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The existence of distinct breast microbiota has been recently established, but their biological impact in breast cancer remains elusive. Focusing on the shift in microbial community composition in diseased breast compared with normal breast, we identified the presence of Bacteroides fragilis in cancerous breast. Mammary gland as well as gut colonization with enterotoxigenic Bacteroides fragilis (ETBF), which secretes B. fragilis toxin (BFT), rapidly induces epithelial hyperplasia in the mammary gland. Breast cancer cells exposed to BFT exhibit “BFT memory” from the initial exposure. Intriguingly, gut or breast duct colonization with ETBF strongly induces growth and metastatic progression of tumor cells implanted in mammary ducts, in contrast to nontoxigenic Bacteroides fragilis. This work sheds light on the oncogenic impact of a procarcinogenic colon bacterium ETBF on breast cancer progression, implicates the β-catenin and Notch1 axis as its functional mediators, and proposes the concept of “BFT memory” that can have far-reaching biological implications after initial exposure to ETBF.Significance:B. fragilis is an inhabitant of breast tissue, and gut or mammary duct colonization with ETBF triggers epithelial hyperplasia and augments breast cancer growth and metastasis. Short-term exposure to BFT elicits a “BFT memory” with long-term implications, functionally mediated by the β-catenin and Notch1 pathways.This article is highlighted in the In This Issue feature, p. 995

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Background and Aim: Obese state is associated with increased breast cancer growth, metastasis, and poor overall survival. In this study, we seek to decipher the underlying molecular mechanisms by which obesity/hyperleptinemia reduces the efficacy of tamoxifen. Methods: The impact of obesity on tamoxifen was evaluated utilizing clonogenicity, high-fat-diet- induced obese mice and leptin-treated xenograft-models. Mechanistic studies involved immunoblotting, real-time PCR, immunocytochemistry, chromatin immunoprecipitation assay, phosphokinase array and in silico analysis. Results: Obese mice with hyperleptinemia exhibit increased tumor progression and respond poorly to tamoxifen compared to non-obese mice. Exogenous leptin abrogates tamoxifen-mediated growth inhibition and potentiates breast tumor growth even in the presence of tamoxifen. Mechanistically, leptin induces nuclear translocation of phosphorylated-ER and increases the expression of ER-responsive genes while reducing tamoxifen-mediated gene repression by abrogating tamoxifen-induced recruitment of corepressors NCoR, SMRT and Mi2. Further, we found that coactivator Med1 potentially associates with 48 (out of 75) obesity-signature genes. Interestingly, leptin upregulates Med1 expression by decreasing miR-205 and increases its functional activation via phosphorylation that is mediated by activation of Her2 and EGFR. It is important to note that Med1 silencing abrogates the negative effects of leptin on tamoxifen efficacy. Additionally, honokiol or adiponectin treatment effectively inhibits leptin-induced Med1 expression and improve tamoxifen efficacy in hyperleptinemic state.Conclusion: In conclusion, these studies show the molecular mechanisms by which obese/hyperleptinemic state may contribute to poor response to tamoxifen implicating leptin-miR205-Med1 and leptin-Her2-EGFR-Med1 axes and present bioactive compound honokiol and adipocytokine adiponectin as agents that can block leptin’s negative effect on tamoxifen. Citation Format: Arumugam Nagalingam, Nethaji Muniraj, Sumit Siddharth, Dimiter Avtanski, Sheetal Parida, Pajamurthy Kuppusamy, Neeraj K Saxena, Nethaji Muniraj. Hyperleptinemia in obese state renders luminal breast cancers refractory to tamoxifen coordinating a crosstalk between Med1, miR205 and Erb B kinases [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-32.

Background and Aim: Triple-negative breast cancer (TNBC) is a very aggressive subtype of breast cancer characterized by the absence of estrogen receptor (ER), progesterone receptor (PR) and HER2 receptor expression. Mortality from TNBC is significantly higher in African American (AA) women in comparison to White American (WA) women (5-year relative survival of only 14% for AA in comparison to 36% for WA) even though the incidence rates are lower in AA women. Irrespective of stage at diagnosis, AA-TNBC is more aggressive with higher metastasis and a poorer survival than WA-TNBC; therefore, it is imperative to understand the molecular determinants that drive aggressive progression of AA-TNBC. Overall aim of this study is to decipher the alterations in the molecular circuitry underlying racial disparity in TNBC progression in AA women compared to WA women. Results: AA-TNBC cells (HCC1806 and HCC1569) exhibited increased growth and higher migration potential; higher expression of stemness factors, increased number of mammospheres and higher CD44+/CD49f+ population in comparison to WA-TNBC (Hs578t, BT549, HCC1937 and HCC1187) cells. We analyzed RNA sequencing data of multiple AA and WA TNBC cell lines to query the status self-renewal pathways (Wnt/βCatenin, GLI1/Shh and YAP-TAZ) and observed significantly higher levels of GLI1 in AA-TNBC cell lines while no significant alterations were observed in other pathway components. Further analysis of TCGA dataset revealed a positive correlation between GLI1 and Notch1 in AA-TNBC with a negative correlation in WA-TNBC. Increased expression of components of GLI1 and Notch1 pathway (SHH, Jagged, cleaved-Notch (NICD), Hes1 and FOXM1) were noted in AA-TNBC compared to WA-TNBC cells. AA-TNBC cells showed increased nuclear localization of GLI1 and NICD as compared to WA-TNBC cells. We observed that GLI1 and NICD co-localize and co-immunoprecipitate in AA-TNBC indicating a direct interaction between these two transcription factors. High expression of GLI1 and Notch1 correlated with poor recurrence free survival in TNBC patients. Analyses of clinical samples revealed higher levels of nuclear NICD and GLI1 in AA-TNBC in comparison to WA-TNBC. Concomitant inhibition of GLI1 and Notch1 using respective small molecule inhibitors, GANT61 and DAPT, along with standard chemotherapeutic agents (Doxorubicin and carboplatin) effectively inhibited AA TNBC tumor growth in mice. Also, ex vivo analyses of tumor cells showed reduced migration and invasion potential as well as downregulation of CD44+/CD49f+ and ADLH1A1+ population in a synergistic manner. Combined treatment with GANT61+ DAPT+ Carboplatin effectively reduced stem cell frequency of AA-TNBC tumors in in vivo limiting dilution assay. Conclusions: In conclusion, these results show that AA-TNBC cells are inherently aggressive with increased growth, migration and stemness potential. We found aberrant activation of GLI1 and Notch pathway and a crosstalk between GLI1 and NICD whose inhibition effectively inhibits AA-TNBC and sensitizes AA-TNBC to standard chemotherapy. Our studies propose a ‘triple drug regimen’ for AA-TNBC tumors based on its molecular circuitry. Citation Format: Sumit Siddharth, Sheetal Parida, Nethaji Muniraj, Shawn M Hercules, Arumugam Nagalingam, Balázs Győrffy, Juliet Daniel, Dipali Sharma. Molecular determinants of racial disparity guide triple-therapy for triple negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS7-86.

Hepatocellular carcinoma (HCC) incidence, as well as related mortality, has been steadily increasing in the USA and across the globe, partly due to the lack of effective therapeutic options for advanced HCC. Though sorafenib is considered standard-of-care for advanced HCC, it only improves median survival by a few months when compared to placebo. Sorafenib is also associated with several unpleasant side effects that often lead to early abatement of therapy. Here, we investigate whether a combination regimen including low-dose sorafenib and a non-toxic dose of anti-diabetic drug metformin can achieve effective inhibition of HCC. Indeed, combining metformin with low-dose sorafenib inhibited growth, proliferation, migration, and invasion potential of HCC cells. We observed a 5.3- and 1.9-fold increase in sub-G1 population in the combination treatment compared to sorafenib alone. We found that the combination of metformin enhanced the efficacy of sorafenib and inhibited the MAPK/ERK/Stat3 axis. Our in vivo studies corroborated the in vitro findings, and mice harboring HepG2-derived tumors showed effective tumor reduction upon treatment with low-dose sorafenib and metformin combination. This work sheds light on a therapeutic strategy aiming to augment sorafenib efficacy or dose-de-escalation that may prove beneficial in circumventing sorafenib resistance as well as minimizing related side effects.

