Sanjay Bansal

Cross talk between mitochondria and superoxide generating NADPH oxidase in breast and ovarian tumors.

Reactive oxygen species (ROS) signal cascades involved in cell growth, cell death, mitogenesis, angiogenesis and carcinogenesis. ROS are produced as a byproduct of oxidative phosphorylation (OXPHOS) in the mitochondria. It is estimated that 2–4% of the oxygen consumed during OXPHOS is converted to ROS. Besides mitochondria, NADPH-oxidase 1 (Nox1) also generates a significant amount of ROS in the cell. In this paper, we tested the hypothesis that mitochondria control Nox 1 redox signaling and the loss of control of this signaling contributes to tumorigenesis. We analyzed Nox1 expression in a mitochondrial gene knockout (?0) cell line and in the isogenic cybrid cell line in which mitochondrial genes were restored by transfer of wild type mitochondria into ?0 cells. Our study revealed, for the first time, that the inactivation of mitochondrial genes leads to down-regulation of Nox1 and that the transfer of wild type mitochondrial genes restores the Nox1 expression to a level comparable to that in the parental cell line. Consistent with Nox1 down-regulation, we found that ?0 cells contained low levels of superoxide anion and that superoxide levels reversed to parental levels in cybrid cells when Nox1 expression was restored by transfer of wild type mitochondria. Increasing mitochondrial superoxide levels also increased the expression of Nox1 in parental cells. Confocal microscopy studies revealed that Nox1 localizes in the mitochondria. Nox1 was highly expressed in breast (86%) and ovarian (71%) tumors and that its expression positively correlated with expression of cytochrome C oxidase encoded by mtDNA. Our study, described in this paper demonstrates the existence of cross talk between the mitochondria and NADPH oxidase. Furthermore, our studies suggest that mitochondria control Nox1 redox signaling and the loss of control of this signaling contributes to breast and ovarian tumorigenesis.

Estrogen Receptor-α Binds p53 Tumor Suppressor Protein Directly and Represses Its Function

Estrogen receptor-α (ERα) promotes proliferation of breast cancer cells, whereas tumor suppressor protein p53 impedes proliferation of cells with genomic damage. Whether there is a direct link between these two antagonistic pathways has remained unclear. Here we report that ERα binds directly to p53 and represses its function. The activation function-2 (AF-2) domain of ERα and the C-terminal regulatory domain of p53 are necessary for the interaction. Knocking down p53 and ERα by small interfering RNA elicits opposite effects on p53-target gene expression and cell cycle progression. Remarkably, ionizing radiation that causes genomic damage disrupts the interaction between ERα and p53. Ionizing radiation together with ERα knock down results in additive effect on transcription of endogenous p53-target gene p21 (CDKN1) in human breast cancer cells. Our findings reveal a novel mechanism for regulating p53 and suggest that suppressing p53 function is an important component in the proproliferative role of ERα.

Estrogen Receptor α Inhibits p53-Mediated Transcriptional Repression: Implications for the Regulation of Apoptosis

Estrogen receptor alpha (ERalpha) and tumor suppressor protein p53 exert opposing effects on cellular proliferation. As a transcriptional regulator, p53 is capable of activating or repressing various target genes. We have previously reported that ERalpha binds directly to p53, leading to down-regulation of transcriptional activation by p53. In addition to transcriptional activation, transcriptional repression of a subset of target genes by p53 plays important roles in diverse biological processes, such as apoptosis. Here, we report that ERalpha inhibits p53-mediated transcriptional repression. Chromatin immunoprecipitation assays reveal that ERalpha interacts in vivo with p53 bound to promoters of Survivin and multidrug resistance gene 1, both targets for transcriptional repression by p53. ERalpha binding to p53 leads to inhibition of p53-mediated transcriptional regulation of these genes in human cancer cells. Transcriptional derepression of Survivin by ERalpha is dependent on the p53-binding site on the Survivin promoter, consistent with our observation that p53 is necessary for ERalpha to access the promoters. Importantly, mutagenic conversion of this site to an activation element enabled ERalpha to repress p53-mediated transcriptional activation. Further, RNA interference-mediated knockdown of ERalpha resulted in reduced Survivin expression and enhanced the propensity of MCF-7 cells to undergo apoptosis in response to staurosporine treatment, an effect that was blocked by exogenous expression of Survivin. These results unravel a novel mechanism by which ERalpha opposes p53-mediated apoptosis in breast cancer cells. The findings could have translational implications in developing new therapeutic and prevention strategies against breast cancer.

