Debasish Boral

Nuclear receptors in the multidrug resistance through the regulation of drug-metabolizing enzymes and drug transporters.

Chemotherapy is one of the three most common treatment modalities for cancer. However, its efficacy is limited by multidrug resistant cancer cells. Drug metabolizing enzymes (DMEs) and efflux transporters promote the metabolism, elimination, and detoxification of chemotherapeutic agents. Consequently, elevated levels of DMEs and efflux transporters reduce the therapeutic effectiveness of chemotherapeutics and, often, lead to treatment failure. Nuclear receptors, especially pregnane X receptor (PXR, NR1I2) and constitutive androstane activated receptor (CAR, NR1I3), are increasingly recognized for their role in xenobiotic metabolism and clearance as well as their role in the development of multidrug resistance (MDR) during chemotherapy. Promiscuous xenobiotic receptors, including PXR and CAR, govern the inducible expressions of a broad spectrum of target genes that encode phase I DMEs, phase II DMEs, and efflux transporters. Recent studies conducted by a number of groups, including ours, have revealed that PXR and CAR play pivotal roles in the development of MDR in various human carcinomas, including prostate, colon, ovarian, and esophageal squamous cell carcinomas. Accordingly, PXR/CAR expression levels and/or activation statuses may predict prognosis and identify the risk of drug resistance in patients subjected to chemotherapy. Further, PXR/CAR antagonists, when used in combination with existing chemotherapeutics that activate PXR/CAR, are feasible and promising options that could be utilized to overcome or, at least, attenuate MDR in cancer cells.

Abstract 3424: Cancer cells lose their expression of lipoxygenases in order to activate pro-survival and angiogenic pathways thereby establishing successful macro-metastases

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Metastasis is the major cause of cancer mortality. We therefore wanted to study the possible genetic changes acquired by these cancer cells in order to be endowed with metastatic capabilities. Several studies have identified genes which are differentially expressed in primary and secondary tumor growths. 15-lipoxygenase (ALOX15B) is one such gene. It encodes for an enzyme that takes part in arachidonic acid metabolism and has been characterized as a functional tumor suppressor in prostate cancer. The expression and activity of ALOX15B is frequently suppressed during the carcinogenic progress in the prostate, lungs, esophagus and sebaceous glands. However the exact role of ALOX15B as a tumor suppressor or the underlying mechanism behind its suppression in metastatic cancer cells remains largely unknown. Our initial in silico analyses from clinical samples showed that ALOX15B expression is attenuated in metastatic prostate cancer, compared to normal tissue or primary prostate cancer. Gene array studies across several cancer cell lines showed that ALOX15B expression is inversely correlated with invasiveness of the cell type. Gene clustering showed that ALOX15B is grouped with genes associated with an epithelial phenotype. ALOX15B expression was knocked down in immortalized non-transformed prostate as well as non/low invasive prostate cancer cell line. This had no effect on the proliferative indices in these cells, neither were the cells more resistant to apoptosis under normal growth conditions. However the cells were endowed with increased resistance to hypoxic stress. This was at least in part achieved by increased production of VEGF-A. Analyses of several pro-survival pathways identified that Akt signaling was hyper activated in these ALOX15B knocked down cells. This in turn led to a decreased FoxO transcriptional activity. Conversely, restoration of ALOX15B in low/non metastatic as well as highly metastatic prostate cancer cell lines decreased their basal level of Akt signaling along with increased FoxO activity and a concomitant decrease in VEGF-A production. Further experiments showed that ALOX15B regulates Akt activity by modulating its phosphorylation status. Luciferase tagged rat prostate cancer cells were engineered to over-express ALOX15B. Implantation of such cells in mice showed that cells with restored expression of ALOX15B showed no difference in growth kinetics of primary tumor growth, but a decrease in luciferase activity in the liver of such mice. To summarize, our data shows that loss of ALOX15B expression in prostate cancer cells endows such cells with pro-survival benefits which allows them to establish successful macro-metastases. 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 3424. doi:1538-7445.AM2012-3424

Cancer Stem Cells in Resistance to Cytotoxic Drugs: Implications in Chemotherapy

The efficacy of cytotoxic chemotherapy is limited by drug resistance presented by some cancer cells. Cancer stem cells (CSCs) are a sub-population of tumor cells that can initiate tumor formation. If chemotherapy kills bulk of cells within a tumor but not CSCs, the surviving CSCs can initiate the formation of recurrent tumors. This article discusses the inherent resistance of CSCs toward cytotoxic chemotherapy and some possible mechanisms involved. Approaches to target CSCs to improve the efficacy of chemotherapy will also be discussed.

