Breast Cancer Tumor-initiating Cells Research Articles

MTSS1 suppresses mammary tumor-initiating cells by enhancing RBCK1-mediated p65 ubiquitination.

Tumor-initiating cells (TICs) are considered the culprits of cancer development and progression. Dysregulation of metastasis suppressor protein 1 (MTSS1) has been widely observed in tumor metastasis, but its functional contribution and mechanism in cancer is poorly understood. Here we report a role of MTSS1 in suppressing TICs in breast cancer. Mtss1 knockout (KO) enhances the mammary epithelial TIC subpopulation in both luminal and basal-like breast cancer mouse models. MTSS1 also suppresses tumorsphere formation in breast cancer cells. Mechanistically, MTSS1 interacts with the E3 ligase RanBP2-type and C3HC4-type zinc finger containing 1 (RBCK1) to facilitate RBCK1-mediated p65 ubiquitination and degradation, thus suppressing the NF-κB signaling pathway and tumorigenesis. In addition, actin beta-like 2 (ACTBL2) competes with RBCK1 for MTSS1 binding, leading to p65 stabilization. Importantly, MTSS1 silencing promotes patient-derived organoid formation and xenograft growth. MTSS1 downregulation in clinical tumors is also linked to worse prognosis. Overall our data reveal a new paradigm of NF-κB regulation and may have important implications in therapeutics targeting TICs.

MMP14 empowers tumor-initiating breast cancer cells under hypoxic nutrient-depleted conditions.

Tumor-initiating cells (TICs) existing in breast cancer are thought to be involved in initiation, progression, and relapse of tumors. In these processes, the epithelial-to-mesenchymal transition (EMT) and proteases are crucial factors that also dependent on the tumor milieu, including hypoxic nutrient-deprived, as well as normoxic nutrient-rich, environments. Therefore, we investigated EMT and proteases in TICs and their response to different environments by means of a newly generated immortalized TIC (iTIC) line. With the use of primary CD24+CD90+CD45- TICs from the mouse mammary tumor virus-polyoma middle T mouse breast cancer model, iTICs were generated by single cell-initiated sphere and subsequent 2-dimensional monolayer culture. Our data demonstrate the possibility to generate iTICs that are highly tumorigenic in culture and in mouse mammary fat pad. Contrasting environmental conditions provide these cells with a phenotypic and molecular plasticity that has a growth-promoting character in nutrient-rich normoxia and a motile character in nutrient-deprived hypoxia. Expression profiling revealed partial and dynamically changing EMT states, as well as a significantly up-regulated proteolytic signature, including many metalloproteinases, such as matrix metalloproteinase 14 ( Mmp14). Inhibitor treatment of metalloproteinases, as well as short hairpin RNA-mediated knockdown of Mmp14 strongly impacted TIC characteristics, including tumor initiation, cell growth, migration, and invasion, especially in starved environments. We conclude that metalloproteinases empower TICs to adapt to changing environments.-Hillebrand, L. E., Wickberg, S. M., Gomez-Auli, A., Follo, M., Maurer, J., Busch, H., Boerries, M., Reinheckel, T. MMP14 empowers tumor-initiating breast cancer cells under hypoxic nutrient-depleted conditions.

Abstract 5753: Cancer stem cell markers allow for immune evasion in triple-negative breast cancer

Abstract Triple-negative breast cancer tumor-initiating cells, or cancer stem cells (CSCs) are identified as a population that is CD44+ and CD24low. These cells are important drivers of distant metastasis and recurrence. An important component of both the recurrence and metastasis functions of CSCs is the capacity to evade immune detection. Our work has identified key members of the antigen presentation machinery required for proper immunosurveillance as being differentially expressed in tumor cells expressing CD44 compared to cells lacking CD44, including a differential response to important immune system cytokines. We are currently working to identify functional differences related to the altered protein levels in these cells. Citation Format: Andrew D. Hoffmann. Cancer stem cell markers allow for immune evasion in triple-negative breast 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 5753.

Polyurethane foam scaffold as in vitro model for breast cancer bone metastasis.

