Choon-Kee Lee

Elevated expression of erbB3 confers paclitaxel resistance in erbB2-overexpressing breast cancer cells via upregulation of Survivin

The coexpression of erbB3 and erbB2 is frequently observed in breast cancer; and erbB3 has a critical role in erbB2 promotion of breast cancer progression and anti-estrogen resistance. In this study, we determine the role of erbB3 in erbB2-mediated paclitaxel resistance in breast cancer cells. The overexpression of exogenous erbB3 via either stable or transient transfection in erbB2-overexpressing, but not epidermal growth factor receptor (EGFR)-expressing, breast cancer cells significantly decreases paclitaxel-induced growth inhibition and apoptosis. Consistently, knockdown of erbB3 expression with a specific short hairpin RNA (shRNA) in breast cancer cells with coexpression of both erbB2 and erbB3 enhances paclitaxel-induced apoptosis evidenced by increased DNA fragmentation, poly (ADP-ribose) polymerase (PARP) cleavage and activation of caspase-3 and -8. Furthermore, while forced overexpression of erbB3 increases, specific knockdown of erbB3 decreases the expression levels of Survivin only in the erbB2-overexpressing breast cancer cells. Targeting Survivin with specific shRNA overcomes paclitaxel resistance without effect on the expression levels of either erbB2 or erbB3. Mechanistic studies indicate that the specific phosphoinositide 3-kinase (PI-3K), Akt and mammalian target of rapamycin (mTOR) inhibitors, but not the mitogen-activated protein kinase kinase (MEK) inhibitor, not only abrogate erbB3-mediated upregulation of Survivin, but also reinforce the erbB2/erbB3-coexpressing breast cancer cells to paclitaxel-induced growth inhibition. These data demonstrate that heterodimerization of erbB2/erbB3 is a prerequisite for erbB2 tyrosine kinase activation; and elevated 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. Our studies suggest that new strategies targeting erbB3 or Survivin may enhance the efficacy of chemotherapeutic agents against erbB2-overexpressing breast cancer.

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.

HDAC Inhibitor SNDX-275 Induces Apoptosis in erbB2-Overexpressing Breast Cancer Cells via Down-regulation of erbB3 Expression

Breast cancer is a highly heterogeneous disease with distinct histologic subtypes. Targeted therapies such as endocrine therapy and growth factor receptor inhibitors have had a significant impact on the treatment of metastatic breast cancer patients. Unfortunately, resistance to these agents eventually occurs, and currently represents a significant clinical problem in the management of breast cancers. Inhibitors of histone deacetylases (HDACi) exhibit anticancer activity in a variety of tumor cell models and have been shown to target mechanisms of resistance to a number of targeted agents. It is unclear, however, if there are specific breast cancer subtypes for which an HDACi may be more or less effective. Here, we report that the class I isoform-selective HDACi entinostat (SNDX-275) preferentially inhibits cell proliferation/survival and inactivates downstream signaling in erbB2-overexpressing compared with basal breast cancer cells. SNDX-275 reduces the levels of both erbB2 and erbB3, as well as significantly decreases P-erbB2, P-erbB3, P-Akt, and P-MAPK in erbB2-overexpressing cells. Additionally, SNDX-275 promotes apoptosis and induces cell cycle arrest predominantly at G(1) phase in erbB2-overexpressing cells, whereas SNDX-275 mainly induces G(2)-M arrest in basal breast cancer cells. The cellular bias of SNDX-275 is shown to be related partly to the levels of erbB3 expression that directly impact the ability of SNDX-275 to inhibit proliferation/survival of the erbB2-overexpressing breast cancer cells. These findings show that SNDX-275 may be developed as a novel therapeutic agent to treat breast cancers with coexpression of both erbB2 and erbB3.

HDAC inhibition synergistically enhances alkylator-induced DNA damage responses and apoptosis in multiple myeloma cells

Histone deacetylase (HDAC) inhibitors induce chromatin destabilization. We sought to determine whether HDAC inhibition may amplify alkylator-induced mitotic cell death in multiple myeloma (MM) cells. The combination of SNDX-275, a class I HDAC inhibitor, with melphalan, showed a powerful synergism on growth inhibition with the combination index ranged from 0.27 to 0.75 in MM1.S and RPMI8226 cells. Their combinations as compared with either agent alone promoted much more caspase-dependent apoptosis. Flow cytometry analysis showed that SNDX-275 had minimal effects on cell cycle progression of MM1.S cells, but clearly increased the percentage of S phase in RPMI8226 cells associated with an upregulation in p21(waf1) and a reduction in cyclin D1 and E2F1. Melphalan alone significantly arrested both MM1.S and RPMI8226 cells at S phase and enhanced expression of p53 and p21(waf1). Furthermore, studies on DNA damage response revealed that phospho-histone H2A.X (gammaH2A.X), a hall marker of DNA double strand break, along with phosphorylated CHK1 (P-CHK1) and CHK2 (P-CHK2) was dramatically induced by SNDX-275 or melphalan. The increase in gammaH2A.X and P-CHK1 was considerably higher on combination than either agent alone. These molecular changes correlated well with the significant increase in mitotic catastrophe. Our data indicate that SNDX-275 synergistically enhances melphalan-induced apoptosis in MM cells via intensification of DNA damage, suggesting that SNDX-275 in combination with melphalan may be a novel therapeutic strategy for MM.

