ErbB2-overexpressing Breast Cancer Cells Research Articles

ErbB2-upregulated HK1 and HK2 promote breast cancer cell proliferation, migration and invasion

ErbB2 is overexpressed in 15-20% of breast cancer, which is associated with malignancy and poor prognosis. We previously reported that ErbB2 supports malignant progression of breast cancer by upregulating lactate dehydrogenase A (LDHA), an important enzyme in glycolysis. However, whether ErbB2 promotes breast cancer progression through other glycolytic enzymes remains unclear. Hexokinase 1 (HK1) and hexokinase 2 (HK2) are the first rate-limiting enzymes of glycolysis and both of them are increased in breast cancer. Here, we aim to investigate whether ErbB2 upregulates HK1 and HK2 and the role of HK1 and HK2 in the malignant progression of ErbB2-overexpressing breast cancer. In current study, we found that the mRNA level of ErbB2 was positively correlated with that of HK1 and HK2, respectively. Moreover, ErbB2 upregulated the protein levels of HK1 and HK2 in breast cancer cells. We also found that both siHK1 and siHK2 significantly inhibited the proliferation, migration and invasion of ErbB2-overexpressing breast cancer cells. Taken together, our findings suggested that ErbB2 promoted the malignant progression of breast cancer cells by upregulating HK1 and HK2, and HK1 and HK2 might serve as promising therapeutic targets for ErbB2-overexpressing breast cancer.

Upregulation of SCD1 by ErbB2 via LDHA promotes breast cancer cell migration and invasion.

The incidence of breast cancer ranks at the top of female malignant tumors in China. Metastasis remains the main cause of death among breast cancer patients. The overexpression of ErbB2 is closely related to the metastasis and poor prognosis of breast cancer patients. Therefore, ErbB2 is an important clinical therapeutic target of breast cancer. However, the molecular mechanism of ErbB2 promoting breast cancer metastasis has not been studied clearly. Stearoyl-CoA desaturase 1 (SCD1) is a key enzyme in catalyzing the conversion of saturated fatty acids (SFAs) into monounsaturated fatty acids (MUFAs). SCD1 is overexpressed in breast cancer, and its overexpression is an indicator of poor prognosis in breast cancer patients. However, the role of SCD1 in ErbB2-overexpressing breast cancer metastasis has not been reported. In this study, we investigated the role of SCD1 in the migration and invasion of ErbB2-overexpressing breast cancer cells and its molecular mechanism. First, we demonstrated that ErbB2 upregulates the expression of SCD1. Second, we found that SCD1 and its catalytic product oleic acid played crucial roles in migration and invasion of ErbB2-overexpressing breast cancer cells. Finally, we found that in breast cancer cells, ErbB2 upregulated SCD1 through lactate dehydrogenase A (LDHA). To sum up, upregulation of SCD1 by ErbB2 via LDHA promotes the migration and invasion of breast cancer cells.

Cerulenin suppresses ErbB2-overexpressing breast cancer by targeting ErbB2/PKM2 pathway.

Cerulenin is a fungal metabolite and a specific inhibitor of fatty acid synthase (FASN), which has shown a potential anticancer activity. 20-25% of breast cancer patients with ErbB2-overexpressing develop resistance to treatment. Therefore, it is urgent to find an effective new target for the treatment of ErbB2-overexpressing breast cancer. Our previous study found that cerulenin inhibits the glycolysis and migration of SK-BR-3 cells, but the effect of cerulenin on other malignant phenotypes of breast cancer is still unknown. Furthermore, the mechanism by which cerulenin displays its inhibitory effects is not fully understood. In this study, we systematically investigate the inhibitory effects of cerulenin on proliferation, migration, invasion and glycolysis of ErbB2-overexpressing breast cancer cells and its molecular mechanism. We found that cerulenin obviously suppresses the proliferation, migration, invasion as well as glycolysis. Through bioinformatic analyses, we found that PKM2 might be a target of cerulenin. In addition, ErbB2 and its signaling pathway upregulated PKM2 protein levels. Furthermore, we demonstrated that cerulenin downregulated the protein levels of ErbB2, PKM2 and EMT markers (MMP9, MMP2 and Snail2) in a dose- and time-dependent manner. Finally, the inhibitory of cerulenin on colony formation, migration, invasion and glycolysis, as well as protein levels of EMT markers were rescued by replenishing with PKM2. These findings illustrated that cerulenin inhibits proliferation, migration, invasion and glycolysis by targeting ErbB2/PKM2 pathway in ErbB2-overexpressing breast cancer cells.

CHIP/STUB1 Ubiquitin Ligase Functions as a Negative Regulator of ErbB2 by Promoting Its Early Post-Biosynthesis Degradation.