Background: 41.9% of adult US population is obese and every 5-unit BMI increase is associated with 12 percent increase in breast cancer risk; it is an independent predictor of poor outcomes in breast cancer. Microbiome has emerged as a central regulator of obesity and we recently discovered that microbes can not only potentiate breast cancer growth and metastasis but also affect therapeutic response. We showed weight-loss by Diet/Exercise/Behavioral intervention (POWER-remote trial) exerts favorable effects. Here, we examine molecular underpinnings of poor therapy response in obese state and explore the gut microbiome to mitigate the same. Methods: We co-implanted lineage determined human White preAdipocytes (hWAT) with MCF7 breast cancer cells in the mammary glands of mice. Changes in tumor microenvironment and tumor cells were evaluated ex situ by RNA sequencing, flow cytometry, Raman spectroscopy, Quantitative phase microscopy, PCR, immunoblotting and functional assays. To inspect the gut microbiome, 16 obese breast cancer patients were subjected to weight loss intervention over 14 weeks. Gut microbiome was analyzed at baseline and post-intervention by microbial shotgun sequencing. Patients were divided into responders and non-responders based on % weight-loss. Results: We found that human breast cancer cells co-implanted with hWAT formed more aggressive tumors compared to controls. RNA sequencing revealed significant upregulation of multiple oncogenic pathways along with robust innate immune response. Tumor dissociated cells from hWAT-MCF7 tumors showed extensive molecular reprogramming rendering them more proliferative, motile and stem-like along with dramatic physiological changes like increased intercellular lipid accumulation. hWAT-MCF7 cells attained resistance to multiple drugs including Tamoxifen, Fulvestrant, Letrozole and Palbociclib. Simultaneous targeting of multiple signaling pathways using pharmacological inhibitors is infeasible in clinical setting hence we explored the obesity-microbiome axis. Obese breast cancer patients underwent POWER-remote intervention and their gut microbiome analysis showed significant differential expression of many microbes. Based on abundance and metabolite profile, F. prausnitzii was selected as the candidate of interest. On treatment with F. prausnitzii growth media, acquired multi-drug resistance in hWAT-MCF7 cells could be reversed suggesting that increase in gut population of the bacterium could be helpful in sensitizing patients unresponsive to therapy. Conclusion: Direct interaction between breast cancer cells and WAT rendered them resistant to multiple drugs. Metabolites produced by weight-loss-associated bacterium F. prausnitzii reversed adiposity-induced multi-drug resistance, hence, can potentially have therapeutic benefits in obese breast cancer patients. Citation Format: Sheetal Parida, Sumit Siddharth, Nethaji Muniraj, Zhenhui Liu, Ishan Barman, Michael Smith, Jenni Sheng, Vered Stearns, Janelle W. Coughlin, Dipali Sharma. Weight loss intervention triggered gut microbiota modulation can positively impact obesity associated endocrine therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5916.

Simple Summary Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer that cannot be treated with endocrine therapy and Her2-targeted therapy. Although its prevalence among newly diagnosed breast cancers is approximately 12.7%, it accounts for 40% of breast cancer-related mortality. Higher mortality rates among TNBC is partly because of the lack of targeted therapies and the development of resistance to chemotherapy. We discuss several important mechanisms that lead to chemoresistance and focus on important pathways and biological features that can be potentially exploited to develop therapies for TNBC. TNBC is currently defined by the absence of ER, PR, and Her2 and the greatest leap for TNBC would be our ability to characterize them with the presence of ‘x’ proteins. With this review, we intend to highlight the key nodes of TNBC and push the field towards connecting the dots between key features of TNBC and novel drug(s). Abstract Metastatic progression and tumor recurrence pertaining to TNBC are certainly the leading cause of breast cancer-related mortality; however, the mechanisms underlying TNBC chemoresistance, metastasis, and tumor relapse remain somewhat ambiguous. TNBCs show 77% of the overall 4-year survival rate compared to other breast cancer subtypes (82.7 to 92.5%). TNBC is the most aggressive subtype of breast cancer, with chemotherapy being the major approved treatment strategy. Activation of ABC transporters and DNA damage response genes alongside an enrichment of cancer stem cells and metabolic reprogramming upon chemotherapy contribute to the selection of chemoresistant cells, majorly responsible for the failure of anti-chemotherapeutic regime. These selected chemoresistant cells further lead to distant metastasis and tumor relapse. The present review discusses the approved standard of care and targetable molecular mechanisms in chemoresistance and provides a comprehensive update regarding the recent advances in TNBC management.

Cancer cells hijack autophagy pathway to evade anti-cancer therapeutics. Many molecular signaling pathways associated with drug-resistance converge on autophagy induction. Honokiol (HNK), a natural phenolic compound purified from Magnolia grandiflora, has recently been shown to impede breast tumorigenesis and, in the present study, we investigated whether breast cancer cells evoke autophagy to modulate therapeutic efficacy and functional networks of HNK. Indeed, breast cancer cells exhibit increased autophagosomes-accumulation, MAP1LC3B-II/LC3B-II-conversion, expression of ATG proteins as well as elevated fusion of autophagosomes and lysosomes upon HNK treatment. Breast cancer cells treated with HNK demonstrate significant growth inhibition and apoptotic induction, and these biological processes are blunted by macroautophagy/autophagy. Consequently, inhibiting autophagosome formation, abrogating autophagosome-lysosome fusion or genetic-knockout of BECN1 and ATG7 effectively increase HNK-mediated apoptotic induction and growth inhibition. Next, we explored the functional impact of tumor suppressor STK11 in autophagy induction in HNK-treated cells. STK11-silencing abrogates LC3B-II-conversion, and blocks autophagosome/lysosome fusion and lysosomal activity as illustrated by LC3B-Rab7 co-staining and DQ-BSA assay. Our results exemplify the cytoprotective nature of autophagy invoked in HNK-treated breast cancer cells and put forth the notion that a combined strategy of autophagy inhibition with HNK would be more effective. Indeed, HNK and chloroquine (CQ) show synergistic inhibition of breast cancer cells and HNK-CQ combination treatment effectively inhibits breast tumorigenesis and metastatic progression. Tumor-dissociated cells from HNK-CQ treated tumors exhibit abrogated invasion and migration potential. Together, these results implicate that breast cancer cells undergo cytoprotective autophagy to circumvent HNK and a combined treatment with HNK and CQ can be a promising therapeutic strategy for breast cancer.

In many solid tumors including triple-negative breast cancer (TNBC), IL-4 receptor (IL-4R) upregulation has been shown to promote cancer cell proliferation, apoptotic resistance, metastatic potential and a Th2 response in the tumor microenvironment (TME). Immunosuppressive cells in the TME including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs) also express the IL4-R. We hypothesized that selective depletion of IL4-R bearing cells in TNBC may have dual cytotoxic and immunotherapeutic benefit. To selectively target IL-4R+ cells, we genetically constructed, expressed and purified DABIL-4, a fusion protein toxin consisting of the catalytic and translocation domains of diphtheria toxin fused to murine IL-4. We found that DABIL-4 has potent and specific cytotoxic activity against TNBC cells in vitro. In murine TNBC models, DABIL-4 significantly reduced tumor growth, splenomegaly and lung metastases, and this was associated with reductions in MDSC, TAM and regulatory T-cells (Tregs) populations with a concomitant increase in the proportion of IFNγ+ CD8 T-cells. The anti-tumor activity of DABIL-4 was absent in IL-4R KO mice directly implicating IL-4R directed killing as the mechanism of anti-tumor activity. Moreover, NanoString analysis of DABIL-4 treated TNBC tumors revealed marked decline in mRNA transcripts that promote tumorigenesis and metastasis. Our findings demonstrate that DABIL-4 is a potent targeted antitumor agent which depletes both IL-4R bearing tumor cells as well as immunosuppressive cell populations in the TME.STATEMENT OF SIGNIFICANCEIn solid tumors like breast cancer, Interleukin-4 receptor (IL-4R) expression in the tumor microenvironment aids tumor growth and metastasis. IL-4R expression upon host immune cells further dampens antitumor immunity. In this study, we have genetically constructed a fusion protein toxin, DABIL-4, composed of the catalytic and translocation domains of diphtheria toxin and murine IL-4. DABIL-4 showed specific cytotoxicity against triple-negative breast cancer (TNBC) cells in vitro. DABIL-4 also markedly inhibited TNBC tumor growth and metastasis in vivo. The primary activity of DABIL-4 was found to be depletion of IL-4R+ immune cells in combination with direct elimination of tumor cells. In conclusion, DABIL-4 targeting of both tumor and immunosuppressive host cells is a versatile and effective treatment strategy for TNBC.

The existence of distinct breast microbiota has been recently established, but their biological impact in breast cancer remains elusive. Focusing on the shift in microbial community composition in diseased breast compared with normal breast, we identified the presence of Bacteroides fragilis in cancerous breast. Mammary gland as well as gut colonization with enterotoxigenic Bacteroides fragilis (ETBF), which secretes B. fragilis toxin (BFT), rapidly induces epithelial hyperplasia in the mammary gland. Breast cancer cells exposed to BFT exhibit “BFT memory” from the initial exposure. Intriguingly, gut or breast duct colonization with ETBF strongly induces growth and metastatic progression of tumor cells implanted in mammary ducts, in contrast to nontoxigenic Bacteroides fragilis. This work sheds light on the oncogenic impact of a procarcinogenic colon bacterium ETBF on breast cancer progression, implicates the β-catenin and Notch1 axis as its functional mediators, and proposes the concept of “BFT memory” that can have far-reaching biological implications after initial exposure to ETBF. Significance: B. fragilis is an inhabitant of breast tissue, and gut or mammary duct colonization with ETBF triggers epithelial hyperplasia and augments breast cancer growth and metastasis. Short-term exposure to BFT elicits a “BFT memory” with long-term implications, functionally mediated by the β-catenin and Notch1 pathways. This article is highlighted in the In This Issue feature, p. 995

Complete eradication of aggressive oral cancer remains a challenge due to the presence of CSCs. They resist conventional chemotherapeutic agents due to their self-renewal, drug efflux, and efficient DNA repair capacity. Here, we formulated a hybrid-nanoparticle (QAuNP) using quinacrine and gold and characterized/investigated its anti-angiogenic and anti-metastatic effect on OSCC-CSCs. QAuNP significantly inhibited cellular proliferation, caused apoptosis in vitro, and disrupted angiogenesis in vivo and tumor regression in xenograft mice model. It not only inhibited crucial angiogenic markers Ang-1, Ang-2 and VEGF but also depleted MMP-2 in H-357-PEMT cells in a p53 and p21-dependent manner. QAuNP also increased the ROS and NO generation in OSCC-CSCs and reduced the mitochondrial membrane potential. It altered the level of inflammatory cytokines IL-6, IL-1β, TNF-α and metastasis-associated markers (CD-44, CD-133) in H-357-PEMT and CM-treated endothelial cells (HUVEC) in p53/p21-dependent manner. Therefore, QAuNP will be a useful therapeutic agent against metastatic OSCC.

