Abstract 227: Manipulation of hypoxia-NFκB pathway to target breast cancer stem cells.

The mechanical and chemical properties of the cellular microenvironment can influence the phenotype of cancer cells. The hypoxic conditions within solid breast tumors can contribute to invasiveness and the formation of breast cancer stem cells (CSCs). Integrin-linked kinase (ILK) is a critical mediator for mechanotransduction, a process that converts exogenous mechanical stimuli into biochemical signals. Yet, it is unclear how the physical properties of the tumor microenvironment, including matrix stiffness and hypoxia, integrate to direct the formation of the breast CSC niche. Using an innovative engineered culture model, we found that stiff and hypoxic microenvironments activate integrin signaling and a stem-like gene signature in breast cancer cells, suggesting that integrin signaling, matrix mechanics and oxygen tension cooperatively promote the formation of breast CSCs. Stiff and hypoxic microenvironments promote integrin signaling and the expression of CSC markers including CD44 and Nanog. Knocking down ILK reverts the CSC phenotype of invasive breast cancer cells on stiff matrix, and abrogates their ability to form mammospheres and colonies in soft agar. In contrast, ectopic expression of ILK enhances CSC properties. ILK promotes integrin signaling and the CSC phenotype through the PI3K/Akt pathway. Stiff microenvironments promote tumor formation and metastasis in ovo. ILK depletion significantly abrogates the tumorigenenic and metastatic potential of invasive breast cancer cells in ovo. Importantly, we found that breast cancer cells expressing ILK and the CSC marker CD44 were only present in the regions of tumors predicted to be stiff in breast cancer patient samples. Our data suggest that ILK act as an essential mechanosensor that regulates breast CSC phenotype in response to matrix mechanics and oxygen tension to regulate the formation of breast CSCs. Citation Format: Mei-Fong Pang, Melody Stallings-Mann, Michael J. Siedlik, Victor D. Varner, Siyang Han, Derek C. Radisky, Celeste M. Nelson. Tissue stiffness and hypoxia regulate breast cancer stem cells through ILK. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4395.

Sulforaphane, a Dietary Component of Broccoli/Broccoli Sprouts, Inhibits Breast Cancer Stem Cells The existence of cancer stem cells (CSCs) in breast cancer has profound implications for cancer prevention. The purpose of this study was to evaluate sulforaphane, a naturally occurring dietary compound from broccoli/broccoli sprouts, for its efficacy to inhibit breast CSCs as well as to elucidate the potential mechanisms of its effect. Aldefluor and mammosphere formation assays were used to assess the effect of sulforaphane on breast CSCs in vitro. A NOD/SCID xenograft model was employed to determine the efficacy of sulforaphane in targeting breast CSCs in vivo. The potential mechanism of sulforaphane was investigated utilizing Western blotting and β-catenin reporter assays. The results showed that sulforaphane (1∼5 µM) decreased aldehyde dehydrogenase (ALDH)-positive cell population by 65%∼80% in human breast cancer cell line (P < 0.01), and reduced the size and number of primary mammospheres by 8∼125-fold and 50%∼75% (P < 0.01), respectively. Daily injection with 50 mg/kg sulforaphane for two weeks reduced ALDH-positive cells by more than 50% in tumors (P = 0.003). Furthermore, sulforaphane eliminated breast CSCs in vivo, thereby abrogating tumor re-growth in secondary mice (P < 0.01). Western blotting showed that sulforaphane down-regulated the Wnt/β-catenin self-renewal pathway. These data suggest that sulforaphane targets breast CSCs through the down-regulation of the Wnt/β-catenin pathway as a potential mechanism. These findings provide a scientific rationale for use of sulforaphane and potentially broccoli/broccoli sprout dietary interventions for breast cancer chemoprevention. 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 4271.