Simple SummaryOverexpressed ErbB2/HER2 receptor drives up to a quarter of breast cancers. One aspect of ErbB2 biology that is poorly understood is how it reaches the cell surface following biosynthesis in the endoplasmic reticulum (ER). Here, the authors show that the CHIP (C-terminus of HSC70-Interacting protein)/STUB1 (STIP1-homologous U-Box containing protein 1) protein targets the newly synthesized ErbB2 for ubiquitin/proteasome-dependent degradation in the ER and Golgi, identifying a novel mechanism that negatively regulates cell surface expression of ErbB2. These findings provide one explanation for frequent loss of CHIP expression is ErbB2-overexpressing breast cancers. The authors further show that ErbB2-overexpressing breast cancer cells with low CHIP expression exhibit higher ER stress inducibility, and ER stress-inducing anticancer drug Bortezomib synergizes with ErbB2-targeted humanized antibody Trastuzumab to inhibit cancer cell proliferation. These new insights suggest that reduced CHIP expression may specify ErbB2-overexpressing breast cancers suitable for combined treatment with Trastuzumab and ER stress inducing agents.Overexpression of the epidermal growth factor receptor (EGFR) family member ErbB2 (HER2) drives oncogenesis in up to 25% of invasive breast cancers. ErbB2 expression at the cell surface is required for oncogenesis but mechanisms that ensure the optimal cell surface display of overexpressed ErbB2 following its biosynthesis in the endoplasmic reticulum are poorly understood. ErbB2 is dependent on continuous association with HSP90 molecular chaperone for its stability and function as an oncogenic driver. Here, we use knockdown and overexpression studies to show that the HSP90/HSC70-interacting negative co-chaperone CHIP (C-terminus of HSC70-Interacting protein)/STUB1 (STIP1-homologous U-Box containing protein 1) targets the newly synthesized, HSP90/HSC70-associated, ErbB2 for ubiquitin/proteasome-dependent degradation in the endoplasmic reticulum and Golgi, thus identifying a novel mechanism that negatively regulates cell surface ErbB2 levels in breast cancer cells, consistent with frequent loss of CHIP expression previously reported in ErbB2-overexpressing breast cancers. ErbB2-overexpressing breast cancer cells with low CHIP expression exhibited higher endoplasmic reticulum stress inducibility. Accordingly, the endoplasmic reticulum stress-inducing anticancer drug Bortezomib combined with ErbB2-targeted humanized antibody Trastuzumab showed synergistic inhibition of ErbB2-overexpressing breast cancer cell proliferation. Our findings reveal new insights into mechanisms that control the surface expression of overexpressed ErbB2 and suggest that reduced CHIP expression may specify ErbB2-overexpressing breast cancers suitable for combined treatment with Trastuzumab and ER stress inducing agents.

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.

Ganetespib targets multiple levels of the receptor tyrosine kinase signaling cascade and preferentially inhibits ErbB2-overexpressing breast cancer cells

Although ErbB2-targeted therapeutics have significantly improved ErbB2+ breast cancer patient outcomes, therapeutic resistance remains a significant challenge. Therefore, the development of novel ErbB2-targeting strategies is necessary. Importantly, ErbB2 is a sensitive client protein of heat shock protein 90 (HSP90), which regulates client protein folding, maturation, and stabilization. HSP90 inhibition provides an alternative therapeutic strategy for ErbB2-targeted degradation. In particular, ganetespib, a novel HSP90 inhibitor, is a promising agent for ErbB2+ cancers. Nevertheless, the anti-cancer efficacy and clinical application of ganetespib for ErbB2+ breast cancer is largely unknown. In our study, we examined the anti-cancer effects of ganetespib on ErbB2+ BT474 and SKBR3 breast cancer cells, and isogenic paired cancer cell lines with lentivirus-mediated ErbB2 overexpression. Ganetespib potently inhibited cell proliferation, cell cycle progression, survival, and activation/phosphorylation of ErbB2 and key downstream effectors in ErbB2+ breast cancer cells. Moreover, ganetespib decreased the total protein levels of HSP90 client proteins and reduced ErbB2 protein half-life. ErbB2-overexpressing cancer cells were also more sensitive to ganetespib-mediated growth inhibition than parental cells. Ganetespib also strikingly potentiated the inhibitory effects of lapatinib in BT474 and SKBR3 cells. Ultimately, our results support the application of ganetespib-mediated HSP90 inhibition as a promising therapeutic strategy for ErbB2+ breast cancer.