Previously, we reported that quinacrine (QC) may cause apoptosis in breast and colon cancer cells by activating the death receptor 5 (DR5), resulting in autophagic cell death through p21 modulation. Here, we systematically evaluated the combined role of p21 and DR5 and their crosstalk in QC-mediated autophagy and apoptosis in breast cancer cells using in vitro and in vivo models. Multiple breast cancer-derived cell lines (MCF-7, ZR-75-1, T47D, MDA-MB-231 and MCF-10A-Tr) and a mouse xenograft model were used. Also, multiple assays, including Western blotting, immunoprecipitation, staining for autophagy and apoptosis, gene silencing, hematoxylin and eosin staining, immunohistochemistry, cell viability assessment, fluorescence imaging and cell sorting were used. We found that QC activates p21 and DR5 in combination with the apoptosis inducer TRAIL in the breast cancer-derived cells tested. Combined TRAIL and QC treatment increased autophagy and apoptosis by increasing the interaction between, and co-localization of, p21 and DR5 in the death-inducing signaling complex (DISC). We found that this combination also inhibited the mTOR/PI3K/AKT signaling cascade and modulated reactive oxygen species (ROS) and nitric oxide (NO) production. Reductions in autophagy and apoptosis in DR5-knockout cells and a lack of change in p21-DR5-silenced cells were noted after TRAIL + QC treatment. This result explains dependence of the death (autophagy and apoptosis) cascade on these two key regulatory proteins. In addition, we found in an in vivo mouse xenograft model that increased expression and enhanced co-localization of p21 and DR5 after TRAIL + QC treatment supported a joint regulatory role of these proteins in the co-prevalence of autophagy and apoptosis. Our data suggest that a combined treatment of TRAIL and QC causes cell death in breast cancer-derived cells via autophagy and apoptosis by increasing the interaction of p21 and DR5, as indicated by both in vitro and in vivo studies.

Each cell in our body is designed with a self-destructive trigger, and if damaged, can happily sacrifice itself for the sake of the body. This process of self-destruction to safeguard the adjacent normal cells is known as programmed cell death or apoptosis. Cancer cells outsmart normal cells and evade apoptosis and it is one of the major hallmarks of cancer. The cardinal quest for anti-cancer drug discovery (bioactive or synthetic compounds) is to be able to re-induce the so called “programmed cell death” in cancer cells. The importance of bioactive compounds as the linchpin of cancer therapeutics is well known as many effective chemotherapeutic drugs such as vincristine, vinblastine, doxorubicin, etoposide and paclitaxel have natural product origins. The present review discusses various bioactive compounds with known anticancer potential, underlying mechanisms by which they induce cell death and their preclinical/clinical development. Most bioactive compounds can concurrently target multiple signaling pathways that are important for cancer cell survival while sparing normal cells hence they can potentially be the silver bullets for targeting cancer growth and metastatic progression.

Background and Aim: The growth and metastatic potential of cancer cells is regulated by the surrounding tumor microenvironment composed of a variety of cells including endothelial cells, fibroblasts, and adipose cells that promote tumor cell growth and invasion. There are two types of adipose tissue within the body. Brown adipocytes (BAT) contains uncoupling protein 1 (UCP1), which burns energy through adaptive thermogenesis. White adipocytes (WAT), which is the most prominent cell type in the breast, stores energy in the form of lipids. White adipocytes have the ability to secrete cytokines, growth factors, and hormones, providing a suitable microenvironment for breast tumor growth and progression. Identifying the mechanism through which adipocytes promote breast tumor progression will allow for the development of novel therapeutic interventions to inhibit adipocyte-breast cancer cell crosstalk during early stages of breast carcinogenesis. Furthermore, development of treatments that target cells within the tumor microenvironment, such as adipose cells, will potentially inhibit primary tumor growth as well as prevent metastatic disease, and could provide a treatment option for breast cancers. Methods: Functional assays including matrigel invasion, transwell migration and growth assays were conducted. Browning of white adipocyte was achieved by the addition of bioactive compounds such as BITC and Honokiol. Results: First, we explored the functional impact of WAT on breast cancer cells and observed that co-culture of WAT and breast cancer cells significantly increases the growth, clonogenicity, invasion and migration potential of breast cancer cells. In addition, breast cancer cells with epithelial-like morphology exhibit an acquisition of mesenchymal-like morphology indicating epithelial-to-mesenchymal conversion. To query the existence of a crosstalk between breast cancer cells and WAT, we examined the secretome of WATs and WATs-co-cultured with breast cancer cells (termed CA-WAT). We observed that CA-WAT exhibit increased levels of pro-inflammatory cytokines (IL-6, MCP, and PAI). Furthermore, we examined if WAT can be converted to BAT using bioactive strategies. We found that successful browning of WAT can be achieved with BITC and Honokiol. The addition of bioactive compounds increases the expression of BAT marker genes (UCP1, PRDM16, EVOL3, COX7a, and CIDEA) in WAT. We discovered that browning of WAT not only abrogates the pro-cancer effects of WAT on breast cancer cells but it also successfully alters the secretome profile of WAT. Conclusion: Our study shows that the conversion of white adipocytes to brown adipocytes with the use of BITC and Honokiol may serve as a novel therapeutic option for altering the breast tumor microenvironment to inhibit the growth and metastatic potential of breast cancer cells. Citation Format: Nethaji Muniraj, Sumit Siddharth, Sheetal Parida, Marey Shriver, Arumugam Nagalingam, Dipali Sharma. Therapeutic browning of white adipose tissue in the tumor microenvironment to inhibit breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2690.

Background: The last decade established significant contributions of microbiome to many organ specific cancers. Existence of distinct breast microbiota has been recently established but their biological impact in breast cancer progression remains elusive. A few recent studies suggested the existence of distinct breast microbiota and a shift in microbial community composition in diseased breast compared to normal breast however, their functional impact and underlying mechanisms are unknown. Present study was designed to examine the contribution of pro-carcinogenic bacteria in breast cancer initiation, progression and metastasis. Utilizing extensive data mining and metagenomic analyses, we discovered the presence of toxin producing enterotoxigenic Bacteroides fragilis (ETBF) in malignant breast. ETBF is a pro-carcinogenic bacteria known for its potential to initiate and/or promote colon cancer and its pathogenicity has been attributed to its unique toxin B. fragilis toxin (BFT)’. Results: Using mammary intraductal model we discovered that ETBF can successfully colonize the breast confirmed by qPCR and Fluorescent in situ hybridization where it induces local inflammation, fibrosis and hyperproliferation of breast epithelial cells. Mice bearing gut ETBF infection exhibit significant circulating BFT confirmed by qPCR and ELISA and distinct morphological alterations in mammary gland as observed from whole breast mounting and histological evaluation. Gut colonization with ETBF rapidly induces hyperplasia in mammary glands with systemic and local breast inflammation validated by flow cytometry, immunohistochemistry and cytokine profiling. While no changes are observed in cell growth and clonogenicity upon BFT treatment, significant increase in migration and invasion potential and decreased adhesion of MCF10A and MCF7 cells are observed. BFT leads to prominent cytoskeletal reorganization, and increase in migration, invasion and stemness potential of breast cancer cells. Our results indicate that breast cancer cells exposed to BFT ensue to exhibit increase tumor growth, form multifocal tumors and show a striking increase in tumor-initiating cells upon in vivo limiting dilution in immunocompromised mice exhibiting retention of ‘BFT memory’ from the initial exposure. Mechanistically, RNA-sequencing shows enrichment of βcatenin and Notch pathway in secondary tumors derived from BFT-exposed breast cancer cells. Inhibitors of βcatenin and Notch axis abrogates BFT-induced migration and invasion potential indicating the functional importance of this axis. Intriguingly, gut colonization with ETBF at a physiologically relevant level strongly induces growth and metastatic progression of 4T1 tumor cells implanted in mammary ducts monitored by whole animal bioluminescent imaging. In vivo and ex vivo analyses of tumors and distant organs reveal a significant induction of lung and liver metastasis of breast cancer by ETBF while gut colonization with non-toxigenic Bacteroides fragilis (NTBF) does not exhibit any tumor-augmenting impact. We mechanistically evaluate the oncogenic impact of alpha bug ETBF on breast cancer progression and its role in promoting liver and lung metastasis using multiple mice models and multiple techniques including multi-color flow cytometry, immunohistochemistry, quantitative PCR, multiplexed ELISA, ex vivo functional assays and western blotting. Conclusion: Collectively, these findings present the first evidence to show that gut colonization with Bacteroides fragilis rapidly induces inflammation, fibrosis and hyperplasia in the breast. In syngeneic breast cancer model, gut colonization with ETBF aggravates breast cancer progression and induces enhanced lung and liver metastasis via systemic immune modulation, cytokine synthesis and activation of pro-oncogenic pathways. Citation Format: Sheetal Parida, Sumit Siddharth, Guannan Wang, Himavanth Gatla, Shaoguang Wu, Brian Ladle, Kathleen Gabrielson, Cynthia L Sears, Dipali Sharma. Gut pathogen, Bacteroides fragilis promotes breast cancer liver and lung metastasis [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-02.