Genomic Loss of Tumor Suppressor miRNA-204 Promotes Cancer Cell Migration and Invasion by Activating AKT/mTOR/Rac1 Signaling and Actin Reorganization

Increasing evidence suggests that chromosomal regions containing microRNAs are functionally important in cancers. Here, we show that genomic loci encoding miR-204 are frequently lost in multiple cancers, including ovarian cancers, pediatric renal tumors, and breast cancers. MiR-204 shows drastically reduced expression in several cancers and acts as a potent tumor suppressor, inhibiting tumor metastasis in vivo when systemically delivered. We demonstrated that miR-204 exerts its function by targeting genes involved in tumorigenesis including brain-derived neurotrophic factor (BDNF), a neurotrophin family member which is known to promote tumor angiogenesis and invasiveness. Analysis of primary tumors shows that increased expression of BDNF or its receptor tropomyosin-related kinase B (TrkB) parallel a markedly reduced expression of miR-204. Our results reveal that loss of miR-204 results in BDNF overexpression and subsequent activation of the small GTPase Rac1 and actin reorganization through the AKT/mTOR signaling pathway leading to cancer cell migration and invasion. These results suggest that microdeletion of genomic loci containing miR-204 is directly linked with the deregulation of key oncogenic pathways that provide crucial stimulus for tumor growth and metastasis. Our findings provide a strong rationale for manipulating miR-204 levels therapeutically to suppress tumor metastasis.

Heat shock protein 90 regulates the expression of Wilms' tumor 1 protein in myeloid leukemias

The aberrant overexpression of Wilms tumor 1 (WT1) in myeloid leukemia plays an important role in blast cell survival and resistance to chemotherapy. High expression of WT1 is also associated with relapse and shortened disease-free survival in patients. However, the mechanisms by which WT1 expression is regulated in leukemia remain unclear. Here, we report that heat shock protein 90 (Hsp90), which plays a critical role in the folding and maturation of several oncogenic proteins, associates with WT1 protein and stabilizes its expression. Pharmacologic inhibition of Hsp90 resulted in ubiquitination and subsequent proteasome-dependant degradation of WT1. RNAi-mediated silencing of WT1 reduced the survival of leukemia cells and increased the sensitivity of these cells to chemotherapy and Hsp90 inhibition. Furthermore, Hsp90 inhibitors 17-AAG [17-(allylamino)-17-demethoxygeldanamycin] and STA-9090 significantly reduced the growth of myeloid leukemia xenografts in vivo and effectively down-regulated the expression of WT1 and its downstream target proteins, c-Myc and Bcl-2. Collectively, our studies identify WT1 as a novel Hsp90 client and support the crucial role for the WT1-Hsp90 interaction in maintaining leukemia cell survival. These findings have significant implications for developing effective therapies for myeloid leukemias and offer a strategy to inhibit the oncogenic functions of WT1 by clinically available Hsp90 inhibitors.

Enhanced sensitivity of colon tumour cells to natural killer cell cytotoxicity after mild thermal stress is regulated through HSF1-mediated expression of MICA

Abstract Purpose: Previously we showed that mild thermal stress increased natural killer (NK) cell-mediated tumour cytotoxicity and that this could be blocked by anti-NKG2D or anti-MICA (major histolocompatability complex (MHC) class I related chain A) antibodies. Here, we investigated the role of the transcription factor heat shock factor 1 (HSF1) in thermal regulation of MICA expression in tumour cells in vitro and in vivo. Materials and methods: Hyperthermia experiments were conducted in vitro and in mice using a target temperature of 39.5 °C. Apoptotic cells and NK cells in situ were visualised by use of the TUNEL assay or expression of NKp46 respectively. Using Colo205 cells, HSF1 message was blocked utilising siRNA while luciferase reporter assays were used to measure the activity of the MICA promoter in vitro. Cell surface MICA was measured by flow cytometry. Results: Following whole body hyperthermia (WBH), tumour tissues showed an increase in NK cells and apoptosis. Mild thermal stress resulted in a transient increase in surface MICA and enhanced NK cytotoxicity of the Colo205 colon cancer cell line. Silencing (mRNA) HSF1 expression in Colo205 cells prevented the thermal enhancement of MICA message and surface protein levels, with partial loss of thermally enhanced NK cytotoxicity. Mutations of the HSF1 binding site on the MICA promoter implicated HSF1 in the thermal enhancement of MICA. Some, but not all, patient-derived colon tumour derived xenografts also exhibited an enhanced MICA message expression after WBH. Conclusions: Up-regulation of MICA expression in Colo205 cells and enhanced sensitivity to NK cell killing following mild thermal stress is dependent upon HSF1.

