Triple-negative Inflammatory Breast Cancer Cell Research Articles

Sensitization of Resistant Cells with a BET Bromodomain Inhibitor in a Cell Culture Model of Deep Intrinsic Resistance in Breast Cancer

We treated highly metabolically adaptable (SUM149-MA) triple-negative inflammatory breast cancer cells and their control parental SUM149-Luc cell line with JQ1 for long periods to determine its efficacy at inhibiting therapy-resistant cells. After 20 days of treatment with 1-2 µM of JQ1, which killed majority of cells in the parental cell line, a large number of SUM149-MA cells survived, consistent with their pan-resistant nature. Interestingly, though, the JQ1 treatment sensitized resistant cancer cells in both the SUM149-MA and SUM149-Luc cell lines to subsequent treatment with doxorubicin and paclitaxel. To measure JQ1-mediated sensitization of resistant cancer cells, we first eradicated approximately 99% of relatively chemotherapy-sensitive cancer cells in culture dishes by long treatments with doxorubicin or paclitaxel, and then analyzed the remaining resistant cells for survival and growth into colonies. In addition, combination, rather than sequential, treatment with JQ1 and doxorubicin was also effective in overcoming resistance. Notably, Western blotting showed that JQ1-treated cancer cells had significantly lower levels of PD-L1 protein than did untreated cells, indicating that JQ1 treatment may reduce tumor-mediated immune suppression and improve the response to immunotherapy targeting PD-L1. Finally, JQ1 treatment with a low 62.5 nM dose sensitized another resistant cell line, FC-IBC02-MA, to treatment with doxorubicin and paclitaxel.

Sensitization of Resistant Breast Cancer Cells with a Jumonji Family Histone Demethylase Inhibitor.

Simple SummaryUsing a cell culture model of resistant breast cancer cells with the phenotype that is often responsible for the early relapse of triple-negative breast cancer, namely, the persistence of these cells in reversible quiescence under a variety of challenges, we found that reprogramming the epigenome by treatment with JIB-04, a small-molecule inhibitor of Jumonji-family histone demethylases, sensitized resistant cells. We used this model of deep intrinsic resistance featuring many molecular mechanisms of achieving this phenotype to perform lengthy evaluations of less cytotoxic doses of JIB-04. We found that resistant cells derived from triple-negative inflammatory breast cancer cell lines were either much more sensitive to JIB-04 than the parental cell line or altered by the treatment such that they became sensitive to the chemotherapeutic drugs paclitaxel and doxorubicin. Notably, JIB-04 exposure increased PD-L1 expression in cancer cells, which means that JIB-04 may have clinical applications in improving the responses of triple-negative breast cancer to anti-PD-L1 therapy.In the present study, we evaluated JIB-04, a small-molecule epigenetic inhibitor initially discovered to inhibit cancer growth, to determine its ability to affect deep intrinsic resistance in a breast cancer model. The model was based on a function-based approach to the selection of cancer cells in a cell culture that can survive a variety of challenges in prolonged, but reversible, quiescence. These resistant cancer cells possessed a variety of mechanisms, including modifications of the epigenome and transcriptome, for generating a high degree of cellular heterogeneity. We found that long pretreatment with JIB-04 sensitized resistant triple-negative inflammatory breast cancer cells and their parental cell line SUM149 to the chemotherapeutic drugs doxorubicin and paclitaxel. Resistant cancer cells derived from another inflammatory breast cancer cell line, FC-IBC02, were considerably more sensitive to JIB-04 than the parental cell line. Investigating a mechanism of sensitization, we found that JIB-04 exposure increased the expression of PD-L1 in resistant cells, suggesting that JIB-04 may also sensitize resistant breast cancer cells to anti-PD-L1 immune therapy. Finally, these results support the usefulness of a cell culture-based experimental strategy for evaluating anticancer agents, such as JIB-04, that may halt cancer evolution and prevent the development of cancer resistance to currently used therapies.