PARP inhibitors in combination with other agents are in clinical trial against cancer, but its effect on cancer stem cells (CSCs) is limited. CSCs are responsible for drug resistance, metastasis and cancer relapse due to high DNA repair capacity. Here, we present preclinical effects of Quinacrine (QC) with ABT-888, a PARP inhibitor, on highly metastatic breast cancer stem cells (mBCSCs). An increased level of Adenomatous polyposis coli (APC) was noted after treatment with ABT-888 in QC pre-treated mBCSCs cells. Increased APC physically interacts with PARP-1 and inhibits PARylation causing the non assembly of base excision repair (BER) multiprotein complex, resulting in an irreparable DNA damage and subsequent apoptosis. Knockdown of APC in mBCSCs inhibited DNA damage, increased BER and PARylation, reduces apoptosis while the over-expression of APC in BT20 (APC low expressing) cells reversed the effect. Thus, combination of QC and ABT-888 decreased mBCSCs growth by activating APC and inhibiting BER within the cells.

Cancer stem cells drive the metastatic cascade by undergoing epithelial to mesenchymal transition (EMT) and again mesenchymal to epithelial transition (MET). Using multiple breast cancer cell lines including cigarette smoke induced breast cancer cells and tumor derived primary cells from patient sample; we developed a breast cancer metastasis model and reported the existence of an adherent, distinct pre-metastatic phase, quiescent-breast cancer stem cells (Q-BCSCs) prior to attaining an EMT. SURVIVIN was found to be expressed in Q-BCSCs. Time dependant biphasic expression of SURVIVIN in Q-BCSCs reveals that Q-BCSCs is a pre-metastatic phase distinct from both epithelial and mesenchymal counterparts. SURVIVIN favours metastasis and up-regulates WNT/β-CATENIN pathway in a PI3 K/AKT-dependant manner for self-renewal. Knockdown of SURVIVIN in Q-BCSCs lost the metastatic property of cells by inhibiting invasion, EMT-MET, PI3 K/AKT/WNT cascade, and induced apoptosis. Thus, our data suggest the existence of a novel pre-metastatic phase (Q-BCSCs) before EMT and SURVIVIN acts as a marker for Quiescent-BCSCs.

Quinacrine (QC) causes apoptosis in breast cancer cells by induction of DNA damage, arrest of cells in S-phase, and by topoisomerase inhibition. Here, we show that QC-mediated apoptosis is not only due to increased DNA damage but also by compromising cell cycle checkpoints and base excision repair (BER) capacity in breast cancer cells. QC decreased CHK1, CDKs (CDC2, MDM2, CDC6), cyclins (B1, E1) and CDC25-A in a dose dependent manner. The expression of basal ATR remains unaltered but pATR (Ser-428) increased after QC treatment. A CHK1 inhibitor, SB218078, was also tested alone and in combination with QC. Like QC, SB218078 caused apoptosis by DNA damage and S-phase arrest. The combination of QC and SB218078 increased apoptosis by blocking the cell cycle in G2/M, which caused a mitotic catastrophe, and induced DNA damage at a higher level in comparison to individual compound treatments. Both drugs individually or in combination decreased the levels of replication protein A (RPA). Measurement of the expression of BER (SP- and LP-BER) proteins and direct in vivo BER activity revealed that the QC/SB218078 combination caused apoptosis in cancer cells by disrupting the induction of BER, which represents a novel means of potentially treating breast cancer.

Background: Checkpoint inhibitors (CPI) have revolutionized the field of cancer immunotherapy. However, CPIs have shown only modest efficacy in clinical trials for TNBC which may be attributed to the presence of immunosuppressive cells both myeloid-derived suppressor (MDSCs) and T regulatory (Treg) cells in the tumor microenvironment (TME). We hypothesized that transient depletion of Tregs and MDSCs along with the administration of checkpoint inhibitors would markedly reduce immunosuppressive signals to the effector arm of the immune system and thereby lead to a more robust anti-tumor response causing tumor regression and a better disease outcome. To target Treg/MDSCs cells, we have developed two diphtheria-toxin based fusion protein toxins and are currently evaluating the activity of these molecules both as monotherapies and dual therapies in combination with CPIs. Methods: To target MDSCs and TNBC tumor cells, we genetically constructed, expressed and purified a diphtheria toxin-based fusion protein, s-DAB389mIL-4 that targets the IL-4 receptor (IL-4Rα) on these cell populations. This fusion protein toxin was tested in vitro against murine TNBC 4T1 cells and in vivo in a syngeneic murine adenocarcinoma model both as a monotherapy and in combination with s-DABIL-2(V6A) which targets Tregs cells through high and intermediate affinity IL-2 receptors. Results: We found that s-DAB389mIL-4 is highly potent against 4T1 cells in vitro (IC50 ranging from 10 pM to ~3 nM), and adversely affects 4T1 cell survival, proliferation, invasion, and migratory abilities. Recombinant IL-4 and anti-IL-4 antibody block the cytotoxic action of s-DAB389mIL-4 confirming that the fusion protein's activity is mediated through the IL-4Rα. s-DAB389mIL-4 treatment in a 4T1 cell-based murine adenocarcinoma model resulted in a significant reduction of (a) tumor volume and weight, (b) MDSCs, (c) splenomegaly, (d) 4T1 metastasis to the lung and, (d) increased infiltration of IFNϒ+ effector T-cells. Sequential combination therapy with both s-DABIL-4 and s-DABIL-2(V6A) resulted in (1) reduction in both tumor volume and weight, (2) transient depletion of activated Tregs, and (3) increased infiltration of T-effector cells in the TME. NanoString analysis of RNA isolated from tumor tissues treated with these two fusion protein toxins show enrichment of gene signatures which enhance the infiltration of anti-tumor immune cells in the TME and concomitant inhibition of processes which encourage tumor growth and metastasis. Conclusions: These findings suggest that s-DABmIL-4 both as a monotherapy and in combination with s-DABIL-2(V6A) results in transient reduction of both MDSCs and Tregs. These observations suggest that these novel fusion protein toxins may serve as an effective immunotherapeutic regimen for the treatment of TNBC. Based upon these results, we are currently testing these fusion protein toxins in combination with checkpoint blockade therapy. Citation Format: Sadiya Parveen, Sumit Siddharth, Laurene Cheung, John R. Murphy, Dipali Sharma, William R. Bishai. Transient and selective depletion of MDSCs and Tregs as an effective immunotherapy against triple-negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6050.

Background and Aim: Triple-negative breast cancer (TNBC) is a very aggressive subtype of breast cancer characterized by the absence of estrogen receptor (ER), progesterone receptor (PR) and HER2 receptor expression. Mortality from TNBC is significantly higher in African American (AA) women in comparison to European American (EA) women (5-year relative survival of only 14% for AA in comparison to 36% for EA) even though the incidence rates are lower in AA women. Irrespective of stage at diagnosis, AA-TNBC is more aggressive with higher metastasis and a poorer survival than EA-TNBC; therefore, it is imperative to understand the molecular determinants that drive aggressive progression of AA-TNBC. Overall aim of this study is to decipher the alterations in the molecular circuitry underlying racial disparity in TNBC progression in AA women compared to EA women. Results: AA-TNBC cells (HCC1806 and HCC1569) exhibited increased growth and higher migration potential; higher expression of stemness factors, increased number of mammospheres and higher CD44+/CD49f+ population in comparison to EA-TNBC (Hs578t, BT549, HCC1937 and HCC1187) cells. We analyzed RNA sequencing data of multiple AA and EA TNBC cell lines for self-renewal pathways (Wnt/βCatenin, GLI1/Shh and YAP-TAZ) and observed significantly higher levels of GLI1 in AA-TNBC cell lines while no significant alterations were observed in other pathway components. Further analysis of TCGA dataset revealed a positive correlation between GLI1 and Notch1 in AA-TNBC with a negative correlation in EA-TNBC. Increased expression of components of GLI1 and Notch1 pathway (SHH, Jagged, cleaved-Notch (NICD), Hes1 and FOXM1) were noted in AA-TNBC compared to EA-TNBC cells. AA-TNBC cells showed increased nuclear localization of GLI1 and NICD as compared to EA-TNBC cells. We observed that GLI and NICD co-localize in AA-TNBC. High expression of GLI1 and Notch1 correlated with poor overall survival in TNBC patients. AA TNBC Tumor samples show higher expression of GLI1 and NICD in comparison to TNBC tumors from EA patients. Concomitant inhibition of GLI1 and Notch1 using respective small molecule inhibitors, GANT61 and DAPT, along with standard chemotherapeutic agents (Doxorubicin and carboplatin) effectively inhibited AA TNBC growth and progression in mice; growth, proliferation, migration and invasion of ex vivo tumor cells and downregulated CD44+/CD49f+ and ADLH1A1+ population in a synergistic manner. Combined treatment with GANT61+ DAPT+ Carboplatin effectively reduced stem cell frequency of AA-TNBC tumors in in vivo limiting dilution assay. Conclusions: In conclusion, these results show that AA-TNBC cells are inherently aggressive with increased growth, migration and stemness potential. We found aberrant activation of GLI and Notch pathway and a crosstalk between GLI1 and NICD whose inhibition effectively inhibits AA-TNBC and sensitizes AA-TNBC to standard chemotherapy. Citation Format: Sumit Siddharth, Anirudh Saxena, Nethaji Muniraj, Sheetal Parida, Arumugam Nagalingam, Dipali Sharma. Aberrant activation of GLI1 and Notch1 contributes to racial disparity in triple-negative breast cancer progression [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-222.