3D in vitro models represent an outstanding alternative in the study of tumor metastases development, compared to traditional 2D in vitro cultures, which oversimplify the 3D tissue microenvironment, and in vivo studies, affected by low reproducibility and ethical issues. Several scaffold-based 3D in vitro models have been proposed to recapitulate the development of metastases in different body sites but, still, the crucial challenge is to correctly mimic the tissue to be modelled in terms of physical, mechanical and biological properties. Here, we prove the suitability of a porous polyurethane foam, synthesized using an appropriate formulaton, in mimicking the bone tissue microenvironment and in reproducing the metastatic colonization derived from human breast cancer, particularly evidencing the devastating effects on the bone extracellular matrix caused by metastatic spreading.

Harnessing protein kinase A activation to induce mesenchymal-epithelial programs to eliminate chemoresistant, tumor-initiating breast cancer cells.

Metastatic dissemination of primary tumors remains the most significant predictor of clinical outcomes of cancer patients, as well as the most lethal characteristic of human malignancies, including those that arise in the breast (1,2). Breast tumors are highly heterogeneous and comprised of numerous subclones that emanate from an array of genetic and epigenetic variants that coalesce in enhancing the fitness of disseminated cells upon their colonization of distant vital organs (3). Moreover, disseminated tumor cells (DTCs) are typically the culprits underlying clinical relapse in breast cancer patients, a process that transpires through the ability of DTCs to acquire tumor-initiating/stem-like and chemoresistant phenotypes, traits that cement DTCs as one of the foremost barriers to eradicating metastatic disease. The molecular mechanisms responsible for metastatic relapse and chemoresistance are multipartite and remain to be fully elucidated; however, recent findings implicate epithelial-mesenchymal transition (EMT) programs as a major driver that (I) induces metastatic cells to detach and egress from the primary tumor; and (II) enables DTCs to surmount the cellular and genotoxic stressors imposed by foreign microenvironments and cytotoxic therapies (4-7). In the most general sense, EMT programs reflect an organized transdifferentiation process whereby polarized epithelial cells shed their immotile behaviors in favor of newly acquired fibroblastoid-like phenotypes characterized by increased invasive and migratory capabilities that compel indolent carcinoma in situ lesions to become highly aggressive invasive lesions (4,7-9).

Abstract 3316: The effects of dopamine receptor 2 on breast cancer tumor initiating cells

Abstract Despite advances in early detection and treatment of breast cancers, metastatic disease remains a significant problem and a large cause of mortality in the US. Although tumors are characterized as a mass of uncontrolled cell division, it is thought that only small populations of cells, known as tumor initiating cells (TICs), are primarily responsible for metastasis. TICs are less differentiated than most tumor cells and many TICs show an epithelial-to-mesenchymal transition (EMT). Recently, the FDA-approved drug thioridazine, a dopamine receptor 2 (DRD2) antagonist, has been shown to inhibit pluripotency gene expression only in transformed stem cells. To test whether DRD2 can regulate TICs in breast cancer cells, two main assays were performed. The tumorsphere assay is used to measure TIC activity within a population of cells in vitro. In this assay, single cells are cultured in suspension, in the absence of serum. Cells must escape anoikis and grow without cell-cell contacts or the assortment of factors contained in serum. However, the gold standard in vivo assay to measure tumor initiation is the transplantation assay at limiting dilutions. Cells are injected into immunocompromised mice in decreasing numbers and the number of tumors each population of cells is capable of forming is measured. In this study the tumorsphere assay and transplantation assay both show that DRD2 supports TICs in breast cancers. Interestingly, our data suggest that DRD2 regulates TICs and cellular differentiation through the activity of the STAT3 transcription factor. Activation of STAT3 has been shown to induce gene expression changes that support TICs and upregulate the pluripotency factor OCT4. STAT3 activity can also induce EMT and resistance to some therapeutics. By inducing the expression of its own activator, IL-6, STAT3 can initiate a feed forward loop to maintain continual expression to promote TICs, EMT, and drug resistance. Halting the continual activation of STAT3 in these cells should be an important objective. Our data supports the concept that DRD2 may represent a new, and druggable target for therapies to decrease metastasis and/or recurrence by interfering with STAT3 and inhibiting TIC-like activities. Citation Format: Matthew Tegowski, Charlene Santos, Albert Baldwin. The effects of dopamine receptor 2 on breast cancer tumor initiating cells. [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 3316.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract B21: DNp63 promotes stem cell activity in mammary gland development and basal-like breast cancer by enhancing Fzd7 expression and Wnt signaling