FGFR inhibitor, AZD4547, impedes the stemness of mammary epithelial cells in the premalignant tissues of MMTV-ErbB2 transgenic mice

The fibroblast growth factor receptor (FGFR) family of receptor tyrosine kinases (RTKs) regulates signaling pathways involved in cell proliferation and differentiation. Currently, the anti-tumor properties of FGFR inhibitors are being tested in preclinical and clinical studies. Nevertheless, reports on FGFR inhibitor-mediated breast cancer prevention are sparse. In this study, we investigated the anti-cancer benefits of AZD4547, an FGFR1-3 inhibitor, in ErbB2-overexpressing breast cancer models. AZD4547 (1–5 µM) demonstrated potent anti-proliferative effects, inhibition of stemness, and suppression of FGFR/RTK signaling in ErbB2-overexpressing human breast cancer cells. To study the in vivo effects of AZD4547 on mammary development, mammary epithelial cell (MEC) populations, and oncogenic signaling, MMTV-ErbB2 transgenic mice were administered AZD4547 (2–6 mg/kg/day) for 10 weeks during the ‘risk window’ for mammary tumor development. AZD4547 significantly inhibited ductal branching and MEC proliferation in vivo, which corroborated the in vitro anti-proliferative properties. AZD4547 also depleted CD24/CD49f-sorted MEC populations, as well as the CD61highCD49fhigh tumor-initiating cell-enriched population. Importantly, AZD4547 impaired stem cell-like characteristics in primary MECs and spontaneous tumor cells. Moreover, AZD4547 downregulated RTK, mTOR, and Wnt/β-catenin signaling pathways in premalignant mammary tissues. Collectively, our data provide critical preclinical evidence for AZD4547 as a potential breast cancer preventative and therapeutic agent.

Human CD3+ T-Cells with the Anti-ERBB2 Chimeric Antigen Receptor Exhibit Efficient Targeting and Induce Apoptosis in ERBB2 Overexpressing Breast Cancer Cells.

Breast cancer is a common malignancy among women. The innate and adaptive immune responses failed to be activated owing to immune modulation in the tumour microenvironment. Decades of scientific study links the overexpression of human epidermal growth factor receptor 2 (ERBB2) antigen with aggressive tumours. The Chimeric Antigen Receptor (CAR) coding for specific tumour-associated antigens could initiate intrinsic T-cell signalling, inducing T-cell activation, and cytotoxic activity without the need for major histocompatibility complex recognition. This renders CAR as a potentially universal immunotherapeutic option. Herein, we aimed to establish CAR in CD3+ T-cells, isolated from human peripheral blood mononucleated cells that could subsequently target and induce apoptosis in the ERBB2 overexpressing human breast cancer cell line, SKBR3. Constructed CAR was inserted into a lentiviral plasmid containing a green fluorescent protein tag and produced as lentiviral particles that were used to transduce activated T-cells. Transduced CAR-T cells were then primed with SKBR3 cells to evaluate their functionality. Results showed increased apoptosis in SKBR3 cells co-cultured with CAR-T cells compared to the control (non–transduced T-cells). This study demonstrates that CAR introduction helps overcome the innate limitations of native T-cells leading to cancer cell apoptosis. We recommend future studies should focus on in vivo cytotoxicity of CAR-T cells against ERBB2 expressing tumours.

Phenformin inhibits growth and epithelial-mesenchymal transition of ErbB2-overexpressing breast cancer cells through targeting the IGF1R pathway.

Reports suggest that metformin, a popular anti-diabetes drug, prevents breast cancer through various systemic effects, including insulin-like growth factor receptor (IGFR) regulation. Although the anti-cancer properties of metformin have been well-studied, reports on a more bioavailable/potent biguanide, phenformin, remain sparse. Phenformin exerts similar functional activity to metformin and has been reported to impede mammary carcinogenesis in rats. Since the effects of phenformin on specific breast cancer subtypes have not been fully explored, we used ErbB2-overexpressing breast cancer cell and animal models to test the anti-cancer potential of phenformin. We report that phenformin (25–75 μM) decreased cell proliferation and impaired cell cycle progression in SKBR3 and 78617 breast cancer cells. Reduced tumor size after phenformin treatment (30 mg/kg/day) was demonstrated in an MMTV-ErbB2 transgenic mouse syngeneic tumor model. Phenformin also blocked epithelial-mesenchymal transition, decreased the invasive phenotype, and suppressed receptor tyrosine kinase signaling, including insulin receptor substrate 1 and IGF1R, in ErbB2-overexpressing breast cancer cells and mouse mammary tumor-derived tissues. Moreover, phenformin suppressed IGF1-stimulated proliferation, receptor tyrosine kinase signaling, and epithelial-mesenchymal transition markers in vitro. Together, our study implicates phenformin-mediated IGF1/IGF1R regulation as a potential anti-cancer mechanism and supports the development of phenformin and other biguanides as breast cancer therapeutics.

Activation of cancerous inhibitor of PP2A (CIP2A) contributes to lapatinib resistance through induction of CIP2A-Akt feedback loop in ErbB2-positive breast cancer cells.