Withaferin A (WFA), a steroidal lactone, negatively regulates breast cancer growth however, its mechanisms of action remain largely elusive. We found that WFA blocks autophagy flux and lysosomal proteolytic activity in breast cancer cells. WFA increases accumulation of autophagosomes, LC3B-II-conversion, expression of autophagy-related proteins and autophagosome/lysosome fusion. Autolysosomes display the characteristics of acidic compartments in WFA-treated cells; however, the protein degradation activity of lysosomes is inhibited. Blockade of autophagic flux reduces the recycling of cellular fuels leading to insufficient substrates for tricarboxylic acid (TCA) cycle and impaired oxidative phosphorylation. WFA decreases expression and phosphorylation of LDHA, the key enzyme that catalyzes pyruvate-to-lactate conversion, reduces ATP levels and increases AMPK activation. AMPK-inhibition abrogates while AMPK-activation potentiates WFA's effect. WFA and 2-deoxyglucose combination elicits synergistic inhibition of breast cancer cells. Genetic-knockout of BECN1 and ATG7 fails to rescue cells from WFA-treatment; in contrast, addition of methyl pyruvate to supplement TCA cycle protects WFA-treated cells. Together, these results implicate that WFA is a potent lysosomal inhibitor; energetic impairment is required for WFA-induced apoptosis and growth-inhibition and combining WFA and 2DG is a promising therapeutic strategy for breast cancer.

Background: The last decade established significant contributions of microbiome to many organ-specific cancers. A few recent studies suggested the existence of distinct breast microbiota and a shift in microbial community composition in diseased breast compared to normal breast; however, their functional impact and underlying mechanisms are unknown. The present study was designed to examine the contribution of procarcinogenic bacteria in breast cancer initiation, growth, and progression. Results: Utilizing extensive data mining and metagenomic analyses, we discovered the presence of toxin-producing Bacteriode fragilis (B. fragilis) in malignant breast. B. fragilis is a procarcinogenic bacteria known for its potential to initiate and/or promote colon cancer, and its pathogenicity has been attributed to its unique toxin “Fragilysin” or “B. fragilis toxin (BFT).” Mice infected with B. fragilis exhibited a significant increase in circulating BFT and distinct morphologic alterations in mammary gland. While no changes were observed in cell growth and clonogenicity upon BFT treatment, significant increase in migration and invasion potential and decreased adhesion of MCF10A and MCF7 cells were observed. BFT-treated cells displayed acquisition of fibroblast-like appearance and increased formation of pseudopodia/microtentacles emanating from the cell membrane along with molecular markers of epithelial-to-mesenchymal transition. Decreased expression of epithelial marker E-cadherin along with elevated levels of mesenchymal markers, N-cadherin, and vimentin were observed. BFT also increased the expression and nuclear translocation of EMT-related transcription factors, Snail, Slug, and Twist. BFT-treated cells attained stem cell-like phenotype exhibiting an increased ability to form secondary and tertiary mammospheres and elevated expression of pluripotency-factors (Oct4, Nanog, and Sox2). Mechanistic studies showed that BFT induced expression and nuclear translocation of NICD (cleaved NOTCH) and βcatenin, resulting in activation of downstream targets. Inhibition of Notch1 and βcatenin using γ-secretase inhibitor and ICG001 successfully inhibited functional effects of BFT. Further, we found that BFT-pretreated MCF7 cells exhibit increased tumor growth and form multifocal tumors in mice. MCF10A-KRas cells, pretreated with BFT, also showed increased tumor progression and multifocal tumors in mice. In vivo limiting dilution assay using breast tumors from BFT-pretreated MCF7 cells exhibited a striking increase in tumor-initiating cells. Follow-up analyses of these tumors demonstrated increased migratory, invasive, and mammospheres-forming behavior, confirming that brief BFT exposure elicits long-term molecular changes. Conclusion: Collectively, these findings present the first in vitro and in vivo evidence to show that Bacteriode fragilis toxin induces EMT, invasion/migration, and stem cell-like phenotype and leads to concomitant activation of Notch and βcatenin axes. This abstract is also being presented as Poster A24. Citation Format: Sheetal Parida, Shaoguang Wu, Nethaji Muniraj, Sumit Siddharth, Arumugam Nagaligam, Christina Hum, Panagiotis Mistriotis, Konstantinos Konstantopoulos, Cynthia Sears, Dipali Sharma. Bacteroides fragilis: A potential pathogen orchestrating EMT and stemness in breast epithelial cells via concomitant activation of Notch and βcatenin axes [abstract]. In: Proceedings of the AACR Special Conference on the Microbiome, Viruses, and Cancer; 2020 Feb 21-24; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2020;80(8 Suppl):Abstract nr PR06.

The last decade established significant contributions of the microbiome to many organ-specific cancers. A few recent studies suggested the existence of distinct breast microbiota and a shift in microbial community composition in diseased breast compared to normal breast; however, their functional impact and underlying mechanisms are unknown. The present study examines the contribution of procarcinogenic bacteria in breast cancer initiation, growth, and progression. Extensive data mining and metagenomic analyses of existing datasets revealed the presence of toxin-producing Bacteroides fragilis in malignant breast. B. fragilis is a procarcinogenic bacteria known for its potential to initiate and promote colon cancer; its pathogenicity has been attributed to its unique toxin “BFT.” B. fragilis-infected mice exhibited a significant increase in circulating BFT and distinct morphologic alterations in mammary gland. In vitro, upon treatment with BFT, no changes were observed in cell growth and clonogenicity. However, significant increase in migration and invasion potential and decreased adhesion of MCF10A and MCF7 cells were observed. BFT-treated cells displayed acquisition of fibroblast-like appearance and increased formation of pseudopodia/microtentacles emanating from the cell membrane along with molecular markers of epithelial-to-mesenchymal transition. Decreased expression of epithelial marker, E-cadherin, along with elevated levels of mesenchymal markers, N-cadherin and vimentin, were observed. BFT also increased the expression of EMT-related transcription factors, Snail, Slug and Twist. BFT-treated cells attained stem cell-like phenotype, exhibiting an increased ability to form secondary and tertiary mammospheres and elevated expression of pluripotency-factors (Oct4, Nanog, and Sox2). Mechanistic studies showed that BFT induced expression and nuclear translocation of NICD (cleaved NOTCH) and β-catenin resulting in activation of downstream targets. Inhibition of Notch1 and β-catenin using γ-secretase and β-catenin inhibitors successfully inhibited functional effects of BFT. Mammary gland implantation and in vivo limiting dilution assays were utilized to corroborate the in vitro findings. BFT-pretreated MCF7 cells exhibit increased tumor growth and form multifocal tumors in mice. MCF10A-KRas cells, pretreated with BFT, also showed increased tumor progression and multifocal tumors in mice. In vivo limiting dilution assay using breast tumors from BFT-pretreated MCF7 cells exhibited a striking increase in tumor-initiating cells. Follow-up analyses of these tumors demonstrated increased migratory, invasive, and mammospheres-forming behavior, confirming that brief BFT exposure elicits long-term molecular changes. Collectively, these findings present the first in vitro and in vivo evidence to show that Bacteroides fragilis toxin induces EMT, invasion/migration and stem cell-like phenotype and leads to concomitant activation of Notch and β-catenin axes. This abstract is also being presented as Poster A66. Citation Format: Sheetal Parida, Shaoguang Wu, Nethaji Muniraj, Sumit Siddharth, Arumugam Nagaligam, Cynthia L. Sears, Dipali Sharma. Bacteroides fragilis toxin induces epithelial-to-mesenchymal transition and stem-like phenotype in breast epithelial cells and concomitantly activates Notch1 and βcatenin axes [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR02.

