients with HER2−overexpressing metastatic breast cancer. Breast cancer stem cells have been shown to express HER2, although its role in the development or maintenance of stem cells is not well characterized. CD44 is one of the proposed markers for breast cancer stem cells. We investigated the role of CD44 in acquired trastuzumab resistance in HER2 positive breast cancer. Methods: CD44 mRNA expression was measured by quantitative real-time PCR. The proportion of CD44 positive cells was measured by fluorescence-activated cell sorting (FACS). Soluble CD44 level was detected by enzyme-linked immunosorbent assay (ELISA). In vitro tumorigenicity was evaluated by soft agar colony formation assay. Cell invasion was tested by using a matrigel invasion assay. In vivo tumor growth was evaluated using a mammary fat pad xenograft mouse model. Results: CD44 mRNA expression, CD44 positive cell population by FACS, and CD44 secretion were significantly increased in both SKBR3 derived trastuzumab resistant SKBR3 clone 3 and BT474 derived trastuzumab resistant HR20 cells compared to parental, trastuzumab sensitive cells. Consistent with the increase in CD44, trastuzumab resistant cells showed an increase in soft agar colony formation and cell invasion, which were blocked by siRNA-induced downregulation of CD44 expression. Tumor growth and incidence were enhanced in mice injected with trastuzumab resistant HR20 cells compared to the mice injected with trastuzumab sensitive parental BT474 cells. Knock-down of CD44 expression by the transfection with CD44-specific siRNA suppressed in vivo tumor growth in the mice injected with trastuzumab resistant HR20 cells. Finally, we found high serum CD44 levels correlated with advanced tumor progression in HER2 positive breast cancer. Conclusion: Our findings indicate that high levels of CD44 mediate trastuzumab resistance in HER2 positive breast cancer cells as well as tumor progression in mice. Circulating CD44 is prognostic in patients with HER2 positive breast cancer. CD44 may be a therapeutic target for HER2 positive breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P2-01-14.

Breast tumor cells isolated from in vitro resistance to trastuzumab remain sensitive to trastuzumab anti-tumor effects in vivo and to ADCC killing.

An understanding of model systems of trastuzumab (Herceptin) resistance is of great importance since the humanized monoclonal antibody is now used as first line therapy with paclitaxel in patients with metastatic Her2 overexpressing breast cancer, and the majority of their tumors has innate resistance or develops acquired resistance to the treatment. Previously, we selected trastuzumab-resistant clonal cell lines in vitro from trastuzumab-sensitive parental BT-474 cells and showed that cloned trastuzumab-resistant cell lines maintain similar levels of the extracellular Her2 receptor, bind trastuzumab as efficiently as the parental cells, but continue to grow in the presence of trastuzumab and display cell cycle profiles and growth rates comparable to parental cells grown in the absence of trastuzumab (Kute et al. in Cytometry A 57:86-93, 2004). We now show that trastuzumab-resistant and trastuzumab-sensitive cells both surprisingly display trastuzumab-mediated growth inhibition in athymic nude mice. This demonstrates that resistance developed in vitro is not predictive of resistance in vivo. The observation that in vitro resistant cells are sensitive to trastuzumab in vivo could be explained by antibody dependent cellular cytotoxicity (ADCC). Therefore, both parental and trastuzumab-resistant cells were assayed for ADCC in real time on electroplates with and without trastuzumab in the presence of a natural killer cell line (NK-92), and granulocyte or mononuclear cellular fractions isolated from human peripheral blood. Mononuclear cells and NK-92 cells were more effective in killing both parental and trastuzumab-resistant cells in the presence of trastuzumab. Both trastuzumab-resistant cells and trastuzumab-sensitive cells showed similar susceptibility to ADCC despite displaying divergent growth responses to trastuzumab. The granulocyte fraction was able to kill these cells with equal efficacy in the presence or absence of trastuzumab. These results support a model of trastuzumab tumor cell killing in vivo mediated primarily by ADCC from the mononuclear fraction of innate immune cells and suggest that in the clinical setting not only should changes in signaling transduction pathways be studied in acquired tumor resistance to trastuzumab, but also mechanisms by which tumors impede immune function should be evaluated.

Both t-Darpp and DARPP-32 can cause resistance to trastuzumab in breast cancer cells and are frequently expressed in primary breast cancers

The clinical use of trastuzumab (Herceptin), a humanized antibody against the HER2 growth factor receptor, has improved survival in patients with breast tumors with ERBB2 amplification and/or over-expression. However, most patients with advanced ERBB2 amplified breast cancers whose tumors initially respond to trastuzumab develop resistance to the drug, leading to tumor progression. To identify factors responsible for acquired resistance to trastuzumab, gene expression profiling was performed on subclones of an ERBB2 amplified breast cancer cell line, BT474, which had acquired resistance to trastuzumab. The most overexpressed gene in these subclones was PPP1R1B, encoding the DARPP-32 phosphatase inhibitor. Western analysis revealed that only the truncated isoform of the DARPP-32 protein, t-Darpp, was overexpressed in the trastuzumab resistant cells. Using gene silencing experiments, we confirmed that t-Darpp over-expression was required for trastuzumab resistance in these cells. Furthermore, transfecting t-Darpp in parental BT-474 cells conferred resistance to trastuzumab, suggesting that t-Darpp expression was sufficient for trastuzumab resistance. We also found that t-Darpp over-expression was associated with Akt activation and that the T75 residue in t-Darpp was required for both Akt activation and trastuzumab resistance. Finally, we found that full-length DARPP-32 and t-Darpp are expressed in a majority of primary breast tumors. Over-expression of full-length DARPP-32 can also confer resistance to trastuzumab and, moreover, is associated with a poor prognostic value in breast cancers. Thus, t-Darpp and DARPP-32 expression are novel prognostic and predictive biomarkers in breast cancer.