Lapatinib, a small molecule ErbB2/EGFR inhibitor, is FDA-approved for the treatment of metastatic ErbB2-overexpressing breast cancer; however, lapatinib resistance is an emerging clinical challenge. Understanding the molecular mechanisms of lapatinib-mediated anti-cancer activities and identifying relevant resistance factors are of pivotal significance. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently identified oncoprotein that is overexpressed in breast cancer. Our study investigated the role of CIP2A in the anti-cancer efficacy of lapatinib in ErbB2-overexpressing breast cancer cells. We found that lapatinib concurrently downregulated CIP2A and receptor tyrosine kinase signaling in ErbB2-overexpressing SKBR3 and 78617 cells; however, these effects were attenuated in lapatinib-resistant (LR) cells. CIP2A overexpression rendered SKBR3 and 78617 cells resistant to lapatinib-induced apoptosis and growth inhibition. Conversely, CIP2A knockdown via lentiviral shRNA enhanced cell sensitivity to lapatinib-induced growth inhibition and apoptosis. Results also suggested that lapatinib downregulated CIP2A through regulation of protein stability. We further demonstrated that lapatinib-induced CIP2A downregulation can be recapitulated by LY294002, suggesting that Akt mediates CIP2A upregulation. Importantly, lapatinib induced differential CIP2A downregulation between parental BT474 and BT474/LR cell lines. Moreover, CIP2A shRNA knockdown significantly sensitized the BT474/LR cells to lapatinib. Collectively, our results demonstrate that CIP2A is a molecular target and resistance factor of lapatinib with a critical role in lapatinib-induced cellular responses, including the inhibition of the CIP2A-Akt feedback loop. Further investigation of lapatinib-mediated CIP2A regulation will advance our understanding of lapatinib-associated anti-tumor activities and drug resistance.

Abstract 1908: FGFR inhibitor, AZD4547, impedes the stemness of mammary epithelial cells in the premalignant tissues of MMTV-ErbB2 transgenic mice

Abstract The fibroblast growth factor receptor (FGFR) family (FGFR1-4) of receptor tyrosine kinases (RTKs) regulates signaling pathways involved in cell proliferation and differentiation. In particular, FGFR1 and FGFR2, which are found in the terminal end buds of developing mammary ducts, play a role in mammary development and glandular morphogenesis involving the regulation of mammary stem cells (MaSCs) in mice. As such, a number of FGFR inhibitors are being tested in preclinical studies and clinical trials for anti-tumor properties. Nevertheless, reports on FGFR inhibitor-mediated breast cancer prevention are sparse. In this study, we aimed to investigate the anti-cancer benefits of AZD4547, a small molecule inhibitor of FGFR1-3, on ErbB2-overexpressing breast cancer models. We particularly focus on the effects of AZD4547 on MaSCs and tumor-initiating cells (TICs) in the premalignant tissues of MMTV-ErbB2 transgenic mice. We first demonstrated the anti-proliferative effects of AZD4547 (1-5 µM) on human ErbB2-overexpressing breast cancer cell lines. We further showed that AZD4547 confers potent inhibition of the stemness of these breast cancer cells, as indicated by significant depletion of ALDH+ cells and impaired tumorsphere formation. To study the in vivo effects of AZD4547 on the stemness of mammary epithelial cells (MECs), MMTV-ErbB2 transgenic mice were administered AZD4547 (2-6 mg/kg/day) for 10 weeks (weeks 8-18 of age) during the ‘risk window’ for mammary tumor development. Histopathological analysis indicated that AZD4547 significantly inhibited ductal branching and MEC proliferation. To examine the effect of AZD4547 on MEC subpopulations and tissue hierarchy dynamics in the premalignant mammary tissues of this model, we performed flow cytometry analyses on the primary MECs using CD24/CD49f and CD61/CD49f cell surface markers. The results showed that AZD4547 treatment substantially reduced MaSC-derived luminal and myoepithelial cell populations. AZD4547 also selectively suppressed the CD61highCD49fhigh cell population, which is enriched with luminal progenitor cells that give rise to TICs during MMTV-ErbB2 mammary tumor development. Mammosphere and colony-forming cell (CFC) assays on primary MECs demonstrated that the stemness of these cells was also blocked by AZD4547 prior to malignant transformation. Consistently, AZD4547 inhibited the anchorage-independent growth of cells from spontaneous tumors. Moreover, we demonstrated that AZD4547 downregulated multiple pathways, including the inactivation of FGFR, EGFR, and Wnt/β-catenin signaling. Collectively, the morphogenic, MaSC/TIC, and signaling regulation associated with AZD4547 treatment provides critical evidence for AZD4547 as a breast cancer preventative and therapeutic agent, which ultimately reveals clues for more effective eradication of refractory mammary tumors. Citation Format: Qingxia Zhao, Amanda B. Parris, Erin W. Howard, Ming Zhao, Ying Xing, Zhikun Ma, Xiaohe Yang. FGFR inhibitor, AZD4547, impedes the stemness of mammary epithelial cells in the premalignant tissues of MMTV-ErbB2 transgenic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1908. doi:10.1158/1538-7445.AM2017-1908

Cooperative but distinct early co-signaling events originate from ERBB2 and ERBB1 receptors upon trastuzumab treatment in breast cancer cells.

