Shruti Desai

Receptor tyrosine kinase ErbB2 translocates into mitochondria and regulates cellular metabolism

It is well known that ErbB2, a receptor tyrosine kinase, localizes on the plasma membrane. Here we describe a novel observation that ErbB2 also localizes in mitochondria of cancer cells and patient samples. We found that ErbB2 translocates into mitochondria through the association with mtHSP70. Additionally, mitochondrial ErbB2 (mtErbB2) negatively regulates mitochondrial respiratory functions. Oxygen consumption and activities of complexes of the mitochondrial electron transport chain were decreased in mtErbB2-overexpressing cells. Mitochondrial membrane potential and the cellular ATP level also were decreased. In contrast, mtErbB2 enhanced cellular glycolysis. The translocation of ErbB2 and its impact on mitochondrial function are kinase dependent. Interestingly, cancer cells with higher levels of mtErbB2 were more resistant to ErbB2 targeting antibody trastuzumab. Our study provides a novel perspective on the metabolic regulatory function of ErbB2 and reveals that mtErbB2 plays an important role in the regulation of cellular metabolism and cancer cell resistance to therapeutics.

Heat Shock Factor 1 (HSF1) Controls Chemoresistance and Autophagy through Transcriptional Regulation of Autophagy-related Protein 7 (ATG7)

Background: HSF1 influences chemoresistance in cancer. Results: Chemotherapy activates HSF1, leading to direct transcriptional regulation of autophagy related gene, ATG7. In vitro findings are supported by patient sample study. Conclusion: HSF1 regulates cytoprotective, heat shock-independent autophagy by directly regulating ATG7, which plays an important role in chemoresistance. Significance: Identification of novel HSF1/ATG7 axis in chemoresistance strongly supports development of robust combination therapies, targeting it in cancer. Heat shock factor 1 (HSF1), a master regulator of heat shock responses, plays an important role in tumorigenesis. In this study we demonstrated that HSF1 is required for chemotherapeutic agent-induced cytoprotective autophagy through transcriptional up-regulation of autophagy-related gene ATG7. Interestingly, this is independent of the HSF1 heat shock response function. Treatment of cancer cells with the FDA-approved chemotherapeutic agent carboplatin induced autophagy and growth inhibition, which were significantly increased upon knockdown of HSF1. Mechanistic studies revealed that HSF1 regulates autophagy by directly binding to ATG7 promoter and transcriptionally up-regulating its expression. Significantly, breast cancer patient sample study revealed that a higher ATG7 expression level is associated with poor patient survival. This novel finding was further confirmed by analysis of two independent patient databases, demonstrating a prognostic value of ATG7. Furthermore, a strong positive correlation was observed between levels of HSF1 and ATG7 in triple-negative breast cancer patient samples, thus validating our in vitro findings. This is the first study identifying a critical role for HSF1 in controlling cytoprotective autophagy through regulation of ATG7, which is distinct from the HSF1 function in the heat shock response. This is also the first study demonstrating a prognostic value of ATG7 in breast cancer patients. These findings strongly argue that combining chemotherapeutic agents with autophagy inhibition by repressing HSF1/ATG7 axis represents a promising strategy for future cancer treatment.

miR-125b Functions as a Key Mediator for Snail-induced Stem Cell Propagation and Chemoresistance

Background: Snail plays an important role in chemoresistance, but the mechanism is still unclear. Results: Up-regulation of microRNA-125b through Wnt signaling by snail enriches cancer stem cells and increases chemoresistance. Conclusion: MicroRNA-125b is a key mediator for Snail-induced stem cell propagation and chemoresistance. Significance: We reveal a novel mechanism for Snail-induced stem cell maintenance and chemoresistance. Chemoresistance is a major obstacle in cancer treatment. Our previous studies have shown that miR-125b plays an important role in chemoresistance. Here we report a novel mechanism that up-regulation of miR-125b through Wnt signaling by Snail enriches cancer stem cells. Overexpression of Snail dramatically increases the expression of miR-125b through the Snail-activated Wnt/β-catenin/TCF4 axis. Snail confers chemoresistance by repressing Bak1 through up-regulation of miR-125b. Restoring the expression of Bak1 or depleting miR-125b re-sensitizes Snail-expressing cancer cells to Taxol, indicating that miR-125b is critical in Snail-induced chemoresistance. Moreover, overexpression of miR-125b significantly increases the cancer stem cell population (CD24-CD44+), while depletion of miR-125b or rescue of the expression of Bak1 increases the non-stem cell population (CD24+CD44+) in Snail-overexpressing cells. These findings strongly support that miR-125b functions as a key mediator in Snail-induced cancer stem cell enrichment and chemoresistance. This novel mechanism for Snail-induced stem cell propagation and chemoresistance may have important implications in the development of strategies for overcoming cancer cell resistance to chemotherapy.