Abstract Emerging evidence suggests that cancer is populated and maintained by tumor initiating cells (TICs) with stem-like properties similar to that of adult tissue stem cells. Despite recent advances, the molecular regulatory mechanisms that may be shared between normal and malignant stem cells remain poorly understood. Here we show that the DNp63 isoform of the Trp63 transcription factor promotes normal mammary stem cell (MaSC) activity by increasing the expression of the Wnt receptor Fzd7 and thereby enhancing Wnt signaling. Importantly, Fzd7-dependent enhancement of Wnt signaling by DNp63 also governs tumor initiating activity of the basal subtype of breast cancer. These findings establish DNp63 as a key regulator of stem cells in both normal and malignant mammary tissues and provide direct evidence that breast cancer TICs and normal MaSCs share common regulatory mechanisms. Citation Format: Rumela Chakrabarti, Satrajit Sinha, Yibin Kang. DNp63 promotes stem cell activity in mammary gland development and basal-like breast cancer by enhancing Fzd7 expression and Wnt signaling. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr B21.

The role of steroid hormones in breast cancer stem cells.

Breast cancer stem cells (BCSCs) are potent tumor-initiating cells in breast cancer, the most common cancer among women. BCSCs have been suggested to play a key role in tumor initiation which can lead to disease progression and formation of metastases. Moreover, BCSCs are thought to be the unit of selection for therapy-resistant clones since they survive conventional treatments, such as chemotherapy, irradiation, and hormonal therapy. The importance of the role of hormones for both normal mammary gland and breast cancer development is well established, but it was not until recently that the effects of hormones on BCSCs have been investigated. This review will discuss recent studies highlighting how ovarian steroid hormones estrogen and progesterone, as well as therapies against them, can regulate BCSC activity.

ABCG2 is a potential marker of tumor-initiating cells in breast cancer.

The existence of tumor-initiating cells (TICs) within solid tumors has been hypothesized to explain tumor heterogeneity and resistance to cancer therapy. In breast cancer, the expression of CD44 and CD24 and the activity of aldehyde dehydrogenase 1 (ALDH1) can be used to selectively isolate a cell population enriched in TICs. However, the ideal marker to identify TICs has not been established. The aim of this study was to evaluate the expression of novel potential markers for TIC in breast carcinoma. We prospectively analyzed the expression of CD44, CD24, ABCG2, and CXCR4, and the activity of ALDH1 by using flow cytometry in 48 invasive ductal carcinomas from locally advanced and metastatic breast cancer patients who were administered primary chemotherapy. A mammosphere assay was employed in 30 samples. The relationship among flow cytometric analyses, ABCG2 gene expression, and clinical and pathological responses to therapy was analyzed. The GSE32646 database was analyzed in silico to identify genes associated with tumors with low and high ABCG2 expression. We observed that the presence of ABCG2(+) cells within the primary tumor was the only marker to predict the formation of mammospheres in vitro (R (2) = 0.15, p = 0.029). Quantitative polymerase chain reaction (qPCR) revealed a positive correlation between ABCG2 expression and the presence of ABCG2(+) cells within the primary tumor. The expression of ABCG2 was predictive of the response to neoadjuvant chemotherapy in our experiments and in the GSE32646 dataset (p = 0.04 and p = 0.002, respectively). The in silico analysis demonstrated that ABCG2(Up) breast cancer samples have a slower cell cycle and a higher expression of membrane proteins but a greater potential for chromosomal instability, metastasis, immune evasion, and resistance to hypoxia. Such genetic characteristics are compatible with highly aggressive and resistant tumors. Our results support the hypothesis that the presence of ABCG2(+) cells in breast carcinomas is a marker of resistance to chemotherapy, and based on in vitro assays and the genetic profile, we show, for the first time, that ABCG2 protein can be used as an independent marker for TIC identification in breast cancer.

Silibinin affects tumor cell growth because of reduction of stemness properties and induction of apoptosis in 2D and 3D models of MDA-MB-468.