HDAC inhibitor SNDX-275 enhances efficacy of trastuzumab in erbB2-overexpressing breast cancer cells and exhibits potential to overcome trastuzumab resistance.

Trastuzumab (or Herceptin), as the first erbB2-targeted therapy, has been successfully used to treat breast cancer patients with erbB2-overexpressing tumors. However, resistances to trastuzumab frequently occur, and novel strategies/agents are urgently needed to abrogate the resistant phenotype. Our current study explores the potential of SNDX-275, a class I HDAC inhibitor, to overcome trastuzumab resistance and investigates the combinational effects of SNDX-275 and trastuzumab on both sensitive and resistant breast cancer cells. Cell proliferation assays showed that SNDX-275 significantly enhanced trastuzumab-induced growth inhibition in trastuzumab-sensitive, erbB2-overexpressing breast cancer cells. Importantly, SNDX-275 at its therapeutic range re-sensitized trastuzumab-resistant cells to trastuzumab-mediated growth inhibition. SNDX-275 in combination with trastuzumab resulted in a dramatic reduction of erbB3 and its phosphorylation (P-erbB3), and inhibition of Akt signaling. Apoptotic-ELISA and western blot analyses confirmed that the combinations of SNDX-275 and trastuzumab as compared to SNDX-275 alone significantly enhanced DNA fragmentation and induced more PARP cleavage and caspase-3 activation in both trastuzumab-sensitive and -resistant breast cancer cells. Furthermore, co-immunoprecipitation assays revealed that SNDX-275 mainly attenuated the interactions of erbB2 and erbB3 receptors, but had no significant effect on erbB2/IGF-1R or erbB3/IGF-1R associations in the trastuzumab-resistant breast cancer cells. These data indicated that SNDX-275 enhanced trastuzumab efficacy against erbB2-overexpressing breast cancer cells, and exhibited potential to overcome trastuzumab resistance via disrupting erbB2/erbB3 interactions and inactivating PI-3K/Akt signaling. SNDX-275 may be included in erbB2-targeted regimen as a novel strategy to treat breast cancer patients whose tumors overexpress erbB2.

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.

Therapeutic potential of cladribine in combination with STAT3 inhibitor against multiple myeloma.

BackgroundCladribine or 2-chlorodeoxyadenosine (2-CDA) is a well-known purine nucleoside analog with particular activity against lymphoproliferative disorders, such as hairy cell leukemia (HCL). Its benefits in multiple myeloma (MM) remain unclear. Here we report the inhibitory effects of cladribine on MM cell lines (U266, RPMI8226, MM1.S), and its therapeutic potential in combination with a specific inhibitor of the signal transducer and activator of transcription 3 (STAT3).MethodsMTS-based proliferation assays were used to determine cell viability in response to cladribine. Cell cycle progression was examined by flow cytometry analysis. Cells undergoing apoptosis were evaluated with Annexin V staining and a specific ELISA to quantitatively measure cytoplasmic histone-associated DNA fragments. Western blot analyses were performed to determine the protein expression levels and activation.ResultsCladribine inhibited cell proliferation of MM cells in a dose-dependent manner, although the three MM cell lines exhibited a remarkably different responsiveness to cladribine. The IC50 of cladribine for U266, RPMI8226, or MM1.S cells was approximately 2.43, 0.75, or 0.18 μmol/L, respectively. Treatment with cladribine resulted in a significant G1 arrest in U266 and RPMI8226 cells, but only a minor increase in the G1 phase for MM1.S cells. Apoptosis assays with Annexin V-FITC/PI double staining indicated that cladribine induced apoptosis of U266 cells in a dose-dependent manner. Similar results were obtained with an apoptotic-ELISA showing that cladribine dramatically promoted MM1.S and RPMA8226 cells undergoing apoptosis. On the molecular level, cladribine induced PARP cleavage and activation of caspase-8 and caspase-3. Meanwhile, treatment with cladribine led to a remarkable reduction of the phosphorylated STAT3 (P-STAT3), but had little effect on STAT3 protein levels. The combinations of cladribine and a specific STAT3 inhibitor as compared to either agent alone significantly induced apoptosis in all three MM cell lines.ConclusionsCladribine exhibited inhibitory effects on MM cells in vitro. MM1.S is the only cell line showing significant response to the clinically achievable concentrations of cladribine-induced apoptosis and inactivation of STAT3. Our data suggest that MM patients with the features of MM1.S cells may particularly benefit from cladribine monotherapy, whereas cladribine in combination with STAT3 inhibitor exerts a broader therapeutic potential against MM.