The Transcription Factor Wilms Tumor 1 Confers Resistance in Myeloid Leukemia Cells against the Proapoptotic Therapeutic Agent TRAIL (Tumor Necrosis Factor α-related Apoptosis-inducing Ligand) by Regulating the Antiapoptotic Protein Bcl-xL

Background: Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) resistance in leukemia is not fully understood. Results: shRNA-targeted knockdown of WT1 oncogene sensitizes TRAIL-resistant leukemia cells to TRAIL-induced cell death. Conclusion: WT1 expression mediates TRAIL resistance in leukemia cells by inducing antiapoptotic protein Bcl-xL. Significance: Approaches to silence WT1 expression can be exploited to overcome TRAIL resistance in myeloid leukemias. Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is considered a promising cancer therapeutic agent due to its ability to induce apoptosis in a variety of cancer cells, while sparing normal cells. However, many human tumors including acute myeloid leukemia (AML) are partially or completely resistant to monotherapy with TRAIL, limiting its therapeutic utility. Therefore, identification of factors that contribute to TRAIL resistance may facilitate future development of more effective TRAIL-based cancer therapies. Here, we report a previously unknown role for WT1 in mediating TRAIL resistance in leukemia. Knockdown of WT1 with shRNA rendered TRAIL-resistant myeloid leukemia cells sensitive to TRAIL-induced cell death, and re-expression of shRNA-resistant WT1 restored TRAIL resistance. Notably, TRAIL-mediated apoptosis in WT1-silenced cells was largely due to down-regulation of the antiapoptotic protein Bcl-xL. Moreover, WT1 expression strongly correlated with overexpression of Bcl-xL in AML cell lines and blasts from AML patients. Furthermore, we found that WT1 transactivates Bcl-xL by directly binding to its promoter. We previously showed that WT1 is a novel client protein of heat shock protein 90 (Hsp90). Consistent with this, pharmacological inhibition of Hsp90 resulted in reduced WT1 and Bcl-xL expression leading to increased sensitivity of leukemia cells to TRAIL-mediated apoptosis. Collectively, our results suggest that WT1-dependent Bcl-xL overexpression contributes to TRAIL resistance in myeloid leukemias.

Putting the brakes on angiogenesis through a novel VEGF–KLH (kinoid) vaccine

Evaluation of: Rad FH, Buanec HL, Paturance S et al. VEGF–kinoid vaccine, a therapeutic approach against tumor angiogenesis and metastases. Proc. Natl Acad. Sci. USA 104(8), 2837–2842 (2007). Angiogenesis, the growth of new blood vessels, is essential for tumor growth and metastasis. Of the several known angiogenic factors, VEGF is an important mediator of tumor-induced angiogenesis and represents a potential target for innovative anticancer therapy. Recently, humanized monoclonal anti-VEGF antibody (bevacizumab) has been approved by the US FDA for combinatorial therapies with cytotoxic drugs in metastatic colorectal cancer. However, adverse side effects and enormous costs are associated with the use and delivery of bevacizumab. In the study under evaluation, Rad et al. demonstrated an alternative approach by using active immunization in mice with a novel VEGF–kinoid vaccine. The authors observed that the antitumor effects elicited by their vaccine were as effective as bevacizumab in xenografted-tumor mouse models.