Silibinin, with a strong antioxidant activity and a weak cytotoxic property, is considered a candidate for cancer prevention. As there is no information on its effect on breast cancer tumor-initiating cells [cancer stem cells (CSCs)] in a 3D culture model, which more closely mimic natural tissues, we carried out this study to determine whether silibinin can target breast CSCs in MDA-MB-468 cells cultured under 3D and 2D conditions. Silibinin was added to culture medium of MDA-MB-468 at a half maximal inhibitory concentration (IC50) dose in 2D and 3D models. Then, stemness properties were assessed using colony and sphere-formation tests. Flow cytometry and real-time PCR were used to determine the different expression levels of stem cell-related marker at protein and mRNA levels under both culture conditions. Our results showed that silibinin inhibits cell growth in a dose-dependent manner by induction of apoptosis, alteration of the cell cycle, reduction of stemness properties and function, and induction of tumoral differentiation. The mechanism of silibinin action and also the response of tumor cells differed when cells were cultured in a 3D model compared with a 2D model. Silibinin may potentially target breast CSCs. Moreover, tumor-initiating cells are more sensitive to silibinin in a 3D culture than in a 2D culture.

Molecular profiling and computational network analysis of TAZ-mediated mammary tumorigenesis identifies actionable therapeutic targets.

Triple-negative breast cancer (TNBC) accounts for approximately 15-20% of all breast cancer (BC) cases and contributes disproportionately to BC mortality. TAZ, a key transducer of the Hippo pathway, has recently been demonstrated to confer breast cancer stem cell (CSC) traits. However, TAZ target genes and the underlying transcriptional regulatory pathways responsible for the CSC phenomenon remain unknown. Here, we demonstrate that the oncogenic activity of TAZ is essential for propagation of the malignant phenotype. We further show that constitutively active TAZ tumor-derived cells exhibit unique tumor-initiating properties, including increased self-renewal and metastatic seeding potential, acquired chemotherapy resistance and the ability to efficiently regenerate tumor formation in vivo. Combined digital RNA expression analysis and computational network approaches identify several signaling pathways that distinguish breast cancer tumor-initiating cells (T-ICs) from bulk tumor cells. We demonstrate the utility of this approach by repositioning the small molecule tyrosine kinase inhibitor, Dasatinib, which selectively targets T-ICs and inhibits TNBC growth in vivo.

Abstract 1248: The Gemini vitamin D analogue BXL0124 inhibits Notch signaling via HES1, resulting in the reduction of CD44+/CD24-/low subpopulation and proliferation of MCF10DCIS cells

Abstract Activation of Notch signaling is correlated with poor prognosis and decreased survival of breast cancer patients. Recent studies reported that Notch signaling plays an important role for the maintenance of tumor-initiating cells in breast cancer. Using MCF10DCIS.com human breast cancer cells (MCF10DCIS), which contain tumor-initiating subpopulation (CD44+/CD24-/low), we demonstrated that the Gemini vitamin D analog, BXL0124, reduced the tumor-initiating subpopulation in vitro and repressed the growth of MCF10DCIS xenograft tumors in vivo. In the present study, we investigated Notch signaling in CD44+/CD24-/low and CD44+/CD24+ subpopulations of MCF10DCIS cells and determined whether BXL0124 represses tumor-initiating subpopulation of breast cancer by targeting Notch signaling. CD44+/CD24-/low subpopulation showed higher Notch1 activation and cell proliferation than CD44+/CD24+ subpopulation in MCF10DCIS cells. To investigate the effects of BXL0124 on Notch signaling, the protein levels of Notch receptors and their ligands were determined. BXL0124 decreased activated Notch1 (c-Notch1) as well as its ligands, Jagged1, Jagged2 and DLL1. However, total Notch1 was not affected by BXL0124, suggesting that BXL0124 inhibits Notch signaling by decreasing Notch ligands. A downstream target of Notch signaling, cMyc, and cell proliferation were reduced by BXL0124. The inhibition of Notch1 activation by BXL0124 was blocked with knock-down of the vitamin D receptor (VDR), indicating that it is a VDR-dependent event. In a time course study, the effects of BXL0124 on the mRNA expression of Notch1, Notch ligands and downstream targets were investigated. BXL0124 decreased the mRNA levels of Jagged1, Jagged2 and DLL1 starting at 8 h which followed by the repression of cMyc mRNA at 16 h. The mRNA level of a transcriptional repressor HES1, which is a key downstream target of Notch signaling, was rapidly induced by BXL0124 as early as 30 min. To determine the involvement of HES1 in Notch signaling, both knock-down and overexpression of HES1 were performed by using HES1 siRNA and HES1-expression vector, respectively. The inhibition of Notch signaling by BXL0124 was reversed by the knock-down of HES1, while the overexpression of HES1 inhibited Notch activation, reduced CD44+/CD24-/low population and decreased cell proliferation of MCF10DCIS cells. These results suggest that HES1 functions as a repressor of Notch signaling, and BXL0124 inhibits Notch signaling in a HES1-dependent manner. The present study demonstrates that BXL0124 inhibits Notch signaling by rapid induction of HES1 which results in the reduction of CD44+/CD24-/low subpopulation and proliferation of MCF10DCIS cells. Our study suggests the Gemini vitamin D analog, BXL0124, as a potential agent to repress the tumor-initiating subpopulation of breast cancer by targeting Notch signaling. Citation Format: Jae Young So, David M. Salerno, Hubert Maehr, Milan Uskokovic, Nanjoo Suh. The Gemini vitamin D analogue BXL0124 inhibits Notch signaling via HES1, resulting in the reduction of CD44+/CD24-/low subpopulation and proliferation of MCF10DCIS cells. [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 1248. doi:10.1158/1538-7445.AM2014-1248