Background: Checkpoint inhibitors (CPI) have revolutionized the field of cancer immunotherapy. However, CPIs have shown only modest efficacy in the clinical trials for TNBC, which could be attributed to the presence of immunosuppressive cells—both myeloid-derived suppressor (MDSCs) and T regulatory (Treg) cells—in the tumor microenvironment (TME). We hypothesize that transient depletion of Tregs and MDSCs along with the administration of checkpoint inhibitors would markedly reduce immunosuppressive signals to the effector arm of the immune system and thereby lead to a more robust antitumor response, causing tumor regression and a better disease outcome. To target Treg/MDSCs cells, we have developed two diphtheria-toxin based fusion protein toxins and are currently evaluating the activity of these molecules both as monotherapies and in combination with CPIs. Methods: To target MDSCs and TNBC tumor cells, we genetically constructed, expressed, and purified a diphtheria toxin-based fusion protein, s-DAB389mIL-4 that targets the IL-4 receptor (IL-4R) on these cell populations. This fusion protein toxin was tested in vitro against murine TNBC 4T1 cells and in vivo in a syngeneic murine adenocarcinoma model both as a monotherapy and in combination with s-DABIL-2(V6A), which targets Tregs cells through high- and intermediate-affinity IL-2 receptors. Results: We found that s-DAB389mIL-4 is highly potent against 4T1 cells in vitro (IC50 ranging from 10 pM to ~3 nM), and adversely affects 4T1 cell survival, proliferation, invasion, and migratory abilities. Recombinant IL-4 and anti-IL-4 antibody block the cytotoxic action of s-DAB389mIL-4, confirming that the fusion protein’s activity is mediated through the IL-4R. s-DAB389mIL-4 treatment in a 4T1 cell-based murine adenocarcinoma model results in a significant reduction of (a) tumor volume, (b) MDSCs, and (c) 4T1 metastasis to the lung. Sequential combination therapy with both s-DABIL-4 and s-DABIL-2(V6A) resulted in (1) reduction in both tumor volume and weight, (2) transient depletion of activated Tregs, and (3) increased infiltration of T-effector cells in the TME. NanoString analysis of RNA isolated from tumor tissues treated with these two fusion protein toxins showed enrichment of those gene signatures, which enhance the infiltration of antitumor immune cells in the TME, and concomitant inhibition of those processes, which lead to tumor growth and metastasis. Conclusions: These findings suggest that s-DABmIL-4 both as a monotherapy and in combination with s-DABIL-2(V6A) results in transient reduction of both MDSCs and Treg. These observations suggest that these novel fusion protein toxins may serve as an effective immunotherapeutic regimen for the treatment of TNBC. Based upon these results, we are currently testing these fusion protein toxins in combination with checkpoint blockade therapy. Citation Format: Sadiya Parveen, Sumit Siddharth, Laurene Cheung, John R. Murphy, Dipali Sharma, William R. Bishai. Transient depletion of MDSCs and Tregs as an effective immunotherapy against triple-negative breast cancer (TNBC) [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A54.

Triple negative breast cancer (TNBC) is a molecularly heterogeneous disease whose incidence is disproportionately higher in African American (AA) women compared to European American (EA) women. Earlier onset, more advanced stage at diagnosis, and aggressive tumor phenotype are some of the characteristic features of TNBC in women with African ethnicity in comparison to EA women, denoting one of the most significant examples of racial disparity in oncology. It is still contentious whether health disparities result in aggressive behavior of TNBC in AA women or it is indeed a molecularly distinct disease. Given the “gaps-in-knowledge” surrounding racial disparity in TNBC, this review discusses various socioeconomic factors and the genetic predispositions contributing to poor prognosis of TNBC in AA women. While socioeconomic factors may contribute to poorer survival, multiple preclinical and clinical studies suggest inherent genetic risk factors and aberrant activation of oncogenic pathways in AA TNBC. Additionally, AA women are more likely to be obese and obesity is known to drive a molecular circuitry resulting in aggressive tumor progression indicating a potential obesity-TNBC axis at work in AA women. Given the multifactorial nature of AA TNBC, a transdisciplinary approach may help bridge the disparity that exists between AA and EA TNBC.

a b s t r a c t Repurposing is the novel means of drug discovery in modern science due to its affordability, safety and availability. Here, we systematically discussed the efficacy and mode of action of multiple bioactive, synthetic compounds and their potential derivatives which are used to treat/prevent malaria and cancer. We have also discussed the detailed molecular pathway involved in anti-cancer potentiality of an antimalarial drug and vice versa. Although the causative agents, pathophysiology and manifestation of both the diseases are different but special emphasis has been given on similar pathways governing disease manifestation and the drugs which act through deregulating those pathways. Finally, a future direction has been speculated to combat these two diseases by a single agent developed using nanotechnology. Extended combination and new formulation of existing drugs for one disease may lead to the discovery of drug for other diseases like an arrow for two sparrows.

Combination therapy using two or more small molecule inhibitors of aberrant signaling cascade in aggressive breast cancers is a promising therapeutic strategy over traditional monotherapeutic approaches. Here, we have studied the synergistic mechanism of resveratrol and curcumin induced apoptosis using in vitro (cigarette smoke condensate mediated transformed breast epithelial cell, MCF-10A-Tr) and in vivo (tumor xenograft mice) model system. Resveratrol exposure increased the intracellular uptake of curcumin in a dose dependent manner and caused apoptosis in MCF-10A-Tr cells. Approximately, ten fold lower IC50 value was noted in cells treated with the combination of resveratrol (3μM) and curcumin (3μM) in comparison to 30μM of resveratrol or curcumin alone. Resveratrol+curcumin combination caused apoptosis by increasing Bax/Bcl-xL ratio, Cytochrome C release, cleaved product of PARP and caspase 3 in cells. Interestingly, this combination unaltered the protein expressions of WNT-TCF and Notch signaling components, β-catenin and cleaved notch-1 val1744, respectively. Furthermore, the combination also significantly decreased the intermediates of Hedgehog-Gli cascade including SMO, SHH, Gli-1, c-MYC, Cyclin-D1, etc. and increased the level of p21(Waf/Cip1) in vitro and in vivo. A significant reduction of Gli- promoter activity was noted in combinational drug treated cells in comparison to individual drug treatment. Un-alteration of the expressions of the above proteins and Gli1 promoter activity in p21(Waf/Cip1) knockout cells suggests this combination caused apoptosis through p21(Waf/Cip1). Thus, our findings revealed resveratrol and curcumin synergistically caused apoptosis in cigarette smoke induced breast cancer cells through p2(Waf/Cip1) mediated inhibition of Hedgehog-Gli cascade.

5-Fluorouracil is the most commonly used drug for the treatment of colon cancer, yet clinical resistance to this drug is frequently observed in patients making this drug ineffective. Thus, identification of gene responsible for 5-FU resistance is of utmost importance. Cellular cytotoxicity and expressions of different protein markers in colon cancer cells were measured by MTT assay and Western blotting, respectively. Cell cycle regulation, migration and colony formation ability were measured by FACS, wound-healing assay and clonogenic assay, respectively. Increased NECTIN-4 expression was observed in 5-FU-resistant (5-FU-R) and 5-FU-exposed HCT-116 cells. A significant increase in the cell proliferation, migration, colony formation, and resistant to 5-FU were noted in 5-FU-R cells, but reverse was observed after silencing of NECTIN-4. Apoptosis caused by 5-FU in 5-FU-R cells after NECTIN-4 knockdown indicates that NECTIN-4 is responsible for 5-FU resistance. Cell survival proteins were upregulated in 5-FU-R and NECTIN-4-over-expressed cells and downregulated in NECTIN-4 knockdown or LY294002-pretreated 5-FU-R cells. Drug combination of BCNU + Resveratrol decreased the cell survival and NECTIN-4 expressions in 5-FU-R cells and NECTIN-4-over-expressed cells. Our data suggest that NECTIN-4 is responsible for 5-FU resistance and BCNU + Resveratrol combination can be used to increase the 5-FU sensitivity.

Cancer stem cells secrete diffusible factors into the microenvironment that bind to specific endothelial cell receptors and initiate an angiogenesis cascade. Tumor-induced angiogenesis is an important parameter of tumorigenesis and is critical for tumor growth and metastasis. A pvrl-4 encoded gene, NECTIN-4, has potential roles in cancer cell growth and aggressiveness, and it is only expressed in cancer cells. There is evidence that nectin-4 plays a role in tumorigenesis, but the function of nectin-4 in tumor angiogenesis has lacked thorough evidence of mechanism. Using highly metastatic breast cancer cells and human umbilical vein endothelial cells (HUVECs), we have developed an excellent angiogenesis model and systematically studied the contribution of nectin-4 to angiogenesis. We also provide in-depth in ovo, in vivo and in vivo evidence that nectin-4 causes angiogenesis. Following hypoxia, metastatic breast cancer stem cells (mBCSCs) driven ADAM-17 expression causes the shedding of the ecto-domain of nectin-4 into the microenvironment, which physically interacts with integrin-β4 specifically on endothelial cells. This interaction promotes angiogenesis via the Src, PI3K, AKT, iNOS pathway and not by Phospho-Erk or NF-κβ pathways. In vitro, in ovo and in vivo induction and abrogation of an angiogenesis cascade in the presence and absence of the nectin-4 ecto-domain, respectively, confirms its role in angiogenesis. Thus, disrupting the interaction between nectin-4 ecto-domain and integrin-β4 may provide a means of targeting mBCSC-induced angiogenesis.

