Jingcao Huang

Functional cooperation of miR-125a, miR-125b, and miR-205 in entinostat-induced downregulation of erbB2/erbB3 and apoptosis in breast cancer cells

We reported that the class I HDAC inhibitor entinostat induced apoptosis in erbB2-overexpressing breast cancer cells via downregulation of erbB2 and erbB3. Here, we study the molecular mechanism by which entinostat dual-targets erbB2/erbB3. Treatment with entinostat had no effect on erbB2/erbB3 mRNA, suggesting a transcription-independent mechanism. Entinostat decreased endogenous but not exogenous erbB2/erbB3, indicating it did not alter their protein stability. We hypothesized that entinostat might inhibit erbB2/erbB3 protein translation via specific miRNAs. Indeed, entinostat significantly upregulated miR-125a, miR-125b, and miR-205, that have been reported to target erbB2 and/or erbB3. Specific inhibitors were then used to determine whether these miRNAs had a causal role in entinostat-induced downregulation of erbB2/erbB3 and apoptosis. Transfection with a single inhibitor dramatically abrogated entinostat induction of miR-125a, miR-125b, or miR-205; however, none of the inhibitors blocked entinostat action on erbB2/erbB3. In contrast, co-transfection with two inhibitors not only reduced their corresponding miRNAs, but also significantly abrogated entinostat-mediated reduction of erbB2/erbB3. Moreover, simultaneous inhibition of two, but not one miRNA significantly attenuated entinostat-induced apoptosis. Interestingly, although the other HDAC inhibitors, such as SAHA and panobinostat, exhibited activity as potent as entinostat to induce growth inhibition and apoptosis in erbB2-overexpressing breast cancer cells, they had no significant effects on the three miRNAs. Instead, both SAHA- and panobinostat-decreased erbB2/erbB3 expression correlated with the reduction of their mRNA levels. Collectively, we demonstrate that entinostat specifically induces expression of miR-125a, miR-125b, and miR-205, which act in concert to downregulate erbB2/erbB3 in breast cancer cells. Our data suggest that epigenetic regulation via miRNA-dependent or -independent mechanisms may represent a novel approach to treat breast cancer patients with erbB2-overexpressing tumors.

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

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

Combination of bendamustine and entinostat synergistically inhibits proliferation of multiple myeloma cells via induction of apoptosis and DNA damage response

Bendamustine, a hybrid molecule of purine analog and alkylator, induces cell death by activation of apoptosis, DNA damage response, and mitotic catastrophe. Entinostat, a selective class I inhibitor of histone deacetylase (HDAC), exerts anti-tumor activity in various cancer types, including multiple myeloma (MM). We sought to determine the combinatorial effects of bendamustine and entinostat on MM cells. Cell growth assays showed that bendamustine or entinostat inhibited proliferation in a dose-dependent manner, and their combinations synergistically induced growth inhibition in all MM cells tested. An apoptotic-ELISA and western blot assays on PARP cleavage and caspase-8 and caspase-3 revealed that bendamustine in combination with entinostat exhibited a much more potent activity than either agent alone to promote the MM cells undergoing apoptosis in a dose-dependent manner. Flow cytometric analysis found that entinostat exhibited distinct effects on cell cycle progression in different lines and bendamustine mainly arrested the cells at S phase, whereas their combinations dramatically blocked the S cells entering G2/M phase. Furthermore, studies on DNA damage response indicated that phospho-histone H2A.X (P-H2A.X), a hall marker of DNA double strand break, along with phosphorylated CHK2 (P-CHK2) was significantly enhanced by the combinations of bendamustine and entinostat as compared to either agent alone. These molecular changes were correlated with the increases in mitotic catastrophe. Collectively, our data demonstrate that bendamustine in combination with entinostat exhibit potent anti-proliferative/anti-survival activity in MM cells via induction of apoptosis and DNA damage response. Regimens consisting of bendamustine and/or entinostat may represent novel therapeutic strategies against MM.

Role of erbB3 receptors in cancer therapeutic resistance

ErbB3 receptors are unique members of the erbB receptor tyrosine kinases (RTKs), which are often aberrantly expressed and/or activated in human cancers. Unlike other members in the family, erbB3 lacks or has impaired kinase activity. To transduce cell signaling, erbB3 has to interact with other RTKs and to be phosphorylated by its interactive partners, of those, erbB2 is the most important one. ErbB3 is frequently co-expressed with other RTKs in cancer cells to activate oncogenic signaling, such as phosphoinositide-3-kinase/protein kinase B (Akt) pathway, mitogen-activated protein kinase kinase (MEK)/mitogen-activated protein kinase (MAPK) pathway, Janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathway, etc. and thereby promote tumorigenesis. Numerous studies have demonstrated that activation of erbB3 signaling plays an important role in the progression of a variety of tumor types, such as erbB2-overexpressing breast cancer, castration-resistant prostate cancer, platinum refractory/resistant ovarian cancer, epidermal growth factor receptor TKI-resistant non-small-cell lung cancer, and others. Basic research on the underlying mechanisms implicated the functions of erbB3 as a major cause of treatment failure in cancer therapy. Thus, concomitant inhibition of erbB3 is thought to be required to overcome the resistance and to effectively treat human cancers. This review focuses on the latest advances in our understanding of erbB3-initiated signaling in the development of resistance to cancer treatments.

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

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/trastuzumab9s 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.