Background: Chemoresistance is the major obstacle for the success of cancer treatment. The advanced breast cancer (BC) is commonly pan-resistant to different anticancer drugs. BC contains a small population of cancer stem cells (CSCs). CSCs express stem cell markers (CD24low/CD44high, ALDH+, CD133+) and are resistant to a wide range of anticancer drugs. Therefore, CSCs may be responsible for the pan-chemoresistance in advanced BC. We have recently developed several acquired chemoresistant BC cell lines. In this study, we investigated CSC traits in these cell lines and examined the cross-resistance of these cell lines to a panel of anticancer drugs. Disulfiram (DS), an antialcoholism drug, shows high cytotoxicity in several different types of cancer cell lines in vitro. It also targets CSCs and reverses pan-chemoresistance. However, the clinical application of DS in cancer treatment is limited by its very short half-life in the bloodstream. In order to increase the bio-stability of DS in the bloodstream, we recently developed a pluronic micelle-encapsulated DS (PM-DS). In this study, we examined the cytotoxicity of a newly developed (PM-DS) in the resistant cell lines. The effect of PM-DS on CSC traits in the resistant BC cell lines was also examined. Cell lines and Methods: Cell lines: MDA-MB-231, MCF7 and T47D and acquired resistant BC cell lines [gemcitabine (MDA-MB-231GEM100), 5-fluouracil (MCF7FU10) and doxorubicin (T47DDOX100nM)]; Methodologies: MTT assay, Western blot, CSC markers detection (ALDH, CD24low/CD44high, CD133), embryonic stem cell markers detection (Nanog, Sox2, Oct 4). Results: The resistant cell lines are highly cross-resistant to 6 conventional anticancer drugs e.g doxorubicin, gemcitabine, docetaxel, cisplatin, 5-fluouracil and vincristine. In comparison with the parental cells, the resistant cell lines have significantly longer doubling time. Significantly higher population of resistant cells expresses high levels of CSC markers e.g. ALDH+, CD24low/CD44high, and CD133+ and embryonic stem cell markers (OCT4, Sox2 and Nanog). DS was successfully encapsulated in pluronic micelles with a high entrapment efficiency of about 90% and our in vitro release studies showed that DS was slowly released from the micelles. Our formulation PM-DS has a relatively small size of about 200nm with high drug loading content (90μgDS/1mgPM). PM-DS was highly toxic to chemoresistant cell lines. It also targeted CSC population and enhanced cytotoxicity of anticancer drugs in the resistant cell lines. Conclusions: The acquired resistant BC cell lines are highly cross-resistant to 6 different kinds of anticancer drugs. CSCs may be responsible for the cross-chemoresistance. PM-DS targets CSC population and reverses the acquired pan-chemoresistance in acquired resistant BC cell lines. Key words: Disulfiram, micelles, breast cancer, stem-like cells. Citation Format: EREBI P. TAWARI, Peng Liu, Zhipeng Wang, Vinodh Kannappan, Christopher Mcconville, Angel Armesilla, John Darling, Juan Irache, Krassimira Yoncheva, Weiguang Wang, Petar Petrov. Pluronic micelle-encapsulated Disulfiram targets cancer stem-like cells and reverses pan-resistance in acquired resistant breast cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4067. doi:10.1158/1538-7445.AM2015-4067