Death receptor 5 (DR5) is an important target for development of anticancer agents against triple-negative breast cancer (TNBC). Recently, we reported the molecular level details for the modulation of TRAIL-DR5 axis by quinacrine (QC) in breast cancer cells. In this work, the DR5 mediated anticancer potential of topoisomerase inhibitor etoposide (ET) and doxorubicin (DOX) against TNBC has been evaluated. ET and DOX enhanced the DR5 expression in TNBC cells, whereas non-topoisomerase inhibitors pifithrin-α (PIF) and dexamethasone (DEX) failed to do so. In the TRAIL pre-treated cells, ET and DOX induced higher apoptosis, indicating their synergistic effect with TRAIL. The molecular docking and molecular dynamics studies showed their ability to stabilize the TRAIL-DR5 complex, whereas PIF and DEX failed to do so. The binding energy for TRAIL-DR5 complexation in the ternary complexes containing ET (−111.08 kcal/mol) and DOX (−76.35 kcal/mol) were higher than reported binding energy of binary complex (−53.70 kcal/mol). The in silico and in vitro mutational studies highlighted the importance of DR5 residue SerB68 in mediating the receptor-drug interaction. ET and DOX failed to enhance apoptosis in DR5 knockdown (DR5-KD) cells. On the other hand, TRAIL+ET exhibited induction of DR5 and subsequent apoptosis in WT-DR5 overexpressed DR5-KD cells, by modulating the mitochondrial intrinsic apoptosis cascade. An induction of apoptosis and DR5 expression was noticed in xenograft mice and in TNBC patient-derived metastatic cells after TRAIL+ET treatment. Thus, data suggests ET and DOX act as DR5 agonistic ligands and enhance the cellular apoptosis in TNBC.

Presences of cancer stem cells (CSCs) in a bulk of cancer cells are responsible for tumor relapse, metastasis and drug resistance in oral cancer. Due to high drug efflux, DNA repair and self-renewable capacity of CSCs, the conventional chemotherapeutic agents are unable to kill the CSCs. CSCs utilizes Hedgehog (HH-GLI), WNT-β catenin signalling for its growth and development. GSK3β negatively regulates both the pathways in CSCs. Here, we have shown that a nano-formulated bioactive small molecule inhibitor Quinacrine (NQC) caused apoptosis in oral cancer stem cells (OCSCs; isolated from different oral cancer cells and oral cancer patient derived primary cells) by down regulating WNT-β catenin and HH-GLI components through activation of GSK3β. NQC activates GSK3β in transcriptional and translational level and reduces β catenin and GLI1 as well as downstream target gene of both the pathways Cyclin D1, C-Myc. The transcription factor activity of both the pathways was also reduced by NQC treatment. GSK3β, β catenin and GLI1 interacts with each other and NQC disrupts the co-localization and interaction between β catenin and GLI1 in OCSCs in a dose dependent manner through activation of GSK3β. Thus, data suggest NQC caused OCSCs death by disrupting the crosstalk between β catenin and GLI1 by activation of GSK3β.

Nectin-4 is well known as a junction protein. Recent reports have implicated it in cancer, but there has been little exploration of its functional significance in metastasis and cancer stem cells. Here, using the breast cancer metastasis model system, we report Nectin-4 is a marker for breast cancer stem cells (BCSCs) and provide experimental evidence suggesting that it utilizes WNT/β-Catenin signaling via Pi3k/Akt axis for self renewal of BCSCs. In vitro, in vivo, ex vivo and clinical pathological data showed upregulated Nectin-4 in breast cancer metastasis and WNT/β-Catenin signaling. Nectin-4 depletion inhibited EMT, metastasis, invasion, and the WNT/β-Catenin pathway; conversely, Nectin-4 overexpression in null cells upregulated EMT and metastasis and also induced WNT/β-Catenin signaling via Pi3k/Akt axis, which in turn, controls cancer stem cell proliferation. Induced Nectin-4 was observed in breast tumor patient samples and in breast tumor metastases to axillary lymph nodes, which indicated that Nectin-4 is not only a BCSC marker but also a breast cancer metastasis marker. The current study provides clear evidence that Nectin-4 is a BCSC marker and is responsible for breast cancer metastasis.

Background: Last decade established significant contributions of microbiome to organ specific cancers. A few recent studies suggested the existence of distinct breast microbiota and a shift in microbial community composition in diseased breast compared to normal breast. However, their functional impact and underlying mechanisms are unknown. Present study was designed to examine the contribution of pro-carcinogenic bacteria in breast cancer initiation, growth and progression. Results: Utilizing extensive data mining and metagenomic analyses, we discovered presence of toxin producing Bacteriodes fragilis in malignant breast. B. fragilis is known for its potential to initiate and/or promote colon cancer. Its pathogenicity has been attributed to its unique toxin BFT. Mice infected with B. fragilis exhibited significant circulating BFT and distinct morphological alterations in mammary gland. While no changes were observed in cell growth and clonogenicity upon BFT treatment, significant increase in migration and invasion potential and decreased adhesion of MCF10A and MCF7 cells were observed. BFT-treated cells displayed acquisition of fibroblast-like appearance and increased formation of pseudopodia/microtentacles emanating from the cell membrane along with molecular markers of epithelial-to-mesenchymal transition. Decreased expression of epithelial marker, E-cadherin along with elevated levels of mesenchymal markers, N-cadherin and vimentin were observed. BFT also increased expression of EMT-related transcription factors, Snail, Slug and Twist. BFT-treated cells attained stem cell-like phenotype exhibiting an increased ability to form secondary and tertiary mammospheres and elevated expression of pluripotency-factors (Oct4, Nanog and Sox2). Mechanistic studies showed BFT induced expression and nuclear translocation of cleaved NOTCH1 and βcatenin resulting in activation of downstream targets. Inhibition of Notch1 and βcatenin using γ-secretase inhibitor and ICG001 successfully inhibited functional effects of BFT. Further, BFT-pretreated MCF7 cells exhibit increased tumor growth and form multifocal tumors in mice. MCF10A-KRas cells, pretreated with BFT, also showed increased tumor progression and multifocal tumors in mice. In vivo limiting dilution assay using breast tumors from BFT-pretreated MCF7 cells exhibited a striking increase in tumor-initiating cells. Follow-up analyses of these tumors demonstrated increased migratory, invasive, and mammospheres-forming behavior confirming that brief BFT exposure elicits long-term molecular changes. Conclusion: Collectively, these findings present the first in vitro and in vivo evidence to show that Bacteriodes fragilis Toxin induces EMT, invasion/migration and stem cell-like phenotype and leads to concomitant activation of Notch and βcatenin axes. Citation Format: Sheetal Parida, Shaoguang Wu, Nethaji Muniraj, Sumit Siddharth, Arumugam Nagaligam, Christina Hum, Panagiotis Mistriotis, Konstantinos Konstantopoulos, Cynthia L. Sears, Dipali Sharma. Bacteroides fragilis: A potential pathogen orchestrating EMT and stemness in breast epithelial cells via concomitant activation of Notch and βcatenin axes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2834.

Background and Aim: Obese state is associated with increased breast cancer growth, metastatic progression, poor overall survival and poor response to therapy. Analyses of clinical trial population showed that obesity associates with poor outcomes in patients with estrogen receptor (ER)-positive breast cancer treated with standard chemohormonal therapy. A characteristic feature of obesity is hyperleptinemia (high leptin levels) which mediates the biological effects of obesity. In this study, we seek to decipher the underlying molecular mechanisms by which obesity/hyperleptinemia reduces the efficacy of tamoxifen. Results: Obese mice models exhibiting hyperleptinemia supported larger tumors and responded poorly to tamoxifen therapy. Kaplan meier analyses showed that high leptin and high leptin receptor levels associates with poor prognosis in ER positive breast cancer. Indeed, leptin treatment abrogated tamoxifen-mediated inhibition of clonogenicity, and soft-agar colony formation. Leptin treatment increased breast tumor growth and significantly reduced the efficacy of tamoxifen. At the molecular level, leptin induced nuclear translocation of pER (ser 118 and ser 167) and increased the expression of ER-responsive genes while reducing tamoxifen-mediated gene repression. Tamoxifen is known to recruit corepressors to ER-responsive genes. ChIP analysis revealed that tamoxifen-induced recruitment of NCoR, SMRT and Mi2 corepressors was abrogated in the presence of leptin. We found that Med1, anchor subunit of human mediator complex, interacts with various key effector molecules of leptin-signaling network and potentially associates with 48 (out of 75) obesity-signature genes indicating its functional importance. Further, we found that leptin upregulated Med1 via decreasing miR-205 and increased its functional activation via phosphorylation, nuclear translocation and promoter-recruitment. Med1 phosphorylation was mediated by activation of Her2 and EGFR in response to leptin. It is important to note that silencing of Med1 abrogated the negative effects of leptin on tamoxifen efficacy. Bioactive strategies using honokiol or adiponectin treatment effectively inhibited leptin-induced Med1 expression and increased the efficacy of tamoxifen even in the presence of leptin. Conclusion: In conclusion, these studies show the molecular mechanisms by which obese state may contribute to poor response to endocrine therapy. We show that activation of Med1 via leptin-induced EGFR and Her2 kinases act as the key node mediating the negative effects of leptin on tamoxifen efficacy. A combination regimen of honokiol and adiponectin with tamoxifen may increase its efficacy in obese state. Citation Format: Arumugam Nagalingam, Nethaji Muniraj, Sumit Siddharth, Dimiter. B Avtanski, Sheetal Parida, Panjamurthy Kuppusamy, Balázs Győrffy, Neeraj Saxena, Dipali Sharma. Hyperleptinemia in obese state renders luminal breast cancers refractory to tamoxifen coordinating a crosstalk between Med1, miR205 and Erb B kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 310.