ΔNp63 promotes stem cell activity in mammary gland development and basal-like breast cancer by enhancing Fzd7 expression and Wnt signalling.

Emerging evidence suggests that cancer is populated and maintained by tumor initiating cells (TICs) with stem-like properties similar to that of adult tissue stem cells. Despite recent advances, the molecular regulatory mechanisms that may be shared between normal and malignant stem cells remain poorly understood. Here we show that the ΔNp63 isoform of the Trp63 transcription factor promotes normal mammary stem cell (MaSC) activity by increasing the expression of the Wnt receptor Fzd7, thereby enhancing Wnt signaling. Importantly, Fzd7-dependent enhancement of Wnt signaling by ΔNp63 also governs tumor initiating activity of the basal subtype of breast cancer. These findings establish ΔNp63 as a key regulator of stem cells in both normal and malignant mammary tissues and provide direct evidence that breast cancer TICs and normal MaSCs share common regulatory mechanisms.

A novel small molecule aurora kinase inhibitor attenuates breast tumor-initiating cells and overcomes drug resistance.

Chemoresistance is a major cause of cancer treatment failure. Tumor-initiating cells (TIC) have attracted a considerable amount of attention due to their role in chemoresistance and tumor recurrence. Here, we evaluated the small molecule Aurora kinase inhibitor AKI603 as a novel agent against TICs in breast cancer. AKI603 significantly inhibited Aurora-A (AurA) kinase and induced cell-cycle arrest. In addition, the intragastric administration of AKI603 reduced xenograft tumor growth. Interestingly, we found that breast cancer cells that were resistant to epirubicin expressed a high level of activated AurA and also have a high CD24(Low)/CD44(High) TIC population. The inhibition of AurA kinase by AKI603 abolished the epirubicin-induced enrichment of TICs. Moreover, AKI603 suppressed the capacity of cells to form mammosphere and also suppressed the expression of self-renewal genes (β-catenin, c-Myc, Sox2, and Oct4). Thus, our work suggests the potential clinical use of the small molecule Aurora kinase inhibitor AKI603 to overcome drug resistance induced by conventional chemotherapeutics in breast cancer.

Canonical and non-canonical NF-κB signaling promotes breast cancer tumor-initiating cells

Canonical and non-canonical NF-κB signaling promotes breast cancer tumor-initiating cells

Abstract IA09: Targeting tumor-initiating cells in xenograft models of human breast cancer