PRDM1 Is Required for Mantle Cell Lymphoma Response to Bortezomib

Mantle cell lymphoma (MCL) is an aggressive form of B-cell lymphoma with a poor disease-free survival rate. The proteasome inhibitor bortezomib is approved for the treatment of relapsed and refractory MCL and has efficacy in about 30% of patients. However, the precise mechanism of action of bortezomib is not well understood. This report establishes a requirement for the transcription repressor PR domain zinc finger protein 1 (PRDM1, Blimp1) in the response to bortezomib. Bortezomib rapidly induces transcription of PRDM1 as part of the apoptotic response in both cell lines and primary MCL tumor cells. Knockdown of PRDM1 blocks activation of NOXA and inhibits apoptosis, whereas ectopic expression of PRDM1 alone leads to apoptosis in MCL. Two novel direct targets of PRDM1 were identified in MCL cells: MKI67 (Ki67) and proliferating cell nuclear antigen (PCNA). Both MKI67 and PCNA are required for proliferation and survival. Chromatin immunoprecipitation and knockdown studies reveal that specific repression of MKI67 and PCNA is mediated by PRDM1 in response to bortezomib. Furthermore, promoter studies and mutation/deletion analysis show that PRDM1 functions through specific sites in the PCNA proximal promoter and an MKI67 distal upstream repression domain. Together, these findings establish PRDM1 as a key mediator of bortezomib activity in MCL. Mol Cancer Res; 8(6); 907–18. ©2010 AACR.

PU.1 regulates positive regulatory domain I-binding factor 1/Blimp-1 transcription in lymphoma cells.

The human positive regulatory domain I-binding factor 1 (PRDI-BF1) and its murine homolog Blimp-1 promote differentiation of mature B cells into Ab-secreting plasma cells. In contrast, ectopic expression of PRDI-BF1 in lymphoma cells can lead to inhibition of proliferation or apoptosis. However, little is currently known about the regulation of PRDM1, the gene encoding PRDI-BF1. This report establishes that in lymphoma cells stimulation through the BCR rapidly induces endogenous PRDM1 at the level of transcription with minor changes in mRNA stability. The induced PRDM1-encoded protein localizes to its target genes in vivo and suppresses their expression. In vivo genomic footprinting of the PRDM1 promoter in unstimulated lymphoma and myeloma cells reveals multiple common in vivo occupied elements throughout the promoter. Further functional and structural analysis of the promoter reveals that the promoter is preloaded and poised for activation in the B cell lines. The transcription factor PU.1 is shown to be required for the BCR-induced expression of PRDM1 in lymphoma cells and in PU.1-positive myeloma cells. Activation of PRDM1 is associated with loss of the corepressor transducin-like enhancer of split 4 from the PU.1 complex. These findings indicate that PRDM1 is poised for activation in lymphoma cells and therefore may be a potential therapeutic target to inhibit lymphoma cell proliferation and survival.

Abstract B55: Role of HSF1 in chemotherapy-induced autophagy and endoplasmic reticulum stress response in breast cancer