Anti‐cancer Effects of Coumestrol in Triple‐Negative Inflammatory Breast Cancer 3D Models: Opportunities for In Vivo Studies

Inflammatory breast cancer (IBC) is the most aggressive subtype of breast cancer. Each molecular subtype is characterized by the presence or absence of two hormonal steroid receptors, estrogen receptor (ERα‐66) and progesterone receptor (PR), and a growth factor receptor called HER2. Particularly, a subtype of breast cancer that lacks these three biomarkers is known as triple‐negative breast cancer (TNBC). However, previous studies showed that TNBC can express alternate estrogen receptors known as ERα‐36 and GPR30, mediating a non‐genomic signaling upon estrogen (E2) treatment. Today, effective targeted therapeutics for triple‐negative inflammatory breast cancer (TN‐IBC) patients, accounting 20‐40% of TN‐IBC cases, is limited. Coumestrol (Cou), a plant derived compound structurally similar to estrogen has shown to induce a cytotoxic effect in TNBC. Till this day, there is no known study of the effects of Cou in three‐dimensional culture models using Matrigel, a solubilized membrane matrix that mimics the tissue microenvironment. Additionally, we want to test Cou’s inhibitory effect on tumor emboli formation, a distinct hallmark of IBC. This tumor cell clusters are known to infiltrate and block the breast lymphatics of patients, presenting a barrier for successful IBC treatment (Arora Etl. 2017). We hypothesize that Cou inhibits cell viability and tumor emboli formation in TN‐IBC cells. First, we performed 3D dose response curves to determine the half‐maximal inhibitory concentration (IC50) of Cou (50µM). Next, 3D proliferationassays were performed using 50µM of Cou. After 10 days, E2 showed a significant increase in colony size in comparison to vehicle control (DMSO). Also, proliferation decreased more than a half after treating the cells with Cou alone and in combination of both treatments. When testing the tumor emboli formation after 72 hours, Cou did not inhibit the formation of emboli, but it decreased the tumor emboli growth rate shown by the decreased in tumor emboli size in both culture conditions. In comparison, E2 promoted a significant increase in the size of the tumor emboli. Lastly, we used Western Blot analysis to validate the decrease in phosphorylation levels of ERK1/2 and AKT, two kinases involved in estrogen non‐genomic signaling, upon Cou treatment. Together, these data demonstrated that Cou decreases cell viability in 3D proliferation assays and decreased growth rate of tumor emboli in TN‐IBC cells. In summary, the anticancer effects of Coumestrol in IBC three‐dimensional models provide the basis for future in vivo studies using animal models.

Molecular Mechanism by which Coumestrol Exerts its Anticancer Activity in Triple‐Negative Inflammatory Breast Cancer

Coumestrol (Cou) is a phytoestrogen present in soy and clover sprouts which is structurally similar to estrogen (E2) and has a cytotoxic effect in breast cancer cell lines. Nevertheless, the molecular mechanism by which Cou can exert its effect in the most aggressive breast cancer subtype, triple‐negative inflammatory breast cancer (TN‐IBC), is still unknown. Several studies show that TN‐IBC cell lines are unresponsive to hormonal therapies, but E2 can activate signaling pathways involved in pro‐oncogenic phenotypes such as motility and invasion. Therefore, the objective of this study was to determine if Cou has an anti‐cancer effect in TN‐IBC cell lines and the molecular mechanism that Cou exerts for its anti‐cancer activity using 2D cell culture models. For this, a dose‐response curve of Cou in 2D models of TN‐IBC cell lines was generated to determine the half‐maximal inhibitory concentration (IC50). Also, relative cell viability was performed in ER‐positive, triple‐negative, TN‐IBC, and HER2‐amplified IBC cell lines. Functional assays were performed in TN‐IBC cell lines to determine the effect of E2 alone, Cou alone, and in a combination of treatments on cell viability, migration, and invasion. Also, the effect on phosphorylation of downstream kinases was analyzed by a proteome profile human phospho‐kinase array. An RNA‐seq was performed in TN‐IBC cell lines to identify transcriptome changes after Cou treatment. Preliminary observations demonstrated that Cou treatment using the IC50 (13µM) caused a decrease in cell viability in TN‐IBC cell lines. Similarly, Cou treatment reduces triple‐negative non‐IBC viability. Also, in comparison with E2, Cou decreases migration and invasion in TN‐IBC cell lines. Finally, Cou treatment reduces the phosphorylation of kinases that promote pro‐oncogenic phenotypes and upregulate genes involved in breast cancer phenotype suppression. In summary, the data gathered in this study will guide us in elucidating a signaling pathway affected by Cou’s anti‐cancer activity. More importantly, it will open the opportunities to design more effective targeted therapeutic strategies to improve the prognosis and survival rates of IBC patients.