Abstract Historically, investigation of treatment resistance in breast cancer has focused on the identification of acquired genetic or epigenetic alterations in tumor epithelial cells that confer resistance to specific agents, or to multiple agents. Our recent data indicate that a unique subpopulation of tumor-initiating cells (TIC), or “cancer stem cells” present in the original tumors appear to possess a form of intrinsic (de novo) resistance to conventional systemic therapies including radiation and chemotherapies, and are therefore likely responsible for tumor initiation and cancer re-growth. Agents that may target TIC cells specifically are becoming attractive experimental therapeutic candidates. Current markers that allow enrichment of breast cancer TIC by fluorescence activated cell sorting (FACS) (e.g. CD44+/CD24low/neg and ALDH activity) are not universally expressed, cannot be monitored in vivo, and typically identify a much larger percentage of cells than the percentage of cells estimated to serve as TIC. Thus, to evaluate TIC therapeutic targeting effectively, new markers must be identified. To investigate the location and function of TIC, and to evaluate agents that may specifically target them therapeutically, we are developing a series of lentiviral fluorescent signaling reporters to identify cells responsive to Wnt, Hedgehog, Notch, and Stat3-mediated signaling – four pathways implicated in TIC self-renewal or regulation. These reporters have been introduced into a recently developed bank of patient-derived human breast cancer xenograft (PDX) models established in our laboratory, as well as traditional cell line xenograft models. Results thus far indicate that both Wnt responsiveness and STAT3-mediated signaling allows significant enrichment for the TIC population by FACS, and has allowed localization of responsive cells in vivo. Given that these pathways have been implicated in TIC regulation, we have begun to target these pathways using a battery of experimental therapeutics both in the preclinical setting, and in the case of Notch signaling, also in the clinical setting. Current data suggest that inhibition of Notch, STAT3, and Hedgehog signaling can enhance the efficacy of systemic chemotherapy, and can impair or alter the function of TIC in limiting dilution transplantation assays. However, in the case of Hedgehog signaling inhibition, the transplantation behavior of the TIC fraction is biphasic suggesting a novel form of inhibitory cell-cell interaction is present in at least some tumors. Overall, results are consistent with the concept that TIC targeted therapeutics may ultimately prove useful clinically. However, our results also indicate that identification and use of additional agents that actually kill TIC will likely be required for eradication of TIC in patients. Citation Format: Michael T. Lewis. Targeting tumor-initiating cells in xenograft models of human breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA09.

Stromal Contributions to Radiation Resistance of Breast Cancer Stem Cells

Breast cancer that is refractory to radiation therapy causes significant morbidity and mortality. Tumor-initiating cells (TICs), also called cancer stem cells (CSCs), display a radioresistant phenotype. Normal stem cells depend on stromal cells for the formation of a stem cell “niche”, but the role of the stromal cells in breast TIC function and treatment resistance is not well understood. We sought to define the effects of stromal fibroblasts on breast TIC self-renewal and resistance to IR (ionizing radiation). For investigations into breast cancer TIC and stromal fibroblast physiology we utilized MMTV-Wnt-1 transgenic mice, which spontaneously generate mammary tumors. These tumors can be dissociated into single cell suspensions and subjected to fluorescence-activated cell sorting (FACS) for in vitro colony assays or molecular analyses. The TIC population in this model has been previously identified based on cell surface markers and has been validated using in vivo limiting dilution analyses. Using this model system, we isolated normal and tumor-associated fibroblasts from primary normal and tumor murine breast tissues by FACS, and their effects on TICs were assayed in a 3D co-culture system. We show that co-culture with primary stromal fibroblasts promotes the expansion of mouse mammary TICs in vitro and maintains their ability to form new tumors in vivo. At clinically relevant doses of IR, DNA double strand breaks are mitigated and clonogenic growth of TICs is enhanced by co-culture with the stromal fibroblasts. Consistent with these observations, the TICs that are grown in co-culture maintain a more basal differentiation phenotype and harbor lower levels of reactive oxygen species (ROS) than TICs alone. The reduction in ROS found in the co-cultured TICs is a consequence of fewer mitochondria and less active oxidative phosphorylation in these cells. Working with primary tissues, we have demonstrated that stromal fibroblasts from mammary tumors play an important role in promoting TIC proliferation and resistance to IR. Our work has led to a better characterization of the unique microenvironment within the TIC-stromal niche. Future endeavors into pharmacological disruption of the supportive cues within the niche could lead to novel radiosensitizers and CSC-directed therapies.