Heat Shock Factor 1 (HSF1), a master regulator of heat shock response, plays an important role in tumorigenesis. In this study we demonstrate that HSF1 is required for chemotherapeutic agents induced cytoprotective autophagy and Endoplasmic Reticulum (ER) stress response. HSF1 can transcriptionally regulate autophagy related gene ATG7 and ER membrane protein IRE1α. Interestingly, most of this regulation is independent of HSF19s heat shock function. Treatment of cancer cells with FDA approved chemotherapeutic agent induced autophagy which was significantly decreased upon HSF1 knockdown. Mechanistic studies revealed that HSF1 regulates autophagy by directly binding to the ATG7 promoter and transcriptionally upregulating its expression. HSF1 also may play a role in regulating autophagy by modulating levels of phosphorylated AMPK, a key protein that can trigger autophagy. Thus HSF1 regulates the process of autophagy in a multi-pronged manner, through regulation of key autophagy protein as well as regulation of key inducer of autophagy. Furthermore, a strong positive correlation was observed between levels of HSF1 and ATG7 in breast cancer patient samples, thus validating our in vitro findings. Similarly, knockdown of HSF1 reduced the induction of ER stress response by chemotherapeutic agents. Mechanistic studies have revealed that HSF1 regulated cytoprotective ER stress response by directly regulating IRE1α expression and sustained activation. IRE1α, a resident ER protein, is a key sensor of ER stress. It activates Xbp1 leading to expression of protein chaperone genes that help alleviate the ER stress response. This HSF1/IRE1α pathway was further validated in cancer patients by immunohistochemistry staining of breast cancer patient tissue array. This study identifies a critical role for HSF1 in controlling cytoprotective stress response pathways of autophagy and ER stress response in tumor cells. HSF1 does this by not only regulating transcription of key proteins required in these pathways but also by regulating activation of key inducers of these pathways. The findings from this study can be further used to design more robust treatment strategies for breast cancer patients thus improving their prognosis. Citation Format: Shruti Desai, Ming Tan. Role of HSF1 in chemotherapy-induced autophagy and endoplasmic reticulum stress response in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B55.

Abstract LB-242: MicroRNA-125b, a key mediator for Snail-induced stem cell propagation and chemoresistance.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC MicroRNAs (miRNAs) are short ribonucleic acid molecules commonly deregulated in various tumors. Our previous studies have shown that miR-125b plays a major role in the chemoresistance of breast cancer. Snail, a transcription factor important in epithelial-mesenchymal transition, has been reported to be responsible for tumor metastasis and recurrence, however, the mechanism by which Snail exerts these effects is not understood. We report a novel mechanism in which upregulation of miR-125b through Wnt signaling by Snail enriches cancer stem cells and confers chemoresistance. We found that Snail is upregulated in chemoresistant cancer cells. Overexpressing Snail increases miR-125b expression by activating the Wnt/beta-catenin/TCF4 axis, while silencing Snail decreases miR-125b expression. Snail activates Wnt signaling by interacting with beta-catenin/TCF4 to activate the expression of target genes. We discovered 5 TCF4 binding sites in the miR-125b promoter. The full length miR-125b promoter showed higher activity with Snail overexpression, and deletion of TCF4 binding sites decreased the promoter activity of miR-125b in a dose-dependent manner. Knockdown of TCF4 decreased miR-125b expression, which confirms that TCF4 is required for Snail to fully activate the miR-125b promoter. Bak1 is a pro-apoptosis molecule and confirmed target of miR-125b. Deregulation of Bak1 is important in cancer development and drug resistance. Since overexpression of Snail confers chemotherapeutic resistance, and Snail was upregulated in Paclitaxel-resistant cells, we examined whether Snail induces drug resistance through Bak1. We found low Bak1 expression in Paclitaxel-resistant cells, which have high expression of Snail. To verify whether Snail regulates Bak1 expression, we expressed Snail in Snail-low expressing cells and saw repression of Bak1 expression. To confirm, we knocked-down Snail in Snail-high expressing cells, which resulted in increased Bak1 expression. Overexpression of Snail or miR-125b increased the resistance of cancer cells to chemotherapy. Inhibiting miR-125b expression or restoring Bak1 expression re-sensitized cells to chemotherapy. These results indicate that Snail increases chemoresistance by repressing Bak1 through miR-125b. Overexpression of miR-125b significantly increases the cancer stem cell population, while depletion of miR-125b or rescue of Bak1 expression increases the non-stem cell population. These findings support that miR-125b functions as a key mediator in Snail-induced cancer stem cells and chemoresistance. This novel mechanism for Snail-induced stem cell propagation and chemoresistance may have important implications in the development of strategies for overcoming chemotherapy resistance. Work supported by: Vincent F. Kilborn Jr. Cancer Research Foundation, NIH Grant R01CA149646, and the Norwegian Hospitalet Legater Project 334003. Citation Format: Zixing Liu, Hao Liu, Shruti Desai, David C. Schmitt, Ming Zhou, Hung T. Khong, Kristine S. Klos, Steven McClellan, Oystein Fodstad, Ming Tan. MicroRNA-125b, a key mediator for Snail-induced stem cell propagation and chemoresistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-242. doi:10.1158/1538-7445.AM2013-LB-242

Abstract 3025: Receptor tyrosine kinase ErbB2/HER2 translocates into mitochondria and regulates cellular metabolism.