Accumulation of amyloid beta (Aβ) and amyloid precursor protein (APP) in tumors formed by a mouse xenograft model of inflammatory breast cancer.

Accumulation of amyloid in breast cancer is a well‐known phenomenon, but only immunoglobulin light‐chain amyloidosis (AL) or transthyretin (TTR) amyloid had been detected in human breast tumor samples previously. We recently reported that another amyloidogenic peptide, amyloid beta (Aβ), is present in an aggregated form in animal and human high‐grade gliomas and suggested that it originates systemically from the blood, possibly generated by platelets. To study whether breast cancers are also associated with these Aβ peptides and in what form, we used a nude mouse model inoculated with triple‐negative inflammatory breast cancer cell (SUM‐149) xenografts, which develop noticeable tumors. Immunostaining with two types of specific antibodies for Aβ identified the clear presence of Aβ peptides associated with (a) carcinoma cells and (b) extracellular aggregated amyloid (also revealed by Congo red and thioflavin S staining). Aβ peptides, in both cells and in aggregated amyloid, were distributed in clear gradients, with maximum levels near blood vessels. We detected significant presence of amyloid precursor protein (APP) in the walls of blood vessels of tumor samples, as well as in carcinoma cells. Finally, we used ELISA to confirm the presence of elevated levels of mouse‐generated Aβ40 in tumors. We conclude that Aβ in inflammatory breast cancer tumors, at least in a mouse model, is always present and is concentrated near blood vessels. We also discuss here the possible pathways of Aβ accumulation in tumors and whether this phenomenon could represent the specific signature of high‐grade cancers.

Phytoestrogen Coumestrol as an Anticancer Therapy Against Triple‐Negative Inflammatory Breast Cancer

Breast cancer is the second leading cause of death in females in the United States, being Inflammatory Breast Cancer (IBC) the most aggressive subtype. The 5‐year survival rate is 40% for IBC as compared to 85% survival rate in patients with other subtypes. The poor prognosis for patients with IBC emphasizes the need to better understand the molecular signature of this disease with the goal of developing effective targeted therapeutics. To this date, there is no effective targeted therapeutics, especially for those patients that account for approximately 20–40% of IBC. Those cases, triple negative IBC (TN‐IBC), express an alternative isoform of estrogen receptor alpha, estrogen receptor alpha‐36, and GPR30. Importantly, several studies have shown that estrogen can activate non‐genomic signaling pathways involved in the acquisition of oncogenic phenotypes such as increased motility and invasion in triple negative (TNBC) and TN‐IBC. In fact, recent studies have shown that phytoestrogen like Cou, structurally similar to estradiol (E2), induces cell cycle arrest and apoptosis in TNBC. We hypothesize (1) that estrogen non‐genomic signaling has an active role in the aggressive metastatic phenotype of IBC, and (2) that Cou has anticancer activity by inhibiting estrogen non‐genomic signaling in IBC. To test this hypothesis, TN‐IBC cells were treated with E2 or Cou and the effect on phosphorylation of downstream kinases (ERK and AKT) was analyzed by Western blot. Also, functional assays had been performed to determine the effect of treatment with E2 alone, Cou alone, and in combination on migration, proliferation and tumor emboli formation. Immunofluorescence in TN‐IBC and Western Blot of breast cancer cell lines showed expression of estrogen receptors. In addition, preliminary observations demonstrated that upon E2 treatment there was an activation of ERK and AKT kinase in both TN‐IBC and TNBC cell lines. Finally, treatment using previously reported IC50 (50μM) of Cou caused a decrease in cell viability in breast cancer cell lines. Also, in comparison to E2, Cou decrease migration, proliferation, and tumor emboli formation in TN‐IBC. In summary, TN‐IBC cells are responsive to E2 treatment making this signaling pathway attractive for the development of innovative therapeutic strategies. Importantly, these Cou preliminary studies show a potential anticancer activity in TN‐IBC cell lines.Support or Funding InformationUPR Fondos Institucionales para la Investigación (FIPI) and RISE program (5R25GM061151‐18)