A novel HER2 inhibitor TAK-285 overcomes trastuzumab resistance of HER2-over-expressing breast cancer.

Abstract Abstract #3143 Background: The anti-HER2 antibody trastuzumab has been proven highly effective in the treatment of HER2-expressing breast cancers clinically. However, the continuous administration often leads to drug resistance, the mechanism of which is not well understood. Here we present a new mechanism for trastuzumab-resistance. We also show TAK-285, a novel small molecule HER2/EGFR kinase inhibitor under clinical evaluation, overcomes the resistance to trastuzumab.
 Methods: A trastuzumab-resistant sub-clone (BT-474TR) of the HER2-positive human breast cancer line, BT-474, was established in house by serial passage in the presence of 5 μg/mL trastuzumab for over 5 months. Gene expression profiles of the clones were assessed using Affymetrix HG-U133 plus 2.0 human gene arrays and confirmed by real-time PCR. Target genes were depleted by siRNA-mediated knockdown and confirmed by real time-PCR and immunoblot. Effects on cell growth were assessed by the CellTiter-Glo luminescent cell viability assay (Promega).
 Results: BT-474TR cells exhibited modified expression of several genes including cofilin, compared to the parental BT-474 cells. Although immunoblotting using anti-cofilin antibody revealed no clear change in protein level, BT-474TR displayed significant increase in phosphorylation of cofilin. Gene-specific knockdown of cofilin-1 led to re-sensitization of BT-474TR to trastuzumab, though the cofilin-2 knockdown did not. Likewise, knockdown of either LIMK1 or PAK1, known to regulate cofilin-phosphorylation, resulted in similar re-sensitization of BT-474TR to trastuzumab. BT-474TR also exhibited increase in phosphorylation of both LIMK1 and PAK1. TAK-285 showed almost equivalent inhibitory activity against both BT-474 and BT-474TR proliferation in cell culture and in xenograft study, while trastuzumab did not affect the proliferation of BT-474TR in either condition
 Conclusions: These data suggest that activation of cofilin signaling pathway is a probable mechanism of trastuzumab resistance. PAK1/LIMK1/cofilin-1 inhibitors might therefore be useful in preventing or reversing trastuzumab resistance. The phosphorylation status of PAK1/LIMK1/cofilin might also serve as a biomarker of Trastuzumab-sensitivity. Moreover a novel HER2 inhibitor, TAK-285, might show benefit in the treatment of trastuzumab-resistant HER2-expressing breast cancers. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3143.

ErbB-Dependent Signaling as a Determinant of Trastuzumab Resistance

The pathogenesis of breast cancer is profoundly influenced by the signaling cascades downstream of the human epidermal growth factor receptor (HER; also known as the ErbB receptor) family of receptor tyrosine kinases and by the HER ligands ([1][1], [2][2]). Abnormalities in the expression and

Autoresistance to ErbB2 kinase inhibitors: Elucidating mechanisms and identfying strategies to prevent its onset in breast cancer

2075 Background: Clinical efficacy of ErbB1/ErbB2 kinase inhibitors is limited by the development of acquired autoresistance. Developing clinically relevant models to study the mechanism(s) of acquired autoresistance will identify therapeutic strategies to prevent its onset. Methods: BT474 cells, an ErbB2-overexpressing/ER+ breast cancer line sensitive to lapatinib-induced apoptosis were chronically exposed to lapatinib, simulating chronic administration of lapatinib in the clinic. Lapatinib-resistant cells (rBT474) and single cell clones were established. Baseline gene expression in parental BT474 and rBT474 cells was compared using Human Affymetrix oligonucleotide arrays. Sequential tumor biopsies were analyzed by quantitative IHC as part of a clinical trial using lapatinib monotherapy (1500mg/d) in patients with metastatic breast cancer. Results: Gene expression and protein analysis indicates that acquired resistance to lapatinib is mediated by increased estrogen receptor (ER) signaling rather than loss of ErbB2 expression or insensitivity of the ErbB2-MAPK-PI3K pathways to lapatinib. Activation of ER signaling results from de-repression of FOXO3a and transient up-regulation of caveolin-1, both facilitating ER transcriptional activity. Gene interference studies confirm that acquired resistance involves a switch in survival dependence and regulation of survivin from ErbB2 to ER. Since ErbB2 signaling is operative in rBT474 cells, treating parental BT474 cells with lapatinib plus (i) fulvestrant, or (ii) estrogen deprivation prevents the onset of acquired lapatinib resistance compared with either therapy alone. Importantly, increased ER signaling occurs in patients with ErbB2-overexpressing/ER+ breast cancers treated with lapatinib monotherapy, consistent with changes in cell lines. Conclusions: Chronic lapatinib monotherapy induces a shift in the dependence of cell survival from ErbB2 to ER. Combining lapatinib with selected anti-estrogens prevents its onset in cell lines and warrants clinical validation. Establishing models of acquired resistance simulating the clinic provides a tool to identify strategies to improve treatment options for patients with ErbB2-overexpressing breast cancers. [Table: see text]