Background: This study aims to repurpose disulfiram (DS), a drug used to treat alcohol dependence, into an effective treatment for non-small cell lung cancer (NSCLC). NSCLC is the leading cause of cancer related death worldwide because of early metastasis and chemoresistance. Cancer stem cells (CSCs) play a key role in resistance and metastasis. Our previous studies indicate that tumor hypoxia induced activation of the nuclear factor-κB (NF-κB) pathway, a pivotal regulator of CSCs. Therefore, development of an NF-κB and CSCs targeting drug will improve NSCLC therapeutic outcomes. New drug development is an expensive and time-consuming procedure. Cytotoxicity of DS is copper (II) (Cu)-dependent. DS/Cu induces reactive oxygen species, inhibits NF-κB activity and demonstrates excellent in vitro anti-CSCs activity in a wide range of cancers. The clinical application of DS as an anticancer drug is impeded by its very short half-life in the bloodstream. To improve the drug delivery efficiency, we developed a poly lactic-co-glycolic-DS (PLGA-DS), which demonstrates strong anti-cancer efficacy in mouse NSCLC models. Methods: NSCLC cell line A549 and H23 cultured as spheroids, hypoxia and PCDNA-3.1 NF-κB p65 transfect A659 cell lines. The cytotoxic effect of DS/Cu analyzed using sphere reformation, clonogenic and MTT assays. Flow cytometric analysis to detect CSC markers ALDH and CD44, and analysis of apoptosis by Annexin V. Western blot analysis to determine expression of embryonic stem cell markers Nanog and OCT4, and identify the importance of the NF-κB pathway together with HIF and including expression of associated apoptotic proteins Bax and Bcl2. Results: CSC and hypoxic cultured cells expressed high levels of CSC markers and were resistant to first and second line NSCLC anticancer drugs (doxorubicin, oxaliplatin, paclitaxel and gemcitabine). High NF-κB expression was detected in CSCs and hypoxia-cultured NSCLC cell lines. After transfection with p65 subunit of NF-κB, A549 cells expressed CSC markers and became resistant to a wide range of anticancer drugs. DS (5-10 nM) supplemented with Cu (10 μM) induced cytotoxicity to hypoxic cultured NSCLC cells, DS (1 μM) in combination with Cu inhibited sphere reformation. DS/Cu effectively inhibited NF-κB activity, abolished the CSC population and synergistically enhanced the cytotoxicity of the above conventional anticancer drugs. Our study also shows that PLGA-DS extends the half-life of free DS in the blood stream from 30 seconds to 7 hours. Intravenous injection of PLG-DS in combination with oral Cu can effectively inhibit the growth of subcutaneous and lung NSCLC xenografts. Conclusion: DS/Cu specifically inhibits NF-κB pathway and targets CSC in NSCLC cell lines. PLGA encapsulation improves delivery of DS which demonstrated very strong anticancer activity in NSCLC xenografts in vivo. Citation Format: Kate Butcher, Vinodh Kannappan, Karim Azar, Rajagopal Kilari, Ogechi Nkeonye, Mark R. Morris, Christopher McConville, Yaohe Wang, Weiguang Wang. Disulfiram targets hypoxia-induced cancer stem cells and reverses chemoresistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1975.

Introduction: Tumors are highly heterogeneous. The heterogeneity of the tumor tissue is rooted in the existence of cancer stem cells (CSCs). Therefore, understanding the mechanism of CSC maintenance and its regulation by the microenvironment is critical to cancer prevention and treatment. Since cancer takes many years to grow, it is important to develop in vitro models to study the molecular basis of tumorigenesis and progression. 3D cell culture systems with biologic materials that support adhesion and growth of many cell types have emerged as another approach to cancer stem cell research. However, it is not possible to isolate individual factors in the microenvironment and the effect on cell response with naturally derived materials. In an effort to control the cell microenvironment, we have developed novel inert permissive gels with controlled physical, mechanical, and biological properties that support the maintenance of CSCs and tumorsphere formation. The objective of this work was to investigate the effect of matrix stiffness and a CD44 binding peptide, conjugated to the inert hydrogel matrix, on tumorsphere formation and CSC maintenance. Methods: Mouse 4T1 and human MCF7 breast cancer cells were encapsulated in the inert PEGDA gel with conjugation of CD44BP. Control groups included dissolved CD44BP and the gel conjugated with mutant CD44BP. Tumorsphere size and density, and expression of CSC markers were determined with incubation time in tumor CSC culture medium. Effect of CD44BP conjugation on breast CSC maintenance was compared with those gels conjugated with integrin binding RGD peptide (IBP) and fibronectin-derived heparin binding peptide (FHBP). For in vivo, cell encapsulated gels were inoculated in syngeneic Balb/C mice and tumor formation was determined with time. Results: The gel stiffness had a strong effect on tumorsphere formation and the effect was bimodal. Tumorsphere formation and expression of CSC markers by the encapsulated cells peaked after 8 days of incubation. 4T1 and MCF7 cells encapsulated in the gel with 5-kPa stiffness formed the largest and highest density of tumorspheres, and had highest expression of breast CSC markers CD44 and ABCG2. Conjugation of CD44 binding peptide to the inert gel inhibited breast tumorsphere formation in vitro and in vivo. The ability of the encapsulated cells to form tumorspheres in the peptide-conjugated gels correlated with the expression of CSC markers. Tumorsphere formation in vitro was enhanced by FHBP while it was abolished by IBP. Conclusion: The PEGDA hydrogel cell culture system provides a novel tool to investigate the individual effect of factors in the microenvironment on CSC maintenance without interference of other factors. This model system can be used to understand the effect of individual factors in the microenvironment on epithelial to mesenchymal transition (EMT) and tumorigenesis. Citation Format: Xiaoming Yang, Samaneh Kamali, Esmaiel Jabbari. Engineered matrix to study the effect of microenvironment on cancer stem cell maintenance. [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 251. doi:10.1158/1538-7445.AM2013-251