Evaluating the Anti-cancer Efficacy of a Synthetic Curcumin Analog on Human Melanoma Cells and Its Interaction with Standard Chemotherapeutics.

Melanoma is the leading cause of skin-cancer related deaths in North America. Metastatic melanoma is difficult to treat and chemotherapies have limited success. Furthermore, chemotherapies lead to toxic side effects due to nonselective targeting of normal cells. Curcumin is a natural product of Curcuma longa (turmeric) and has been shown to possess anti-cancer activity. However, due to its poor bioavailability and stability, natural curcumin is not an effective cancer treatment. We tested synthetic analogs of curcumin that are more stable. One of these derivatives, Compound A, has shown significant anti-cancer efficacy in colon, leukemia, and triple-negative inflammatory breast cancer cells. However, the effects of Compound A against melanoma cells have not been studied before. In this study, for the first time, we demonstrated the efficacy of Compound A for the selective induction of apoptosis in melanoma cells and its interaction with tamoxifen, taxol, and cisplatin. We found that Compound A induced apoptosis selectively in human melanoma cells by increasing oxidative stress. The anti-cancer activity of Compound A was enhanced when combined with tamoxifen and the combination treatment did not result in significant toxicity to noncancerous cells. Additionally, Compound A did not interact negatively with the anti-cancer activity of taxol and cisplatin. These results indicate that Compound A could be developed as a selective and effective melanoma treatment either alone or in combination with other non-toxic agents like tamoxifen.