ERBB2 receptor belongs to the ERBB tyrosine kinase receptor family. At variance to the other family members, ERBB2 is a constitutively active orphan receptor. Upon ligand binding and activation, ERBB receptors form homo- or hetero-dimers with the other family members, including ERBB2, promoting an intracellular signaling cascade. ERBB2 is the preferred dimerization partner and ERBB2 heterodimers signaling is stronger and longer acting compared to heterodimers between other ERBB members. The specific contribution of ERBB2 in heterodimer signaling is still undefined.Here we report the formation of circular dorsal ruffles (CDRs) upon treatment of the ERBB2-overexpressing breast cancer cell lines SK-BR-3 and ZR751 with Trastuzumab, a therapeutic humanized monoclonal antibody directed against ERBB2. We found that in SK-BR-3 cells Trastuzumab leads to surface redistribution of ERBB2 and ERBB1 in CDRs, and that the ERBB2-dependent ERK1/2 phosphorylation and ERBB1 expression are both required for CDR formation. In particular, in these cells CDR formation requires activation of both the protein regulator of actin polymerization N-WASP, mediated by ERK1/2, and of the actin depolymerizing protein cofilin, mediated by ERBB1. Furthermore, we suggest that this latter event may be inhibited by the negative cell motility regulator p140Cap, as we found that p140Cap overexpression led to cofilin deactivation and inhibition of CDR formation.In conclusion, here we show for the first time an ERBB2-specific signaling contribution to an ERBB2/ERBB1 heterodimer, in the activation of a complex biological process such as the formation of CDRs.

Construction and evaluation of pH-sensitive immunoliposomes for enhanced delivery of anticancer drug to ErbB2 over-expressing breast cancer cells

1,5-Dihexadecyl N,N-diglutamyl-lysyl-L-glutamate (GGLG) liposomes were previously developed to enhance drug delivery efficiency in tumor cells owing to its pH-responsive properties. Herein, we report the modification of GGLG liposomes by conjugating a Fab′ fragment of an ErbB2 antibody to the terminus of PEG (polyethylene glycol)-lipid (Fab′-GGLG liposomes). The conjugation of Fab′ fragments did not affect the antibody activity, drug (doxorubicin, DOX) encapsulation efficiency, stability during storage or pH-sensitivity. However, the binding affinity of Fab′-GGLG liposomes was enhanced to ErbB2-overexpressing HCC1954 cells specifically, and the cell association increased 10-fold in comparison to GGLG liposomes. Consequently, intracellular DOX delivery was enhanced, with an increased cytotoxicity in HCC1954 cells (i.e., IC50 of 1.17 and 3.08 μg/mL for Fab′-GGLG-DOX and GGLG-DOX liposomes, respectively). Further, a significantly enhanced tumor growth inhibition was obtained in an ErbB2-overexpressing breast cancer-bearing mouse model. Therefore, a potent anticancer drug delivery system was constructed by the immunological modification of pH-sensitive liposomes.

Abstract 4091: Mesenchymal stem cells drive paclitaxel-resistance in erbB2-overexpressing breast cancer cells via paracrine of NRG-1