As a key mediator of integrin signaling, focal adhesion kinase (FAK) regulates cellular responses to extracellular matrix interactions. Amplification and overexpression of FAK have been observed in aggressive human cancers including breast cancer. FAK has been implicated in multiple steps in carcinogenesis including tumor growth, metastasis and angiogenesis. We now demonstrate the importance of FAK in breast cancer stem cell function, and the reduction of cancer stem cell function by the selective FAK inhibitors VS-4718 and VS-5095. VS-4718 and VS-5095 are potent and selective FAK inhibitors which were optimized following high throughput screening. Both VS-4718 and VS-5095 block fibronectin-stimulated FAK autophosphorylation of Tyr397 with low nanomolar cellular potency and are highly selective for FAK among a panel of protein kinases. Consistent with their mechanism of action, VS-4718 and VS-5095 showed greater inhibitory potency on the growth of multiple cancer cell lines in 3D matrigel culture as compared to conventional 2D culture. To determine if FAK plays a role in the biology of breast cancer stem cells in addition to its reported function in normal mammary stem cell biology, the effects of these FAK inhibitors were characterized using two different in vitro assays. It was previously demonstrated that immortalized mammary epithelial cells (HMLEs) driven to undergo epithelial to mesenchymal transition (EMT) by knockdown of E-cadherin (HMLE-shECad) exhibit many of the characteristics of cancer stem cells and can be used to identify agents that selectively target cancer stem cells. VS-4718 exhibited 25-fold greater potency against proliferation of mesenchymal HMLE-shECad cells as compared to epithelial HMLE-shGFP control cells, suggesting preferential effects on breast cancer stem cells. Furthermore, pre-treatment of SUM159 triple negative breast cancer cells with VS-5095 in matrigel attenuated secondary tumorsphere formation, suggesting that FAK is important for the self-renewal function of breast cancer stem cells. The role of FAK in breast cancer stem cell renewal was further corroborated by the observation that FAK shRNA inhibited tumorsphere formation by SUM159 cells. The in vivo efficacy of the FAK inhibitor VS-5095 was evaluated in the MDA-MB-231 human triple negative breast cancer xenograft model. By oral administration, VS-5095 induced significant dose-dependent tumor growth inhibition. In summary, these results demonstrate the importance of FAK in the self-renewal of breast cancer stem cells, and support the clinical development of the selective FAK inhibitors VS-4718 and VS-5095 to target breast cancer stem cells for the treatment of triple negative breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-192. doi:1538-7445.AM2012-LB-192

Cancer stem cells initiate tumorigenesis and are the causes of metastasis and relapse. Cancer stem cells, rather than other differentiated cells in tumors display resistance to cytotoxicity of anticancer drugs, but we do not know whether by-product of chemotherapy can induce cancer stem cells leading to chemotherapy failure. Glucosylceramide synthase (GCS) is a limiting enzyme regulating the synthesis of glycosphingolipids that play an essential role in the maintenance of embryonic stem cells. The purpose of this study was to identify and characterize the effect of ceramide glycosylation in the formation and maintenance of breast cancer stem cells (BCSCs). We have found that GCS overexpression was interrelated to the increment BCSCs and drug resistance in human breast cancer MCF-7 cell lines after doxorubicin induction. In MCF-7/Dox (doxorubicin-resistant) cells, GCS protein was increased by 39-fold accompanied with enhanced enzyme activity, as compared to wild-type MCF-7 cells. The BCSCs with CD24-/CD44+/ESA+ phenotype was increased by 5-fold in MCF-7/Dox cells as compared to MCF-7 cells. Silencing GCS by using mixed-backbone oligonucleotide (MBO-asGCS) significantly decreased the numbers of BCSCs more than 2-fold in MCF-7/Dox cells. In the soft agar colony formation assay, MCF-7/Dox cells showed significantly higher colonies formed as compare to the MCF-7 cells. BCSCs sorted from MCF-7/Dox cells displayed significantly higher GCS enzyme activity more than 3-fold and formed 2-fold more colonies, as compare with other non-stem cell subsets. The BCSCs cells showed significantly higher tumorigenicity and metastasibility as compared to non-stem cells (CD24-/CD44-) in athymic nude mice. More interestingly, doxorubicin treatment (1 mg/kg, once a week, and 40 days) considerably increased BCSCs 2-fold in tumors, and MBO-asGCS treatment eliminated the tumor growth and reduced metastasis due to reduction of BCSCs more than 2-fold in vivo. Comparison of glycosphingolipids and gene profile of stem cells indicated GCS or globo-series glycosphingolipids upregulates FGF1, FGFR1, ALDH2, ISL1, MSX1, SIGMAR1, PPARD and downregulates CXCL12, FRAT1, CCND1 to accumulate BCSCs during the course of chemotherapy. These results, for the first time, demonstrate that ceramide glycosylation by GCS accumulates BCSCs formation and causes aggressive tumor progression. Silencing of GCS that eliminates BCSCs can prevent and treat drug-resistant tumors and relapse. 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 4365. doi:10.1158/1538-7445.AM2011-4365