Abstract P5-17-06: Immune modulation with humanized anti-EGFR antibody panitumumab in an immunocompetent mouse model for inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is the most lethal and aggressive form of breast cancer and there are no approved targeted therapies specifically for IBC. Targeting the epidermal growth factor receptor (EGFR) pathway is a promising therapeutic target for patients with triple-negative IBC (TN-IBC) with a reported pathological complete response rate of 42% (JAMA Oncology, 2018). The tumor microenvironment (TME) is a critical contributor to the aggressiveness of IBC. Delineating cross-talk between EGFR-targeted therapies and TME components, which define IBC, could inform more efficient combination regimens and novel clinical trial designs for IBC. However, such studies have not been conducted due to the lack of a syngeneic IBC mouse model. Here we report the establishment of an IBC immunocompetent mouse model and the effects of panitumumab (PmAb) on IBC tumor growth and the TME. Methods: TN-IBC cell lines, SUM149 or FC-IBC-02, were mixed with 50% Matrigel and inoculated into mammary fat pads of hu-NSG-SGM3 mice engrafted with hematopoietic stem cells (The Jackson Laboratory). SUM149 tumor growth in hu-NSG-SGM3 mice treated with either IgG2 (isotype control, 4 mg/kg) or PmAb (1 mg/kg and 4 mg/kg) was measured. The percentages of TME components, including human CD4+ T, CD8+ T, regulatory T (Tregs), and natural killer (NK) cells, and M1 or M2 macrophages, in the peripheral blood and tumor tissues treated with IgG2 and PmAb for 7 weeks were measured by flow cytometry. Results: Hu-NSG-SGM3 mice supported the growth of TN-IBC SUM149 and FC-IBC-02 xenografts. These humanized mouse models were named SUM149-huSGM3 and FC-IBC-02-huSGM3, respectively. Analysis of the blood cells showed that SUM149-huSGM3 mice display human CD4+ T, CD8+ T, Tregs, M1 and M2 macrophages. T cell infiltration and M1 and M2 macrophages were also detected in SUM149-huSGM3 tumors. NK cells were not detected in both peripheral blood and tumors. PmAb treated SUM149-huSGM3 mice had significantly reduced SUM149 tumor growth, compared with mice that received the IgG2 control. PmAb treatment increased the percentage of CD8+ T cells and reduced the percentage of Tregs in peripheral blood. A similar analysis of tumor infiltrating lymphocytes isolated from each group showed an increase in percent CD8+ T cells in mice treated with PmAb. There were no significant changes of M1 or M2 macrophages following PmAb treatment. These results suggest that the increase in percentage of CD8+ T cells in peripheral blood and IBC tumors, and the decrease in percentage of Tregs in peripheral blood may contribute to the therapeutic efficacy of PmAb. Conclusion: We established the first immunocompetent mouse model to study the TME and immune response in IBC, which provides the premise for conducting a diversity of novel preclinical therapeutic studies. The mechanism of how immune responses of TN-IBC xenografts mediates the therapeutic efficacy of PmAb in IBC tumors needs to be further investigated. Our study also suggests that combination therapy with immune checkpoint inhibitors may potentiate the efficacy of anti-EGFR therapy in IBC. The therapeutic efficacy of PmAb and anti-PD-L1 combination in SUM149 humanized mice is in progress. Citation Format: Wang X, Shao S, Pearson T, Cheng Y, Reuben JM, Tripathy D, Ueno NT. Immune modulation with humanized anti-EGFR antibody panitumumab in an immunocompetent mouse model for inflammatory breast cancer [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-17-06.

Abstract P1-10-09: EPHA2-targeting enhances eicosapentaenoic acid cytotoxicity against triple-negative inflammatory breast cancer via ABCA1 inhibition–mediated membrane rigidity