Abstract 1700: TLR4 as a novel determinant of paclitaxel response in metastatic breast cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Background: Paclitaxel elicits both cytotoxic and pro-survival responses in tumor cells. The tumor-promoting effect of paclitaxel is a currently unrecognized determinant for decreasing the apoptotic effect of paclitaxel therapy. The likely mechanism for paclitaxel-dependent tumor-activating effects is the ability of paclitaxel to activate Toll-like Receptor-4 (TLR4) pathway. TLR4 is often over-expressed in malignant epithelial cells in which its signaling hyper-activates NF-kB, MAPK and PI3K pathways providing a pro-survival benefit to the residual cancer cells. The goal of this project was to identify the TLR4 role in paclitaxel resistance by correlating the expression profile of TLR4 pathway in a panel of breast carcinoma cells with sensitivity to paclitaxel therapy. Methods: TLR4 mRNA levels were determined in 18 breast carcinoma lines by qRT-PCR. The functionality of TLR4 pathway was determined by measuring mRNA of downstream products (IL-6, IL-8, MCP-1 and TNFα) after stimulating cells with a natural TLR4 ligand, lipopolysaccharide (LPS), or paclitaxel. Protein levels were measured by ELISA. Cytotoxic assays were used to correlate the level of TLR4 expression and responsiveness to paclitaxel. TLR4-positive (MDA-MB-231) and negative (HCC1806) lines were engineered to stably down-regulate and over-express TLR4, respectively. The target expression in modified clones was determined by qRT-PCR and Western Blot. The modified lines were analyzed for functionality and altered responsiveness to paclitaxel using the aforementioned methods. Results: TLR4 was expressed in 60% of human breast cancer cell lines. TLR4 receptor in MDA-MB-231 line was functional as demonstrated by up-regulation of inflammatory cytokines. LPS increased IL-6 and MCP-1 by 8-10 fold whereas IL-8 and TNF-alpha were increased by 120-300 fold. Paclitaxel had 1/10 to 1/3 of the LPS activity. MDA-MB-231 line was ∼5 fold more resistant to paclitaxel than HCC1806 lacking TLR4. Paclitaxel treatment not only drastically increases secretion of NF-kB dependent cytokines but also up-regulated the expression levels of their receptors suggesting establishment of novel autocrine pro-survival and proliferative positive loops. Anti-TLR4 antibody inhibited paclitaxel stimulating effects by nearly 100%. Conclusions: These data show that paclitaxel up-regulates both inflammatory cytokines and their receptors in human breast carcinoma cells, likely through activation of the TLR4 pathway. Inflammatory pathway signaling increases survival and proliferation in TLR4-positive cells, suggesting that activation of this pathway in malignant cells maintain chronic inflammation and promote tumor growth and metastasis through both paracrine and autocrine loops. This study suggests that tumor resistance to paclitaxel might be determined by TLR4 expression, and that blocking TLR4 might significantly improve tumor response to paclitaxel therapy. 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 1700. doi:10.1158/1538-7445.AM2011-1700

Abstract 5285: Protein phosphatases modulate kinase signaling mediated Wnt activation

Multiple growth factors, signaling through their respective kinase cascades, phosphorylate beta-catenin at specific Serine residues to cause its nuclear sequestration thereby fueling Wnt driven tumor cell proliferation. Our present work identifies a group of phosphatase genes that attenuate such growth factor mediated activation of Wnt signaling in colo-rectal cancer (CRC) cells. This proposed mechanism proposes an additional mode of regulation of Wnt activity in CRC cells. Using a lentiviral TCF-LEF reporter system, we isolated distinct SW480 (primary) and SW620 (metastatic) cell fractions based on their endogenous level of Wnt activity. Receptor Tyrosine Kinase (RTK) antibody arrays using cell lysates from these cell fractions identified that FGFR3, ALK and RET signaling pathways have a positive correlation with enhanced Wnt activity. Simultaneously qPCR arrays performed on these same cellular fractions identified a total of 10 phosphatase genes whose expression levels are significantly decreased with heightened Wnt activity, out of which 4 genes PHLPP, PHLPPL, PTPRK and PTPN6 are known to regulate the aforementioned RTK pathways. Of these phosphatase genes, PTPRK has been reported to undergo gene fusion events with RSPO3 (a non-canonical activator of Wnt) in CRC patients. Since the fusion gene product provides no additional Wnt activation that the wild-type RSPO3, we theorized that these fusion events occur either to allow CRC cells to utilize the enhanced promoter activity of the PTPRK gene promoter, or to nullify the Wnt attenuating properties of PTPRK. To test this hypothesis, we have presently cloned and identified PTPRK and RSPO3 gene promoter regions and wish to test their activity in distinct Wnt responsive cell fractions. Our work will allow a better understanding of how phosphatase genes may be involved in controlling the Wnt pathway and how their inactivation may contribute to CRC cell proliferation. Citation Format: Debasish Boral, Daotai Nie. Protein phosphatases modulate kinase signaling mediated Wnt activation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5285. doi:10.1158/1538-7445.AM2014-5285

Abstract 5006: SOX2 inhibits Wnt driven colon cancer cell proliferation.