INTRODUCTION: Despite the multitude of drug options, most patients with metastatic colorectal cancer (mCRC) die within 3 years of diagnosis. Response to systemic therapy is not durable (< 1year) and drug resistance is inevitable. There is accumulating evidence for the existence of cancer stem cells (CSCs) in CRC, which are now believed to mediate chemo-resistance. The molecular mechanisms that regulate the CSC phenotype in CRC have not been fully elucidated. The metalloproteinase ADAM17 [also known as TNFα-converting enzyme (TACE)], an enzyme involved in Notch pathway activation, has been shown to be overexpressed in CRC and mediate cell proliferation and chemo-resistance in CRC cells. However, the role of ADAM17 in mediating the CSC phenotype in CRC has not been characterized. The present study sought to determine the role of CRC-associated ADAM17 in mediating chemo-resistance of CRC cells by regulating the CSC phenotype. METHODS: Most preclinical studies used established cancer cell lines that have been cultured in vitro for decades. Our laboratory has shown that these cell lines are not suitable for consistent isolation of CSCs (Fan et al, 2014, BJC in press). To circumvent this problem, we developed a new model using CRC cells either freshly isolated from patient-derived xenografts (PDX) or early passage of newly established Human CRC Primary cell lines (HCP) derived from the PDXs. With this newly established HCP cell line model, we used siRNA knockdown or a synthetic peptide inhibitor TAPI-2, to determine the effect of blocking ADAM17 on the CSC phenotype and chemo-resistance in CRC cells. RESULTS: ADAM17 inhibition, either by siRNA or by TAPI-2, reduced the protein levels of cleaved Notch1 (NICD) and its downstream target HES-1 in CRC cells. Levels of proteins that are involved in other CSC-associated pathways were not altered by ADAM17 inhibition, suggesting that the Notch pathway is the major stem-cell pathway activated by ADAM17. Furthermore, ADAM17 inhibition caused a decrease in the CSC-phenotype in CRC cells as determined by the sphere formation assay and the Aldefluor assay (a standard assay for identifying CSCs). In addition, the lethal dose 50 (LD50) of 5-fluorouracil (5-FU) was decreased by 3 fold when pre-incubating CRC cells with the ADAM17 inhibitor TAPI-2. We also showed that the protein levels of ADAM17 were higher in our chemo-resistant cells (cells that survive continuous exposure to chemotherapy) compared to control. CONCLUSION: Our studies demonstrated the role of ADAM17 in promoting the CSC phenotype and chemo-resistance in CRC cells. Utilization of drugs that inhibit ADAM17 activity may increase the therapeutic benefit to patients with mCRC, and potentially other solid malignancies. Citation Format: Rui Wang, Fan Fan, Delphine Boulbes, Rajat Bhattacharya, Xiang-Cang Ye, Ling Xia, Lee Ellis. ADAM17 mediation of cancer stem cell-ness and chemo-resistance in colorectal cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2337. doi:10.1158/1538-7445.AM2015-2337