Abstract Breast cancer remains the most frequently diagnosed cancer and the leading cause of cancer death in women, both worldwide and in less developed countries. Surgery in conjunction with adjuvant chemotherapy is the main treatment of choice for patients with locally advanced breast cancer, leading to reduce cancer-related symptoms and prolong survival. Paclitaxel as a critical drug in the treatment of breast cancer patients, intrinsic and acquired resistance to paclitaxel represents a significant clinical problem. We had previously demonstrated that increased expression of erbB3 is required for erbB2-mediated paclitaxel resistance in breast cancer cells via PI-3K/Akt/mTOR signaling pathway-dependent upregulation of Survivin. Mesenchymal stem cells (MSCs) are emerging as an important component of tumor microenvironment, which may play an essential role in regulating cancer cell growth, motility, invasion and therapeutic resistance. In the present study, we have explored the possible role of MSCs in regulating the chemo-sensitivity of erbB2-overexpressing breast cancer cells. We show that both human umbilical cord and bone marrow-derived MSCs express significantly higher level of Neuregulin-1 (NRG-1, also Heregulin-β1) as compared with erbB2-overexpressing breast cancer cells themselves. Coculture or treatment with conditioned medium of MSCs not only decreases the anti-proliferation effect of paclitaxel on erbB2-overexpressing breast cancer cells, but also significantly inhibits paclitaxel-induced apoptosis. We further demonstrate that this MSCs-drived paclitaxel-resistance in erbB2-overexpressing breast cancer cells could be attributed to paracrine effect of NRG-1/erbB3 signaling, as specific nutralizaion of NRG-1 or blocking of erbB3 resensitizes erbB2-overexpressing breast cancer cells to paclitaxel treatment. Moreover, overexpression of erbB3 enhances, while knockdown expression of erbB3 abrogates MSCs-drived paclitaxel-resistance. Taken together, our current data indicate that paracrine of NRG-1 by MSCs induce resistance of paclitaxel in erbB2-overexpressing breast cancer cells through activation of erbB3 signaling. Our findings suggest that simultaneously targeting mesenchymal stem cells in tumor microenviroment may be a novel strategy to overcome chemotherapeutic resistance. Citation Format: Ling Zhu, Jin Wang, Weimin Zuo, Rong Lin, Tingting Lin, Yan Lei, Bingshuang Ren, Jun Lu, Huiyue Dong, Lingjing Lin, Lianghu Huang, Qinghua Wang, Yujie Ma, Hui Lyu, Bolin Liu, Jianming Tan, Shuiliang Wang. Mesenchymal stem cells drive paclitaxel-resistance in erbB2-overexpressing breast cancer cells via paracrine of NRG-1. [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 4091.

Marked enhancement of lysosomal targeting and efficacy of ErbB2-targeted drug delivery by HSP90 inhibition.

Targeted delivery of anticancer drugs to tumor cells using monoclonal antibodies against oncogenic cell surface receptors is an emerging therapeutic strategy. These strategies include drugs directly conjugated to monoclonal antibodies through chemical linkers (Antibody-Drug Conjugates, ADCs) or those encapsulated within nanoparticles that in turn are conjugated to targeting antibodies (Antibody-Nanoparticle Conjugates, ANPs). The recent FDA approval of the ADC Trastuzumab-TDM1 (Kadcyla®; Genentech; San Francisco) for the treatment of ErbB2-overexpressing metastatic breast cancer patients has validated the strong potential of these strategies. Even though the activity of ANPs and ADCs is dependent on lysosomal traffic, the roles of the endocytic route traversed by the targeted receptor and of cancer cell-specific alterations in receptor dynamics on the efficiency of drug delivery have not been considered in these new targeted therapies. For example, constitutive association with the molecular chaperone HSP90 is thought to either retard ErbB2 endocytosis or to promote its recycling, traits undesirable for targeted therapy with ANPs and ADCs. HSP90 inhibitors are known to promote ErbB2 ubiquitination, targeting to lysosome and degradation. We therefore hypothesized that ErbB2-targeted drug delivery using Trastuzumab-conjugated nanoparticles could be significantly improved by HSP90 inhibitor-promoted lysosomal traffic of ErbB2. Studies reported here validate this hypothesis and demonstrate, both in vitro and in vivo, that HSP90 inhibition facilitates the intracellular delivery of Trastuzumab-conjugated ANPs carrying a model chemotherapeutic agent, Doxorubicin, specifically into ErbB2-overexpressing breast cancer cells, resulting in improved antitumor activity. These novel findings highlight the need to consider oncogene-specific alterations in receptor traffic in the design of targeted drug delivery strategies. We suggest that combination of agents that enhance receptor endocytosis and lysosomal routing can provide a novel strategy to significantly improve therapy with ANPs and ADCs.

Abstract 3557: Cancerous inhibitor of PP2A is a novel molecular target and resistance factor of Lapatinib