Novel post-transcriptional and post-translational regulation of pro-apoptotic protein BOK and anti-apoptotic protein Mcl-1 determine the fate of breast cancer cells to survive or die

Deregulation of apoptosis is central to cancer progression and a major obstacle to effective treatment. The Bcl-2 gene family members play important roles in the regulation of apoptosis and are frequently altered in cancers. One such member is pro-apoptotic protein Bcl-2-related Ovarian Killer (BOK). Despite its critical role in apoptosis, the regulation of BOK expression is poorly understood in cancers. Here, we discovered that miR-296-5p regulates BOK expression by binding to its 3’-UTR in breast cancers. Interestingly, miR-296-5p also regulates the expression of anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1), which is highly expressed in breast cancers. Our results reveal that Mcl-1 and BOK constitute a regulatory feedback loop as ectopic BOK expression induces Mcl-1, whereas silencing of Mcl-1 results in reduced BOK levels in breast cancer cells. In addition, we show that silencing of Mcl-1 but not BOK reduced the long-term growth of breast cancer cells. Silencing of both Mcl-1 and BOK rescued the effect of Mcl-1 silencing on breast cancer cell growth, suggesting that BOK is important for attenuating cell growth in the absence of Mcl-1. Depletion of BOK suppressed caspase-3 activation in the presence of paclitaxel and in turn protected cells from paclitaxel-induced apoptosis. Furthermore, we demonstrate that glycogen synthase kinase (GSK3) α/β interacts with BOK and regulates its level post-translationally in breast cancer cells. Taken together, our results suggest that fine tuning of the levels of pro-apoptotic protein BOK and anti-apoptotic protein Mcl-1 may decide the fate of cancer cells to either undergo apoptosis or proliferation.

Abstract 2336: Novel regulatory mechanisms for Bcl2-related Ovarian Killer (BOK) expression in breast cancer

Deregulation of apoptosis is central to cancer progression and a major obstacle to effective treatment. The Bcl-2 gene family members play important roles in the regulation of apoptosis and are frequently altered in cancers. One such member is Bcl-2-related Ovarian Killer (BOK), which is a pro-apoptotic protein. Despite its critical role in apoptosis, the regulation of BOK expression is poorly understood in cancers. Here, we discovered that miR-296-5p, regulates BOK expression by binding to its 3’UTR in breast cancers. Furthermore, we show that depletion of BOK by either miR-296-5p or siRNA against BOK protected breast cancer cells from undergoing paclitaxel-induced apoptosis. Interestingly, miR-296-5p also regulates the expression of Mcl-1, which is an anti-apoptotic protein and is highly expressed in breast cancers. Our results reveal that Mcl-1 is important for suppression of BOK function as ectopic BOK expression induced Mcl-1, while silencing of BOK resulted in reduced Mcl-1 levels in breast cancer cells. In addition, we show that specific silencing of Mcl-1 reduced the long-term growth of breast cancer cells, whereas BOK inhibition didn’t have any effect on the growth of breast cancer cells. Surprisingly, silencing of both Mcl-1 and BOK rescued the effect of Mcl-1 silencing on breast cancer cell growth, suggesting that BOK is important for attenuating cell growth in the absence of Mcl-1, and also showing a tight feedback regulatory loop between BOK and Mcl-1 in breast cancer cells. Furthermore, we demonstrated that BOK protein level is regulated post-translationally by GSK3α and to some extent GSK3β as GSK3 inhibitor (CHIR99021) or silencing of GSK3 significantly increased BOK protein levels in breast cancer cells. Notably, we found that Mcl-1 interacts with GSK3α/β and silencing of Mcl-1 using siRNA significantly attenuated endogenous GSK3α/β levels in breast cancer cells. Taken together, our results suggest that fine tuning (either post-transcriptionally by miR-296-5p or post-translationally by GSK3) of the levels of pro-apoptotic protein BOK and anti-apoptotic protein Mcl-1 decide the fate of cancer cells to either undergo Apoptosis or proliferation. Citation Format: Benjamin Chidi Onyeagucha, Panneerdoss Subbarayalu, Subapriya Rajamanickam, Nourhan Abdelfattah, Santosh Timilsina, Rosa M. Guzman, Carla Zeballos, Vijay Eedunuri, Sanjay Bansal, Hima Bansal, Tabrez A. Mohammad, Yidong Chen, Manjeet K. Rao. Novel regulatory mechanisms for Bcl2-related Ovarian Killer (BOK) expression in breast cancer [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 2336. doi:10.1158/1538-7445.AM2017-2336