Abstract Background: Effective treatment options for triple-negative inflammatory breast cancer (TN-IBC), the most aggressive form of breast cancer, are currently lacking. We previously reported that mediators of inflammation promote the growth of TN-IBC xenografts. Eicosapentaenoic acid (EPA), an omega-3 fatty acid (fish oil) with anti-inflammatory properties, is an emerging FDA-approved therapeutic with a favorable toxicology profile. Here we aimed to develop a novel approach to enhance EPA efficacy against TN-IBC by identifying a kinase inhibitor that synergizes with EPA's antitumor activity. Methods and Results: Using a high-throughput siRNA screen in the TN-IBC cell line SUM149PT, we identified inhibition of ephrin type-A receptor 2 (EPHA2), an oncogenic receptor tyrosine kinase, as a target that sensitizes TN-IBC cells to EPA therapy. To determine the clinical relevance of EPHA2, we investigated a meta-analysis of breast cancer mRNA expression data sets and found that high EPHA2 tumor expression, compared with low expressing, correlated significantly with poor overall survival in TN-IBC patients (P = 0.01), while not with other subtypes. Similar findings were observed in vitro, were EPHA2 protein and mRNA overexpression occurred predominantly in the TN subtypes among 49 and 51 breast cancer cell lines (63% and 47%, respectively), highlighting EPHA2 translational potential. Functional expression studies using proliferation and apoptosis assays in vitro, and xenografts in vivo, were performed in two EPHA2-expressing TN-IBC cell lines, SUM149PT and BCX010, to validate EPHA2 as a synergistic combinational target with EPA. EPHA2 gene silencing in combination with EPA significantly reduced cell growth, and enhanced apoptosis, compared with untreated and monotherapy in vitro (P < 0.05), and in vivo (P < 0.001). To translate our findings to the clinic, we validated dasatinib, an FDA-approved small molecule inhibitor of EPHA2, in combination to EPA to significantly enhance apoptosis of TN-IBC cells in vitro (P < 0.05) and in vivo (P < 0.05), compared with untreated and monotherapies. Using membrane fluidity assessment and cholesterol quantification we determined that apoptosis induction after combination therapy was due to increased membrane rigidity and cholesterol concentrations in the plasma membrane of TN-IBC cells (P < 0.05, compared with monotherapies). Finally, we discovered by western blot and gain/loss-of-expression studies that combination therapy inhibited the cholesterol efflux protein ATP-binding cassette sub-family A member 1 (ABCA1), which plays a significant role mediating increased cellular cholesterol (P < 0.05), cell membrane rigidity (P < 0.05), and induction of apoptosis (P < 0.05) in TN-IBC after EPA and EPHA2-targeting combination therapy. Conclusions: This is the first study demonstrating that EPA can enhance conventional targeted therapy against breast cancer. Our study provides molecular and preclinical evidence to support the development of an EPA/EPHA2-inhibition–based phase I clinical trial for patients with EPHA2-positive TN-IBC; our study further suggests the use of EPHA2 and ABCA1 protein expression as biomarkers for patient selection and therapeutic response. Citation Format: Torres-Adorno AM, Vitrac H, Qi Y, Tan L, Levental KR, Fan Y-Y, Yang P, Chapkin RS, Eckhardt BL, Ueno NT. EPHA2-targeting enhances eicosapentaenoic acid cytotoxicity against triple-negative inflammatory breast cancer via ABCA1 inhibition–mediated membrane rigidity [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-10-09.

Abstract P3-07-15: Treatment with a Jumonji demethylase inhibitor JIB-04 sensitizes resistant breast cancer cells to chemotherapeutic drugs in an in vitro model of intrinsic resistance

Abstract We have recently developed a usable model of panresistance in triple-negative breast cancer. We have shown that only 0.01% cells survive a metabolic challenge involving lack of glutamine in culture medium of SUM149 triple-negative Inflammatory Breast Cancer cell line. These cells, designated as SUM149-MA for metabolic adaptability, are resistant to chemotherapeutic drugs, and they efficiently metastasize to multiple organs in nude mice. The MA cells can survive a variety of challenges in the metastasis process because of their embryo-like nature and high adaptability. Epigenetic state is a key player in cancer evolution. Therefore, a variety of compounds are being developed that would target epigenome for anticancer therapy. We believe that the identification of potentially effective epigenetic drugs can benefit from their evaluation in a usable system of panresistant cancer cells such as our model system of MA cells. This is particularly true for the compounds that may affect cellular adaptability upon a long treatment rather than affecting cell proliferation or apoptosis in a short period. In this study we evaluated JIB-04, a pan-inhibitor of Jumonji family of histone demethylases, for its ability to affect intrinsic resistance in MA cells. First, we found that MA cells are more resistant to JIB-04 treatment in a comparative evaluation with the parental SUM149 cells line. A treatment with 125-250 nM JIB-04 for 18-20 days killed all parental cells but not all MA cells; at least 20 MA cells survived and formed colonies after a 14-days recovery. Next, we asked whether JIB-04 treatment would affect the intrinsic resistance of MA cells, e.g., sensitize them to treatment with chemotherapeutic drugs. In order to assess the most resistant cells in population, these experiments involved treatment of MA cells with 125 nM JIB-04 for 10 days followed by a recovery in drug-free medium for 7 days; then we treated the cells with 5 nM paclitaxel or 100 nM doxorubicin for 6-8 days followed by a recovery for 1-12 days. We found that JIB-04 pre-treatment sensitized MA cells and the parental SUM149-Luc cell line to both these chemotherapeutic drugs as assessed by a dramatic drop in the number of colonies as compared to those obtained after treatment of control cells with the same chemotherapeutic drugs. To consider how our results may apply in the context of evolving disease in patients who are not taking chemotherapeutic drugs, we asked whether JIB-04 treatment would alter expression of surface molecules on resistant breast cancer cells such that they can be destroyed by immune system. In this regard, we specifically found that JIB-04 treatment increased the expression of PD-L1 in MA cells. Published results from a clinical study suggest that PD-L1 expression on breast cancer cells renders them responsive to various therapies, not just immune checkpoint blockade targeting PD-L1. In conclusion, our results suggest a novel approach for evaluating potential anticancer agents such as JIB-04 that would halt cancer evolution and prevent development of resistance to currently offered therapies. Supported by a State of Texas Grant for Rare and Aggressive Cancers. Citation Format: Singh B, Washburn LJ, Kinne HE, Milligan RD, Lucci A. Treatment with a Jumonji demethylase inhibitor JIB-04 sensitizes resistant breast cancer cells to chemotherapeutic drugs in an in vitro model of intrinsic resistance [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-07-15.