Hyaluronic acid hydrogels with defined crosslink density for the efficient enrichment of breast cancer stem cells

SY01-02: Targeting stem cell pathways in human cancer

Chromoprotein homologue 7 (CBX7), a member of the polycomb repressive complex (PRC)-1, has been involved in several biological processes including stem cell regulation and cancer development, but its roles in cancer stem cells (CSCs) and breast tumorigenicity remain unknown. Here, we demonstrated that CBX7 negatively regulates breast CSCs. CD44+/CD24-/ESA+ cells, a population of breast CSCs, showed diminished CBX7 expression compared with non-CSCs. Furthermore, small hairpin RNA (shRNA)-mediated CBX7 knockdown in breast epithelial and cancer cell lines increased breast CSC population and reinforced in vitro self-renewal and in vivo tumor initiating ability. Similarly, CBX7 overexpression showed reduced stemness of breast CSCs. We also found that CBX7 inhibits the Wnt/β-catenin/TCF pathway by enhancing the expression of Dickkopf-1 (DKK-1), a Wnt antagonist, to impede self-renewal of breast CSCs. Particularly, CBX7 increased DKK-1 transcription by cooperating with p300 acetyltransferase and subsequently enhancing the histone acetylation of DKK-1 promoter. Furthermore, pharmacological inhibition of DKK-1 in CBX7 overexpressing cells showed recovery of the Wnt signaling and consequent rescue of breast CSC activity. Taken together, these findings indicated that CBX7-mediated epigenetic induction of DKK-1 is crucial for inhibition of breast CSC activity, suggesting that CBX7 could be a potential tumor suppressor as a novel negative regulator of CSCs in human breast cancer. Citation Format: Hey-Yon Kim, Ji-Hye Park, Hee-Young Won, Jeong-Yeon Lee, Gu Kong. CBX7 inhibits breast cancer stem cell activity and tumorigenicity through DKK-1-mediated suppression of the Wnt/β-catenin pathway. [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 LB-52. doi:10.1158/1538-7445.AM2014-LB-52

Many cancers, including breast cancer are maintained by a subpopulation of cells that display stem cell properties, mediate metastasis and contribute to treatment resistance. These cancer stem cells are regulated by the interaction of cell intrinsic pathways as well as extracellular signals originating from the tumor microenvironment. Developing tumors recruit a diverse collection of cells that make up the tumor microenvironment and through iterative interactions both the tumor cell and its microenvironment co-evolve. The tumor microenvironment is composed of multiple cell types including mesenchymal cells, fibroblasts, endothelial cells and immune cells. We have utilized in vitro and mouse xenograft models to examine the interaction between breast cancer stem cells and cells within the microenvironment. We previously demonstrated that bone marrow derived mesenchymal stem cells like tumor stem cells are hierarchically organized and are recruited to the growing sites of breast cancers via cytokines such as interlukin-6 (IL-6). Mesenchymal stem cells interact with cancer stem cells forming “niches” which influences the growth and differentiation of both cellular compartments. Both IL-8 and IL-6 play seminal roles in the regulation of breast cancer stem cells. IL-6 also plays an important role in mediating resistance of breast cancer stem cells to a variety of therapeutic agents. These include development of resistance of HER2 overexpressing breast cancers to trastuzumab-targeted therapy. Although inactivation of the PTEN gene has been implicated in the development of resistance to HER2 targeting antibody trastuzumab, the mechanisms mediating this resistance have remained elusive. We have found that development of trastuzumab resistance in PTEN deleted cells is mediated by activation of an IL-6 inflammatory loop leading to expansion of the cancer stem cell population. Interestingly, long term trastuzumab treatment generates highly enriched cancer stem cells with an epithelial mesenchymal (EMT) phenotype which secrete over 100-fold more IL-6 than parental cells. The IL-6 receptor antibody tociluzumab was able to interrupt this inflammatory loop reducing the cancer stem cell population resulting in decreased tumor growth and metastasis in mouse xenografts. These studies demonstrate that trastuzumab resistance may be mediated by an IL-6 inflammatory loop and suggests that blocking this loop represents a novel strategy to overcome trastuzumab resistance. Another inflammatory cytokine intimately involved in breast cancer stem cell regulation is IL-8. The IL-8 receptor CXCR1 is selectively over expressed in breast cancer stem cell populations. Recombinant IL-8 stimulates breast cancer self-renewal and a small molecule CXCR1 inhibitor, reparaxin is able to block this signaling pathway. Reparaxin is able to target breast cancer stem cells in mouse xenograft models reducing tumor growth and metastasis. Based on these preclinical models a clinical trial utilizing reparaxin in combination with chemotherapy for the treatment of women with advanced breast cancers is currently being planned. Together, these studies suggest that strategies aimed at blocking the interaction of breast cancer stem cells with the microenvironment may prove an effective strategy to target this important cell population.