Abstract A070: The erbB3 blocking antibody MM-121 resensitizes trastuzumab-resistant breast cancer cells to trastuzumab-mediated antitumor activity in vitro and in vivo

Background: Amplification and/or overexpression of erbB2 occur in approximately 25% of invasive breast cancer and are significantly associated with a worse prognosis for breast cancer patients. The erbB3 receptor plays a critical role in erbB2-overexpressing breast cancer, and its elevated expression induces resistance to therapeutic agents, including trastuzumab. Our recent studies indicate that erbB3 interacts with both erbB2 and IGF-1 receptor to form a heterotrimeric complex in trastuzumab-resistant breast cancer cells. Herein, we investigate the antitumor activity of MM-121, a humanized anti-erbB3 antibody, against erbB2-overexpressing breast cancer cells resistant to trastuzumab in vitro and in vivo. Material and Methods: Cell Titer AQueous Non-Radioactive Cell Proliferation Assay (MTS) to determine cell proliferation. Flow Cytometry analyses to examine cell cycle distribution. Western blot analysis to evaluate protein expression and activation upon treatment. Tumor xenograft model to explore the combinatorial effects of MM-121 and trastuzumab on tumor growth of trastuzumab-resistant breast cancer cells in vivo. Immunohistochemistry assays to assess the treatment effects on expression of erbB3, erbB2, Ki-67, and cleaved capase-3 in vivo. Results: Cell proliferation assays showed that MM-121 significantly enhanced trastuzumab-induced growth inhibition in both sensitive and resistant breast cancer cells. MM-121 in combination with trastuzumab mainly resulted in a dramatic reduction of phosphorylated erbB3 (P-erbB3) and the downstream signaling P-Akt. MM-121 combined with trastuzumab did not lead to apoptosis in trastuzumab-resistant cells in in vitro cell culture condition, rather induced cell cycle G1 arrest which was associated with a slight decrease of E2F-1 and upregulation of p27kip1. Interestingly, in the mouse tumor xenografts model established from trastuzumab-resistant cells, MM-121 in combination with trastuzumab as compared to either agent alone significantly inhibited tumor growth correlated with a significant reduction of Ki-67 staining and increase of cleaved caspase-3 in the tumor tissues. These data suggest that the combination of MM-121 and trastuzumab not only inhibits tumor cell proliferation, but also promotes the resistant cells undergoing apoptosis in vivo. Conclusions: We demonstrate that MM-121 exhibits potent antitumor activity when combined with trastuzumab in vitro and in vivo. MM-121 may be added into the treatment regimens for breast cancer patients whose tumors overexpress erbB2 and become resistant to trastuzumab. Citation Format: Jingcao Huang, Hui Lyu, Youngseok Lee, Bolin Liu. The erbB3 blocking antibody MM-121 resensitizes trastuzumab-resistant breast cancer cells to trastuzumab-mediated antitumor activity in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A070.

Abstract LB-473: The HDAC inhibitor entinostat targets erbB2/erbB3 receptors via functional cooperation of miR-125a, miR-125b, and miR-205 in breast cancer cells

Breast cancer is highly heterogeneous and has been classified into five distinct subtypes, among which erbB2-overexpressing (erbB2+) and basal breast cancers have the least favorable prognosis and express erbB receptors that are excellent targets for anti-cancer therapies. We previously reported that the class I HDAC inhibitor, entinostat (also known as MS-275, SNDX-275, Syndax Pharmaceuticals, Inc., Waltham, MA) selectively inhibited proliferation and induced apoptosis in erbB2+ breast cancer cells. Entinostat preferably targeted erbB2 and erbB3 receptors rather than the epidermal growth factor receptor (EGFR). Here, we study the molecular mechanism through which entinostat downregulates erbB2/erbB3 and promotes the breast cancer cells undergoing apoptosis. We showed that entinostat dramatically reduced the protein levels of erbB2/erbB3, whereas it had no significant effect on their mRNA expression in MDA-MB-453 and BT474 breast cancer cells. In addition, entinostat specifically decreased the endogenous, but not the exogenous erbB2/erbB3, suggesting that it did not promote erbB2/erbB3 protein degradation. Therefore, we hypothesizes that entinostat reduces erbB2/erbB3 receptors through transcription-independent mechanism; it may inhibit erbB2/erbB3 protein translation via induction of specific miRNA. Quantitative real-time (qRT) PCR revealed that entinostat significantly upregulated the expression of three erbB2/erbB3-targeting miRNAs, miR-125a, miR-125b, and miR-205 in erbB2+ breast cancer cells. Specific miRNA inhibitors were used to determine whether these miRNAs played a causal role in entinostat-induced downregulation of erbB2/erbB3 and apoptosis. While inhibition of any one of the three miRNAs failed to block entinostat action, simultaneous inhibition of any two of them significantly abrogated entinostat-induced erbB2/erbB3 reduction and apoptosis. Collectively, these data demonstrate that entinostat targets erbB2/erbB3 receptors via functional cooperation of miR-125a, miR-125b, and miR-205. Our findings suggest that miRNA-mediated epigenetic regulation may represent a novel strategy targeting erbB2/erbB3 to treat the aggressive erbB2+ breast cancer. The combinations of entinostat and erbB2-targeted therapy may be more efficacious for breast cancer patients whose tumors overexpress erbB2. This hypothesis will be directly tested by a recently initiated clinical study determining the activity of entinostat in combination with lapatinib in breast cancer patients that are erbB2+ and progressed on trastuzumab. 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 LB-473. doi:1538-7445.AM2012-LB-473