Abstract 2416: Important role of FTO in the survival of rare panresistant triple-negative inflammatory breast cancer cells under a severe metabolic challenge

Abstract Cancer is an evolution-like process. Effectively dealing with the currently untreatable component of breast cancer (the cells that drive metastasis) is vital. In this regard, we have developed a model of panresistant cancer cells that allows us to evaluate therapeutic agents to eradicate them. We have previously shown that only 0.01% cells survive a metabolic challenge involving lack of glutamine in culture medium of SUM149 triple-negative Inflammatory Breast Cancer (TN-IBC) cell line. These cells, designated as SUM149-MA for metabolic adaptability, are resistant to chemotherapeutic drugs, and they efficiently metastasize to multiple organs in nude mice. The MA cells can survive a variety of challenges in the metastasis process because of their embryo-like nature. We hypothesized that obesity-related molecular networks, which normally help in cellular and organismal survival under metabolic challenges, may help in the survival of MA cells. We found that fat mass and obesity-associated protein FTO is overexpressed in MA cells. It was reported recently that obesity-associated cis-acting elements in non-coding region of FTO regulate the expression of IRX3 gene, thus activating obesity networks. Here we found that IRX3 protein is significantly overexpressed in MA cells (4 to 5-fold) as compared to the parental SUM149 cell line, supporting our hypothesis. We utilized MO-I-500, a pharmacological inhibitor of FTO to investigate its role in MA cells. When included in a glutamine-starvation medium in 1.5 to 2 μM range, MO-I-500 significantly (>90%) inhibited survival and/or colony formation of SUM149-MA cells as compared to untreated cells or those treated with a control compound MO-I-100. Interestingly, MO-I-500 treatment had no significant effect on cell growth of either the SUM149 or SUM149-MA cell line when added to a complete medium containing glutamine that does not pose a metabolic challenge. Furthermore, SUM149-MA cells that were initially selected in a glutamine-free medium were not affected by a subsequent treatment with MO-I-500, even in glutamine-free medium. These results are significant as they reveal linkages between tumor adaptability, the embryonic nature of cancer cells, and obesity-like networks at the roots of therapy-resistant TN-IBC. They also suggest a novel approach for evaluating potential anticancer agents that would halt cancer evolution and prevent development of resistance to currently offered therapies. Supported by a State of Texas Grant for Rare and Aggressive Cancers. Citation Format: Balraj Singh, Hannah E. Kinne, Ryan D. Milligan, Laura J. Washburn, Mark Olsen, Anthony Lucci. Important role of FTO in the survival of rare panresistant triple-negative inflammatory breast cancer cells under a severe metabolic challenge. [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 2416.