Abstract Deregulation of erbB-2 and EGFR is frequently detected in breast cancer, which has been associated with poor prognosis. Lapatinib, an orally active small molecule acting as a dual inhibitor of erbB-2 and EGFR, has been used for the treatment of patients with advanced or metastatic erbB-2+ breast cancer. Lapatinib resistance, however, is emerging as a clinical challenge. Understanding the molecular mechanisms of Lapatinib-mediated anti-cancer activities and identifying relevant resistance factors are of pivotal significance in breast cancer treatment. Cancerous inhibitor of PP2A (CIP2A) is a recently identified oncoprotein that is overexpressed in breast and other types of cancers. Through interacting with protein phosphatase 2A (PP2A), CIP2A regulates c-Myc and factors involved in cell proliferation and survival. This study aimed to investigate the role of CIP2A in Lapatinib-mediated inhibition of erbB-2 overexpressing breast cancer cells. Our data showed that Lapatinib downregulated CIP2A in erbB-2 overexpressing SK-BR-3 and 78617 cells, which was correlated with concurrent inactivation of erbB-2, EGFR, Akt, Erk1/2 and mTOR. Overexpression of CIP2A rendered the cells resistant to Lapatinib induced apoptosis and growth inhibition. Conversely, CIP2A knockdown via siRNA sensitized the cells to Lapatinib-mediated effects. We also demonstrated that Lapatinib-induced downregulation of CIP2A can be abolished by LY294002, suggesting that CIP2A downregulation was regulated by Akt. Analysis with cycloheximide (CHX) and MG132 indicated that Lapatinib modulates CIP2A protein stability and promotes its degradation through prosteasome pathway. More importantly, we found that Lapatinib induced differential downregulation of CIP2A between the parental and Lapatinib-resistant (LR) BT-474 cells. Decrease in CIP2A downregulation in BT-474-LR cells suggests its association with Lapatinib resistance. Knockdown of CIP2A via lentiviral siRNA system significantly sensitized Lapatinib induced growth inhibition and apoptosis. Taken together, our results demonstrate that CIP2A is a molecular target of Lapatinib, which plays a critical role in Lapatinib-induced cellular responses. Lapatinib-induced downregulation of CIP2A involves the inhibition of the Akt-CIP2A feedback loop. Correlation between Lapatinib resistance and decreased CIP2A downregulation, as well as CIP2A knockdown-mediated sensitization of BT-474-LR cells indicate that CIP2A is a resistance factor of Lapatinib. Further investigation on Lapatinib-mediated regulation of CIP2A will advance our understanding of Lapatinib-associated antitumor activities and drug resistance. Citation Format: Ming Zhao, Amanda Blackwelder, Harry Lee, Xiaohe Yang. Cancerous inhibitor of PP2A is a novel molecular target and resistance factor of Lapatinib. [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 3557. doi:10.1158/1538-7445.AM2015-3557

Abstract 1975: Role and activation mechanisms of myeloid zinc finger-1 (MZF1) in ErbB2-induced breast cancer invasion

Abstract ErbB2 overexpression gives rise to a highly invasive, aggressive phenotype associated with an increased activity of lysosomal cysteine cathepsins B and L and with a pericellular accumulation of lysosomes at the close vicinity of the cell membrane, enabling lysosomal exocytosis and secretion of lysosomal contents to the extracellular space. We recently showed that ErbB2 expression level correlates positively with cysteine cathepsin B and L levels in primary breast cancer and that ErbB2 regulates the expression of cathepsins B and L leading to their increased activity in several ErbB2-positive breast cancer cell lines. We found that the increased cysteine cathepsin activity is essential for the invasion of ErbB2 overexpressing breast cancer cells in 3-dimensional (3D) Matrigel cultures and that it is mediated by a novel signaling network that involves TGFβR, ERK2-MAPK, PAK4, PKCα and CDC42bpβ kinases and two transcription factors, Myeloid Zinc Finger 1 (MZF1) and ETS1, which regulate the ErbB2-induced expression of cathepsins B and L. We currently work on understanding the mechanisms that increase the transcriptional activity of MZF1 in breast cancer. Thus far we have identified two basic mechanisms that can increase MZF1 target gene expression: cancer-induced, increased levels of MZF1 and ErbB2 signaling-induced, increased transcriptional activity of MZF1. Our results thus far show that the level of MZF1 can be directly downregulated by specific, non-coding RNAs. The cancer-induced increase in the MZF1 level is most likely due to changes in the expression of these non-coding RNAs. Preliminary data employing two-dimensional SDS-PAGE as well as the factual involvement of several kinases downstream of ErbB2 for its transcriptional activity, suggests that MZF1, a prominent phosphoprotein, is activated by phosphorylation in response to ErbB2 signaling. Towards this end we have set up various experiments to verify the phosphorylation of MZF1 in response to ErbB2 signaling. We now aim at the identification of the ErbB2-regulated phosphorylation sites. With this work we expect to collect more information about the molecular mechanisms underlying ErbB2-induced invasion. Increased knowledge of the mechanisms is expected to enable the development of novel therapies and biomarkers for aggressive, ErbB2-positive breast cancer. Citation Format: Tuula Kallunki, Ditte M. Brix, Knut Kristoffer B. Clemmensen, Siri A. Tvingsholm, Bo Rafn, José Moreira, Irina Gromova, Marja Jäättelä. Role and activation mechanisms of myeloid zinc finger-1 (MZF1) in ErbB2-induced breast cancer invasion. [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 1975. doi:10.1158/1538-7445.AM2015-1975

Abstract B20: Role of ErbB3 in tumorigenesis and drug resistance in ErbB2-driven breast cancer