It is well accepted that ErbB2, an important oncoprotein, localizes on the plasma membrane as a receptor tyrosine kinase. This study describes a novel observation that ErbB2 localizes in mitochondria of multiple cancer cell lines and patient samples. We identified an endogenous mitochondrial targeting sequence in ErbB2 and found that ErbB2 translocates into mitochondria through the association with mtHSP70, a key player in the canonical mitochondrial protein importation pathway. Additionally, we observed that mitochondrial ErbB2 (mtErbB2) negatively regulates mitochondrial respiratory functions. Oxygen consumption and activities of complex I, II and IV of the mitochondrial electron transport chain were decreased in mtErbB2-overexpressing cells. Mitochondrial membrane potential and the cellular ATP level also were decreased by mtErbB2. In contrast, mtErbB2 enhanced cellular glycolysis. The translocation of ErbB2 and its impact on mitochondrial function are kinase dependent. Mitochondrial ErbB2 regulates the phosphorylation and activity of COX II and consequently the cytochrome c release and apoptosis. Additional studies showed that cancer cells with higher levels of mtErbB2 were more resistant to Trastuzumab and deletion of the mitochondrial targeting sequence of ErbB2 sensitized the cells to Trastuzumab. Our study provides a novel perspective on the metabolic regulatory functions of ErbB2 and reveals that mtErbB2 plays an important role in the regulation of cellular metabolism and cancer cell resistance to therapeutics. Since ErbB2 plays important roles in multiple organs and multiple types of cancers, the study may have broad impact on the fields of cancer biology. The work was supported by The Vincent F. Kilborn Jr. Cancer Research Foundation and National Institutes of Health Grant RO1CA149646. Citation Format: Yan Ding, Zixing Liu, Shruti Desai, Lewis Pannell, Hong Yi, Elizabath Wright, Laurie Owen, Windy Dean-Colomb, Oystein Fodstad, Jianrong Lu, Ming Tan. Receptor tyrosine kinase ErbB2/HER2 translocates into mitochondria and regulates cellular metabolism. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3025. doi:10.1158/1538-7445.AM2013-3025

Abstract LB-488: Induction of autophagy by heat shock factor 1 promotes breast cancer resistance to chemotherapy

Breast cancer is the most common type of cancer as well as one of the leading causes of cancer-related deaths in women. The increasing emergence of drug resistance to commonly used chemotherapeutic agents has warranted the development of novel and robust treatment options for cancer patients. Carboplatin, a DNA damaging agent, is one of the therapeutic agents which has shown promising results in breast cancer clinical trials. Carboplatin is a second generation platinum drug, increasingly used in clinic to treat cancer patients because of its reduced toxicity and side effects when compared to Cisplatin. Heat Shock Factor 1 (HSF1) is the master regulator of heat shock responses which has also been shown to regulate certain non-heat shock reponse genes. As a transcription factor HSF1 can function as both an activator and repressor of its target genes, resulting in regulation of critical cellular processes invovled in managing cellular stress. The focus of this study is to identify the role of HSF1 in Carboplatin mediated-sensitivity in breast cancer cells, to provide novel insights to improve treatment strategies for cancer patients. Carboplatin treatment in multiple breast cancer cell lines induced growth inhibition which was significantly enhanced upon HSF1 knockdown. Interestingly, the treatment also induced autophagy in these cells. Autophagy is a type II programmed cell death, which is induced in response to metabolic stress and has been demonstrated to mediate drug resistance, since it can play a protective role and allow cells to survive under stressful conditions. Knockdown of HSF1 along with inhibition of autophagy by 3-Methyladenine (3-MA) led to increase in apoptosis in these cells. Furthermore, HSF1 was required for the induction of autophagy in Carboplatin treated cells. Knockdown of HSF1 by siRNA (transient) and shRNA (stable), inhibited induction of autophagy in the presence of Carboplatin, as measured by the punctate EGFP-LC3 structures as well as acridine orange staining. Chromatin immunoprecipitation (ChIP) assay along with Real time- PCR analysis revealed that HSF1 can regulate autophagy by directly regulating the expression of autophagy related genes in the presence of Carboplatin. These findings indicate that HSF1 activates autophagy in response to the Carboplatin induced cellular stress thus leading to cell survival and reducing sensitivity to Carboplatin. Our data strongly support a critical role for HSF1 in promoting chemoresistance through induction of autophgy. This finding has important implications in the development of targeted therapeutics for overcoming Carboplatin resistance in breast cancer. 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-488. doi:1538-7445.AM2012-LB-488