Abstract B25: Inhibition of autocrine signaling as a strategy to target tumor-initiating breast cancer cells

Abstract The epithelial-mesenchymal transition (EMT), a pleiotropic cellular program, has been associated with the acquisition of metastatic ability, self-renewal traits and resistance to chemotherapeutic drugs in breast cancer and other carcinomas. During normal development and tumor progression, this change in cell phenotype is induced by contextual signals that epithelial cells receive from their microenvironment, thereby promoting cellular heterogeneity. The signaling context responsible for inducing an EMT and maintaining the resulting cellular states has been unclear. We describe three signaling pathways, involving transforming growth factor (TGF)-beta and canonical/beta-catenin dependent and noncanonical Wnt signaling, that collaborate to induce activation of the EMT program and thereafter function in an autocrine fashion to maintain the resulting mesenchymal and stem cell-like state. Importantly, the induction of the EMT program and associated autocrine signaling is partly enabled by the downregulation of endogenous inhibitors of autocrine signals secreted by epithelial cells prior EMT, indicating a homeostatic mechanism. These inhibitors include secreted frizzled-related protein 1 (SFRP1), which has been shown by others to be a rate-limiting determinant of Wnt signaling, and Bone Morphogenetic Proteins, which appear to inhibit TGF-beta signaling. Conversely, disruption of autocrine signaling by adding back endogenous inhibitors of autocrine signaling as recombinant proteins inhibits migration and self-renewal of breast cancer cells in vitro and reduces tumorigenicity and spontaneous metastasis in vivo. Together, our results indicate that ongoing autocrine signaling is required for the maintenance of mesenchymal and stem cell traits in breast cancer cells. In the longer term, we intent to develop a cocktail small-molecule inhibitors to therapeutically interrupt multiple autocrine pathways, thereby forcing tumor-initiating cells of breast and other carcinomas to exit the mesenchymal, stem cell-like state. Since this cellular state is associated with resistance to chemotherapeutic drugs, we predict that such a treatment protocol might render breast cancer cells that have passed through an EMT more sensitive to standard chemotherapy, thereby serving as a therapeutic strategy to overcome tumor heterogeneity and relapse. Citation Format: Christina Scheel, Diana Dragoi, Elinor Ng Eaton, Sophia Hsin-Jung Li, Ferenc Reinhardt, Robert A. Weinberg. Inhibition of autocrine signaling as a strategy to target tumor-initiating breast cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr B25.

Abstract 3949: Hypoxia induces the conversion of tumor initiating cells from stationary to invasive by promoting cell surface trafficking of matrix metalloproteinase-14.

Abstract Emerging experimental and clinical evidence has demonstrated that quiescent, stationary tumor initiating cells (TICs) can become invasive and metastatic. The mechanism underlying the conversion of TICs from stationary to invasive remains to be characterized. A better understanding of this conversion is crucial for the design of inhibitors aimed at preventing cancer metastasis. We report here for the first time that hypoxia, a low oxygen environment that often occurs in solid tumors, induces translocation of the membrane anchored protease, matrix metalloproteinase-14 (MMP-14) from the cytoplasmic storage pool to the cell surface resulting in conversion of stationary TICs to invasive TICs. Exposure of minimally invasive breast cancer TICs, SK-3rd, that displays CD44high/CD24low with high mammosphere-forming and tumorigenic capacities, to hypoxic conditions (1% O2) results in markedly enhanced cell invasive ability. This phenotypic change is also observed in SK-3rd TICs in the presence of cobalt chloride (CoCl2), an agent that mimics hypoxia. Given the role of MMP-14 in cancer invasion/metastasis, we explored a possible involvement of MMP-14 in hypoxia-induced TIC invasion. Silencing of MMP-14 by a shRNA approach resulted in diminution of hypoxia-induced cell invasion in vitro and metastasis in vivo. When SK-3rd cells were cultured under hypoxic conditions, MMP-14 redistributed from cytoplasmic storage pools to the cell surface of TICs, which coincides with the increased cell invasion. In addition, CD44, a cancer stem-like cell marker, inversely correlated with increased cell surface MMP-14. Interestingly, cell surface MMP-14 gradually disappeared when the hypoxia-treated cells were switched to normoxia, suggesting the plasticity of TICs in response to oxygen content. Furthermore, we dissected the pathways leading to upregulated MMP-14 in cytoplasmic storage pools under normoxic conditions. We demonstrated a cascade involving Twist1-miR10b-HoxD10 leading to enhanced MMP-14 expression in SK-3rd TICs. These observations suggest that MMP-14 is a key molecule capable of executing conversion of stationary TICs to invasive TICs under hypoxic conditions and thereby controlling metastasis. Citation Format: Jian Cao. Hypoxia induces the conversion of tumor initiating cells from stationary to invasive by promoting cell surface trafficking of matrix metalloproteinase-14. [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 3949. doi:10.1158/1538-7445.AM2013-3949