Abstract A49: Combination of bendamustine and the HDAC inhibitor entinostat enhances apoptosis and DNA damage response in multiple myeloma cells

Bendamustine (Cephalon, Inc., West Chester, PA), a hybrid molecule of purine analog and alkylator, induces cell death by activation of DNA damage response and apoptosis, inhibition of mitotic checkpoints, and induction of mitotic catastrophe. Entinostat (also known as MS-275 or SNDX-275, Syndax Pharmaceuticals, Inc., Waltham, MA), a selective class I inhibitor of histone deacetylase (HDAC), induces apoptosis in multiple myeloma (MM) cells. We sought to determine whether the combination of bendamustine with entinostat enhances cell death of MM cells, and the potential mechanisms. Cell growth assay showed that bendamustine inhibited proliferation of MM cells in a dose-dependent manner. The IC50 of bendamustine for RPMI8226, U266 and MM1.S cells was approximately 662.6, 295.5, and 159.6 umol/L. Apoptotic-ELISA indicated that either bendamustine or entinostat was able to induce apoptosis in all three myeloma cells, and their combinations exhibited a much more potent activity to promote the MM cells undergoing apoptosis. Further studies with western blot analyses revealed that bendamustine in combination with entinostat dramatically induced PARP cleavage and activation of caspase-3 in all three MM cell lines, and also reduced the expression levels of cyclin D1, phospho-Stat3 (P-Stat3) and Stat3 in U266 cells. Moreover, studies on DNA damage response showed that phosphohistone H2A.X, a hall marker of DNA double strand break, was significantly induced by the combinations of bendamustine and entinostat as compared to either agent alone in all three MM cell lines. Our data suggest that bendamustine and its combination with entinostat possess anti-MM activity in vitro. Regimens consisting of bendamustine and/or entinostat may represent novel therapeutic strategies against MM.

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

Abstract 1710: Expression of erbB3 upregulates Survivin via transcription-independent mechanism in erbB2-overexpressing breast cancer cells to confer Paclitaxel resistance

The erbB2 receptor is often activated via dimerization with another erbB receptor, such as EGFR or erbB3. Studies show that co-expression of erbB3 and erbB2 is frequently observed in breast cancer; and erbB2 requires erbB3 to stimulate breast cancer cell proliferation. Increased resistance to therapeutic agents such as paclitaxel is one of the mechanisms by which erbB2 contributes to breast tumorigenesis. While several molecular mechanisms contributing to paclitaxel resistance have been proposed, a series studies demonstrate that overexpression of erbB2 inhibits paclitaxel-induced apoptosis in breast cancer cells. However, the role of erbB3 in paclitaxel resistance remains unclear. Our current studies focus on determining the role(s) of erbB3 in erbB2-mediated paclitaxel resistance in breast cancer cells. Overexpression of exogenous erbB3 via either stable or transient transfection in erbB2-overexpressing, but not EGFR-expressing, breast cancer cells significantly decreases paclitaxel-induced growth inhibition and apoptosis. Consistently, knockdown of erbB3 expression with a specific shRNA in breast cancer cells with coexpression of both erbB2 and erbB3 enhances paclitaxel-induced apoptosis evidenced by increased DNA fragmentation, PARP cleavage, and activation of caspase-3, and -8. Furthermore, while forced overexpression of erbB3 increases, specific knockdown of erbB3 decreases the protein but not mRNA expression levels of Survivin only in the erbB2-overexpressing breast cancer cells. Targeting Survivin with specific shRNA overcomes paclitaxel resistance without effect on the expression levels of either erbB2 or erbB3. Mechanistic studies indicate that the specific PI-3K, Akt, and mTOR inhibitors, but not the MEK inhibitor, not only abrogate erbB3-mediated upregulation of Survivin, but also reinforce the erbB2/erbB3-coexpressing breast cancer cells to paclitaxel-induced growth inhibition. Our studies demonstrate that heterodimerization of erbB2/erbB3 results in paclitaxel resistance in breast cancer cells via PI-3K/Akt/mTOR signaling pathway. Elevated expression of erbB3 upregulates Survivin through transcription-independent mechanism in erbB2-overexpressing cancer cells, suggesting that novel strategies targeting erbB3 or Survivin protein translation may enhance the efficacy of chemotherapeutic agents against breast cancer with erbB2-overexpressing tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1710. doi:10.1158/1538-7445.AM2011-1710