The dissemination of metastases from the primary tumor accounts for over 90% of cancer-associated mortality. Despite advances on treatment modalities involving chemotherapeutic drugs, radiotherapy, and surgery, metastasis almost invariably arises by persistence of unresolved cancer cells following initial treatment. One intriguing explanation for this resiliency is the cancer stem-cell (CSC) hypothesis. According to the CSC model, phenotypic heterogeneity exists within the tumor, and a minority fraction of cells are able to reconstitute the tumor following ablation by current treatments. In like manner to their “normal” stem-cell counterparts, CSCs are capable of unlimited self-renewal, and the ability to “differentiate” to reconstitute the phenotypic heterogeneity of the initial tumor. Recent studies have sought to identify cancer stem cells in numerous types of solid tumors, including breast and prostate. Among the cell surface antigens utilized to identify the CSC subpopulations are CD24, CD44, CD133, and other factors associated with epithelial-to-mesenchymal transition (EMT). In this study, we sought to investigate the phenotypic changes associated with cell-cycle in breast (BCa-SC) and prostate (PCa-SC) cancer. We characterized in vitro the malignant and osteolytic human breast cancer cell line MDA-MB-231 and human prostate cancer cell line PC3, with the goal of identifying an optimal checkpoint where the CSC subpopulation would be most vulnerable to subsequent targeted chemotherapy. We found that common CSC markers are expressed in a time-dependent manner due to changes in the cell cycle. Our hypothesis is that temporal inhibition of the cell cycle when the CSC phenotype is least prominent will circumvent the chemo-resistance inherent to CSC. Citation Format: Joseph Feduska, Carnellia Lee, Selvarangan Ponnazhagan. Cell cycle regulation influences the phenotype of breast and prostate cancer stem cells in vitro. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2539.

Although conventional chemotherapies eliminate differentiated cancer cells in the bulk of tumors, they are ineffective against cancer stem cells (CSCs), which initiate tumors and result in cancer recurrence. The purpose of this study is to investigate the efficacy of HDAC inhibition by SAHA on breast CSCs through epigenetic modifications in cancer stem cells. Two different breast cancer cell lines with basal (SUM159) and luminal (MCF7) subtypes were used in this study. In order to examine HDAC mediated CSC regulation, we utilized a number of in vitro and in vivo CSC assays including tumorsphere formation, Aldefluor assay, CD44+/CD24- phenotype and mouse xenografts. Breast cancer stem cells (CSC) were monitored after treatment of different concentrations of SAHA (0.5-2µM). The ability of HDAC inhibitor to suppress the self-renewal capability of CSCs was evaluated by tumorsphere assay from a single Aldefluor-positive cell in a 96-well Ultra-low attachment plate under the suspension condition. The tumorsphere formation rate was used to evaluate the self-renewal capability of CSCs. The mouse xenograft model bearing with SUM159 cells and primary breast tumors (MC1) was used to test the inhibition of SAHA on CSCs and impairment of their self renewal in vivo. Microarray gene expression analysis was performed to elucidate molecular mechanisms of HDAC underlying the CSC inhibition, following the treatment of SAHA. SAHA reduced SUM159 Aldefluor-positive cells in a concentration-dependent manner with significant reduction (>50%) at 1µM. SAHA dramatically reduced the CSC population (CD44+/CD24 low) by 90% in MCF7 cells. Since CSCs are able to expand its population through the self-renewal, a CSC-targeting drug to inhibit both self-renewal and to eliminate CSCs is highly desired. Tumorsphere formation assay showed that SAHA (0.5µM) reduced the tumorsphere formation rate by 77%. In addition, the sustaining tumorsphere reductions (∼80%) in second and third passages in the absence of SAHA suggest the the impaired self-renewal capability of breast CSCs by SAHA is not repairable. Furthermore, the size of SAHA-treated tumorspheres was substantially smaller than that of the control, suggesting that SAHA also inhibited the proliferation of progenitor cells. In SUM159 tumor xenograft model, SAHA significantly inhibited the tumor growth by 45% and 84% in SUM159 and MC1 tumor xenografts, respectively, compared to the vehicle-treated group. SAHA reduced Aldefluor-positive cells in SUM159 and MC1 tumors by 50-90% following 2-week treatment. Tumor reimplantation showed that SAHA was able to abolish the tumor engraftment ability of cells derived from drug-treated mice in secondary mice. These data suggest that SAHA was able to target breast CSCs in vitro and in vivo. SAHA not only diminished the CSC population but also attenuated their self-renewal ability of the residual CSCs. 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 3354. doi:10.1158/1538-7445.AM2011-3354