APC mutation/s resulting in aberrant Wnt signaling, initiates intestinal polyposis and drives adenoma to carcinoma progression in human colon. This represents a target for specific therapeutic intervention and has generated extreme interest among the scientific community. However, it is becoming increasingly apparent that though Wnt signaling may cause robust proliferation, it is the quiescent cell population, defined by stem-ness related gene signatures, that evades chemo-radio therapy targeted at rapidly dividing cells, and perpetuates self-renewal ultimately causing relapse at local site and/or recurrence as distant metastases. We report that SOX2, an embryonic stem cell transcription factor, counteracts Wnt driven tumor cell proliferation and maintains quiescence in colo-rectal cancers (CRC). SOX2 expression increases with progressive staging of colon cancer patients. Using colon cancer cell lines transduced with Wnt reporter constructs, we identified distinct sub-populations with varying degrees of Wnt activity and found that SOX2 expression and Wnt activity are mutually exclusive in SW480 primary CRC cell line, but co-expressed in the isogenic SW620 metastatic CRC cell line. Knockdown of SOX2 in SW620 cell increased anchorage independence in vitro and tumor growth in xenograft models. This was accompanied by an increase of cells in S-phase and increased Ki67 index in IHC of xenograft tissue. Over-expression of SOX2 in SW480 cells resulted in decreased colony formation in soft agar and primary tumor growth in mice. Over-expression also caused increased fraction of cells in G0-G1 phase and decreased Ki67 staining in tumor tissue. In SW620 cell line, SOX2 knockdown not only increased basal Wnt activity, but also caused acquisition of Wnt activity in hitherto Wnt negative cells. Intriguingly, over-expression of SOX2 in SW480 cells, reversed the enhanced Wnt activity gained by loss-of-function APC mutation. Experiments conducted with both SOX2 silenced and over-expressed cell lines revealed that SOX2 inhibits phosphorylation of β-catenin at Serine 552 and 675 thereby decreasing its nuclear sequestration and activation. Screening with protein kinase and phosphatase inhibitors identified PHLPP and PHLPPL as candidate genes that are controlled by SOX2 and are responsible for its effects on β-catenin. Western Blot and qPCR analyses showed that SOX2 enhances PHLPP/L expression. Reporter assays demonstrated that SOX2 controls PHLPP/L promoter activity while ChIP assays confirmed binding of SOX2 to various regions of the respective promoters. IHC of SOX2 silenced xenograft tissue showed decreased PHLPP/L and increased pβ-catenin staining while SOX2 over-expression showed reciprocal changes. Our findings delineate the mechanism by which CRC cells suppress Wnt activity thereby maintaining a pool of quiescent cancer cells, and lay the basis for further research to identify and target these cells. Citation Format: Debasish Boral, Man-Tzu Wang, Daotai Nie. SOX2 inhibits Wnt driven colon cancer cell proliferation. [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 5006. doi:10.1158/1538-7445.AM2013-5006

Abstract 3852: Combination treatment of nab-paclitaxel and bevacizumab in a new model of triple-negative breast cancer

Background: Tumors that lack estrogen, progesterone, and Her2/nu (triple-negative) are one of the most aggressive, therapy-resistant and highly metastatic subtypes of breast cancer (BC). Most currently available models do not recapitulate expression profile of triple-negative BC, thus making it difficult to devise new treatments that target this tumor type. Here, we established and characterized a new model of triple-negative BC. We also tested the novel combination of nab-paclitaxel with bevacizumab, which has been successful in treating other metastatic cancer types. Methods: HCC1806 cells were obtained stably transfected with Red Fluorescent Protein (RFP) and Renilla luciferase to establish dual reporter termed RR (RFP and Renilla luciferase). The new derivative was designated as HCC1806-RR and characterized in proliferation and cytotoxicity assays. HCC1806-RR was also orthotopically implanted into mammary fat pads of immunodeficient mice to determine tumor cell sensitivity to chemotherapy alone or in combination with anti-VEGF-A antibody. Mice bearing orthotopic HCC1806-RR tumors of 150mm 3 in size were treated with saline (control), bevacizumab (4mg/kg. i.p., twice a week, for 10 weeks), nab-paclitaxel (10mg/kg, i.v., qdx5), or with combination of nab-paclitaxel and bevacizumab. Tumor growth rate was monitored by using calipers. Metastasis was analyzed by measuring luciferase activity in the lymph nodes (LN) and lungs. Results: The parental HCC1806 and its derivative HCC1806-RR had identical morphology, proliferation rates and sensitivity to nab-paclitaxel. Luciferase measurements and imaging in vivo showed that HCC1806-RR tumors have predominant LN metastasis with lungs being a second metastatic site. Bevacizumab and nab-paclitaxel inhibited tumor growth by 0% and 90%, respectively. Combination therapy inhibited tumor growth by 100%. This result was highly significant compared with either control (P Conclusions: The HCC1806-RR is a new model double-tagged with RFP and Renilla luciferase. These tags allow for quantitative assessment of metastatic spread. This model can be used to study the biology of triple-negative breast cancer and for designing new approaches to treat this type of cancer. Preliminary studies demonstrated high sensitivity of HCC1806-RR to nab-paclitaxel combined with bevacizumab suggesting that this therapy could significantly improve the health outcome for patients with triple-negative breast cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3852.