Background and Aim: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer characterized by the absence of estrogen receptor, progesterone receptor and HER2 expression. Mortality from TNBC is significantly higher in African American (AA) women compared to European American (EA) women (5-year relative survival of 14% for AA compared to 36% for EA). Irrespective of stage at diagnosis, AA-TNBC is aggressive with higher metastasis and poorer survival than EA-TNBC; hence, it is imperative to understand the molecular determinants that drive aggressive progression of AA-TNBC. Overall this study aims to decipher the alterations in the molecular circuitry underlying racial disparity in TNBC progression. Results: AA-TNBC cells (HCC1806 and HCC1569) exhibited increased growth and higher migration potential in comparison to EA-TNBC (Hs578t, BT549, HCC1937 and HCC1187) cells. AA-TNBC cells also exhibited higher expression of stemness factors, increased number of mammospheres and higher CD44+/CD49f+ population. To decipher the molecular mechanism underlying these functional differences, we analyzed RNA sequencing data of multiple AA and EA TNBC cell lines for self-renewal pathways and observed significantly higher levels of GLI1 in AA-TNBC cell lines while no significant alterations were observed in other pathway components. Further analysis of TCGA dataset revealed a positive correlation between GLI1 and Notch1 in AA-TNBC (Pearson coefficient = 0.308303) with a negative correlation in EA-TNBC (Pearson coefficient = -0.06695). Immunoblot analyses showed increased expression of components of GLI1 and Notch1 pathway (SHH, Jagged, NICD, Hes1 and FOXM1) in AA-TNBC compared to EA-TNBC cells. AA-TNBC cells showed increased nuclear localization of GLI1 and NICD as compared to EA-TNBC cells. We observed that GLI and NICD co-localize in AA-TNBC cells and their interaction was confirmed using co-immunoprecipitation assays. High expression of GLI1 and Notch1 correlated with poor overall survival in TNBC patients. Concomitant inhibition of GLI1 and Notch1 using respective small molecule inhibitors, GANT61 and DAPT, along with standard chemotherapeutic agents (Doxorubicin and Carboplatin) effectively inhibited AA TNBC growth and progression in mice; proliferation, migration and invasion of ex vivo tumor cells and downregulated CD44+/CD49f+ and ALDH1+ population in a synergistic manner. Combined treatment with GANT61+ DAPT+ Carboplatin effectively reduced stem cell frequency of AA-TNBC tumors in in vivo limiting dilution assay. Conclusions: In conclusion, these results show that AA-TNBC cells are inherently aggressive with increased growth, migration and stemness potential. We found aberrant activation of GLI and Notch pathway and a crosstalk between GLI1 and NICD whose inhibition effectively inhibits AA-TNBC and sensitizes AA-TNBC to standard chemotherapy. Citation Format: Sumit Siddharth, Nethaji Muniraj, Sheetal Parida, Arumugam Nagalingam, Dipali Sharma. Aberrant activation of gli1 and notch1 contributes to racial disparity in triple negative breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3468.

Background and Aim: Honokiol, a natural phenolic compound isolated from an extract of seed cones from Magnolia grandiflora, is widely known for its therapeutic potential as an antioxidant, anti-inflammatory, antithrombosis, and anti-depressant agent. Our recent studies show that honokiol impedes breast carcinogenesis. Cancer cells undergo cytoprotective autophagy and evade chemotherapy therefore many clinical trials are investigating the efficacy of autophagy inhibition in combination with chemotherapy. In the present study, we investigated the involvement of autophagic process in honokiol-mediated functional networks in breast cancer and explored the efficacy of combination regimens involving honokiol and chloroquine. Methods: Autophagy studies were conducted utilizing immunoblot, RT-PCR, immunofluorescence analyses, confocal imaging and transmission electron microscopy for autophagy markers. Autophagic flux was analyzed using a plasmid tfLC3B and acridine orange staining. The fusion of autophagosome and lysosome was examined by using GFP-LC3/LysoTracker-red. Functional impact of autophagic process was evaluated using genetic knockout (KO) of AGT7 and BECN1 in MCF7 cells as well as combined treatment with autophagy inhibitors (3-MA, Baf-1 and CQ) and honokiol. Alterations in ATP levels were measured by ATPliteTM luminescence assay. In vivo studies using mammary gland implantation of cancer cells in NOD-SCID mice were conducted to evaluate the efficacy of combination regimen of Honokiol and Chloroquine. Results: We found that Honokiol induces autophagy flux, increases accumulation of autophagosomes and elevates LC3B-II-conversion. Utilizing tandem-mCherry-GFP-LC3B assay and LysoTracker Red-staining, we observed that honokiol increases the autophagosome/lysosome fusion in breast cancer cells. We found that Honokiol induces autophagic response in a STK11-dependent manner as STK11-null breast cancer cells do not exhibit LC3B-II-puncta in response to Honokiol. Next, we explored the functional impact of autophagy in Honokiol mediated breast cancer inhibition and found that inhibiting autophagosome formation, abrogating autophagosome-lysosome fusion or genetic-knockout of BECN1/Beclin1 and ATG7 effectively increases the efficacy of Honokiol. These results clearly showed the cytoprotective nature of Honokiol-mediated autophagy and put forth the notion that a combined strategy of autophagy-inhibition with Honokiol would be more effective. Indeed, our in vivostudies showed that a combined treatment with Honokiol and Chloroquine can effectively inhibit primary tumor growth as well as metastatic progression. Conclusion: Together, these results implicate that honokiol is a potent inducer of cytoprotective autophagy and a combined treatment of honokiol and chloroquine is a promising therapeutic strategy for breast cancer. Citation Format: Nethaji Muniraj, Marey Shriver, Arumugam Nagalingam, Sumit Siddharth, Sheetal Parida, Neeraj K Saxena, Dipali Sharma. Induction of STK11-dependent cytoprotective autophagy in breast cancer cells upon Honokiol treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4271.

Adiponectin is one of the most important adipocytokines secreted by adipocytes and is called a “guardian angel adipocytokine” owing to its unique biological functions. Adiponectin inversely correlates with body fat mass and visceral adiposity. Identified independently by four different research groups, adiponectin has multiple names; Acrp30, apM1, GBP28, and AdipoQ. Adiponectin mediates its biological functions via three known receptors, AdipoR1, AdipoR2, and T-cadherin, which are distributed throughout the body. Biological functions of adiponectin are multifold ranging from anti-diabetic, anti-atherogenic, anti-inflammatory to anti-cancer. Lower adiponectin levels have been associated with metabolic syndrome, type 2 diabetes, insulin resistance, cardiovascular diseases, and hypertension. A plethora of experimental evidence supports the role of obesity and increased adiposity in multiple cancers including breast, liver, pancreatic, prostrate, ovarian, and colorectal cancers. Obesity mediates its effect on cancer progression via dysregulation of adipocytokines including increased production of oncogenic adipokine leptin along with decreased production of adiponectin. Multiple studies have shown the protective role of adiponectin in obesity-associated diseases and cancer. Adiponectin modulates multiple signaling pathways to exert its physiological and protective functions. Many studies over the years have shown the beneficial effect of adiponectin in cancer regression and put forth various innovative ways to increase adiponectin levels.

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related mortality, and despite recent advances in early diagnosis and therapeutics, HCC related morbidity and mortality rate continue to rise. Clearly, it is imperative to develop novel effective therapies for HCC to improve long-term survival of HCC patients. We found that Withaferin A (WFA), a bioactive compound derived from Withania somnifera, is an effective agent for HCC inhibition. Interestingly, we observed that in addition to inducing apoptotic cell death, WFA also induces autophagy in HCC cells. Utilizing mRFP-EGFP-LC3B, LC3B-GFP/Lysotracker and LC3B-GFP/Rab7-RFP, we show that WFA induces autophagosomes-lysosomes fusion. WFA-induced autolysosomes exhibit intact protein degradation activity as evident with cathepsin-D activation and DQ-BSA assays. Importantly, we present that inhibiting WFA-induced autophagy either by blocking autophagosome-formation or by elevating lysosomal pH (Chloroquine and Bafilomycin) enhances WFA-induced growth-inhibition and apoptosis, indicating the presence of cytoprotective autophagy. Indeed, WFA and CQ combination shows synergism and higher efficacy in comparison to either monotherapy. Collectively, we reveal that the efficacy of WFA is somewhat diminished by the concomitant induction of cytoprotective autophagy which can be successfully conquered by cotreatment with CQ, and we pave the way for development of a novel combination therapeutic strategy for HCC.