A considerable body of evidence indicates that many cancers are hierarchically organized and driven by cancer stem cells (CSCs) that have the ability to self-renew and to generate heterogeneous populations of tumor bulk. Preclinical studies have also demonstrated that CSCs mediate tumor metastasis and resistance to chemotherapy and radiation therapy. As a result of substantial studies to identify CSCs markers and related pathways, agents developed to target CSCs are in early stage clinical trials. Given the clinical importance of CSCs, there is an urgency to develop the methodology to assay these cells. In this study, we explored CSCs heterogeneity at the single cell level using C1 and BioMark HD technologies. Cancer stem cells from MCF7 and SUM159 breast cancer cell lines were sorted by MoFlo XDP based CSCs marker including CD44, CD24 and ALDH1 immunophenotyping. Sorted cells were then separately loaded onto the C1 chip and studied by C1 instrument that isolates up to 96 single cells and performs lysis, RNA release, reverse transcription, and finally cDNA preamplification of up to 96 gene transcripts for each single cell. We selected the 96 genes based on important genetic pathways, EMT and CSCs markers, and also genes identified in the expression array data to be differentially expressed in CD44+/CD24- and or ALDH+ primary breast tumor samples. Following C1 preamplification, we analyzed the mRNA expression levels of 96 genes on each of the isolated single cells by BioMark HD instrument which generates nearly 10,000 qPCR data points in a single run using a 96x96 chip. Results showed that more than one population exist with significant differences in mRNA expression levels of certain genes such as CAPRIN2, TM4SF1, NECAB3, PPL, TSPAN6, STAP2, FAM46C, TCEA3, TPSB2 and FCN1, as well as IL6, GATA3, KI67, PCNA, EZH2, and MET in seemingly homogenous populations of ALDH+ and ALDH-(CD44+/CD24- in MCF7 cells) CSCs. In addition, a distinct pattern of gene expression for sorted luminal MCF7 in comparison to basal SUM159 cells was observed for epithelial versus mesenchymal markers such as CDH1, CDH2, KRT7, KRT19, EpCAM, and Vimentin genes. We have recently reported that breast cancer stem cells (BCSCs) exist in inter-convertible EMT/MET states characterized by distinct genes and biomarker expression. Using this methodology of single cell analysis, we found that these CSC populations still display significant heterogeneity in mRNA expression. This technique of single cell multiplex gene expression analysis, along with a microfluidic method for a label-free cell sorting is currently under optimization to be used for isolation and molecular characterization of circulating tumor cells (CTCs) from blood samples of breast cancer patients as a means of liquid biopsy. Altogether, results of this and similar studies on single CSCs have considerable clinical implications in cancer diagnosis and prognosis, as well as in the successful targeted therapy of cancer stem cells. Citation Format: Ebrahim Azizi, Shamileh Fouladdel, Yadwinder S. Deol, Jonathan Bender, Sean McDermott, Hui Jiang, Mary Sehl, Shawn G. Clouthier, Sunitha Nagrath, Max S. Wicha. Exploring cancer stem cells heterogeneity via single cell multiplex gene expression analysis. [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 1943. doi:10.1158/1538-7445.AM2014-1943