Abstract Introduction: Overexpression of erbB3 is frequently observed in erbB2 altered breast cancers. Numerous studies indicate that the expression of erbB3 plays an important role in erbB2-driven breast cancer formation and progression. In this study, we take advantage of the mammary tumors derived from MMTV-neu transgenic mice and the tumor xenografts established from human erbB2-overexpressing (erbB2+) breast cancer cells to further elucidate the role of erbB3 in tumorigenesis and resistance to chemotherapy and erbB2-targeted therapy. Methods: Immunohistochemical analyses were performed to study the expression and phosphorylation of rat c-neu/ErbB2 and mouse erbB3 in mammary tumors. Co-immunoprecipitation (IP) analysis was employed to examine the heterodimerization between erbB2 and erbB3. Cell growth (MTS) assays were used to determine cell viability. Lentiviral vector containing shRNA was used to specifically knockdown erbB3. Flow cytometric assays were performed to define cell cycle distribution. Western blot analyses were performed to assess the expression and activation of proteins. The tumor xenografts were established by inoculation of BT474-HR20 cells (trastuzumab-resistant subline derived from BT474 cells) into nude mice. The tumor-bearing mice were treated with paclitaxel/trastuzumab and/or an anti-erbB3 monoclonal antibody (Ab, MM-121/SAR256212) to determine whether an erbB3-targeted therapy may enhance paclitaxel/trastuzumab's antitumor activity. Results: The expression status of erbB2 and/or erbB3 had no effect on tumor latency, however, the mammary tumors with co-expression of both erbB2 and erbB3 receptors grew significantly faster than those with either erbB2 or erbB3 expression alone. The tumors with co-expression of both receptors showed higher levels of phosphorylated-erbB2 (P-erbB2) and P-erbB3. The interaction between the rat c-neu transgene encoded erbB2 and mouse erbB3 was confirmed via IP assays in the mammary tumors. Elevated expression of erbB3 enhanced cell proliferation, whereas knockdown of erbB3 inhibited proliferation of erbB2+ breast cancer cells. The combinations of trastuzumab with an erbB3 blocking Ab significantly reduced cell viability in several erbB2+ breast cancer cell lines. Furthermore, MM-121 in combination with paclitaxel or trastuzumab (as compared to either alone) dramatically inhibited tumor growth in a mouse xenograft model. Conclusion: Activation of the erbB3 signaling plays a pivotal role in the development of erbB2-driven breast cancer. Strategies against erbB3 abrogates erbB2-mediated paclitaxel / trastuzumab resistance in breast cancer. Our data provide a strong basis to explore the therapeutic potential of erbB3-targeted therapy in combination with paclitaxel/ trastuzumab in breast cancer patients whose tumors overexpress erbB2. Citation Format: Hui Lyu, Jingcao Huang, Susan M. Edgerton, Ann D. Thor, Bolin Liu. Role of ErbB3 in tumorigenesis and drug resistance in ErbB2-driven breast cancer. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B20.

Polypeptide-based nanogels co-encapsulating a synergistic combination of doxorubicin with 17-AAG show potent anti-tumor activity in ErbB2-driven breast cancer models

ErbB2-driven breast cancers constitute 20–25% of the cases diagnosed within the USA. The humanized anti-ErbB2 monoclonal antibody, Trastuzumab (Herceptin™; Genentech), with chemotherapy is the current standard of treatment. Novel agents and strategies continue to be explored, given the challenges posed by Trastuzumab-resistance development in most patients. The HSP90 inhibitor, 17-allylaminodemethoxygeldanamycin (17-AAG), which induces ErbB2 degradation and attenuates downstream oncogenic signaling, is one such agent that showed significant promise in early phase I and II clinical trials. Its low water solubility, potential toxicities and undesirable side effects observed in patients, partly due to the Cremophor-based formulation, have been discouraging factors in the advancement of this promising drug into clinical use. Encapsulation of 17-AAG into polymeric nanoparticle formulations, particularly in synergistic combination with conventional chemotherapeutics, represents an alternative approach to overcome these problems. Herein, we report an efficient co-encapsulation of 17-AAG and doxorubicin, a clinically well-established and effective modality in breast cancer treatment, into biodegradable and biocompatible polypeptide-based nanogels. Dual drug-loaded nanogels displayed potent cytotoxicity in a breast cancer cell panel and exerted selective synergistic anticancer activity against ErbB2-overexpressing breast cancer cell lines. Analysis of ErbB2 degradation confirmed efficient 17-AAG release from nanogels with activity comparable to free 17-AAG. Furthermore, nanogels containing both 17-AAG and doxorubicin exhibited superior antitumor efficacy in vivo in an ErbB2-driven xenograft model compared to the combination of free drugs. These studies demonstrate that polypeptide-based nanogels can serve as novel nanocarriers for encapsulating 17-AAG along with other chemotherapeutics, providing an opportunity to overcome solubility issues and thereby exploit its full potential as an anti-cancer agent.