Important Role of FTO in the Survival of Rare Panresistant Triple-Negative Inflammatory Breast Cancer Cells Facing a Severe Metabolic Challenge.

We have previously shown that only 0.01% cells survive a metabolic challenge involving lack of glutamine in culture medium of SUM149 triple-negative Inflammatory Breast Cancer cell line. These cells, designated as SUM149-MA for metabolic adaptability, are resistant to chemotherapeutic drugs, and they efficiently metastasize to multiple organs in nude mice. We hypothesized that obesity-related molecular networks, which normally help in cellular and organismal survival under metabolic challenges, may help in the survival of MA cells. The fat mass and obesity-associated protein FTO is overexpressed in MA cells. Obesity-associated cis-acting elements in non-coding region of FTO regulate the expression of IRX3 gene, thus activating obesity networks. Here we found that IRX3 protein is significantly overexpressed in MA cells (5 to 6-fold) as compared to the parental SUM149 cell line, supporting our hypothesis. We also obtained evidence that additional key regulators of energy balance such as ARID5B, IRX5, and CUX1 P200 repressor could potentially help progenitor-like TNBC cells survive in glutamine-free medium. MO-I-500, a pharmacological inhibitor of FTO, significantly (>90%) inhibited survival and/or colony formation of SUM149-MA cells as compared to untreated cells or those treated with a control compound MO-I-100. Curiously, MO-I-500 treatment also led to decreased levels of FTO and IRX3 proteins in the SUM149 cells initially surviving in glutamine-free medium as compared to MO-I-100 treatment. Interestingly, MO-I-500 treatment had a relatively little effect on cell growth of either the SUM149 or SUM149-MA cell line when added to a complete medium containing glutamine that does not pose a metabolic challenge. Importantly, once selected and cultured in glutamine-free medium, SUM149-MA cells were no longer affected by MO-I-500 even in Gln-free medium. We conclude that panresistant MA cells contain interconnected molecular networks that govern developmental status and energy balance, and genetic and epigenetic alterations that are selected during cancer evolution.

Abstract 1203: Metastasis-associated oncogene RhoC as a regulator of glutamine metabolism in the inflammatory breast cancer cell line SUM149

Abstract Metabolic reprogramming is increasingly recognized as a fundamental hallmark of cancer. While the Warburg effect and normal proliferative metabolism are similar, they are not equivalent. We hypothesize that there are key drivers of malignant metabolism that can be modulated to impede cancer proliferation without substantial effects on normal tissue growth. Using 13C-labeled glucose and glutamine tracers in combination with mass spectrometry and measurements of extracellular glucose, lactate, and glutamine flux, we have characterized system level differences in a series of breast cancer cell lines as well as normal-like breast epithelial cells. We observed an increase in the reductive carboxylation of glutamine-derived citrate and alpha-ketoglutarate in the triple-negative inflammatory breast cancer cell line SUM149. We also observed that the SUM149 exhibit high levels of HIF-1α and low levels of oxygen consumption under normoxia, suggesting that the cell line is highly adapted to hypoxia. Surprisingly, the stable depletion of HIF-1α via shRNA had no significant effect on the metabolic profile of these cells. Previous work by our lab and others has demonstrated that the GTPase RhoC is a driver of the metastatic phenotype exhibited by inflammatory breast cancer. Activation of RhoC is known to induce cytoskeletal rearrangements and increase invasive potential. The Rho GTPase family of proteins has also recently been linked to metabolism, specifically regulation of glutaminase activity. Here we show that stable knockdown of RhoC in SUM149 cells results in a marked decrease in the rate of both glutamine uptake and intracellular reductive carboxylation. This work reinforces the role of RhoC as an important driver of inflammatory breast cancer metastatic potential. We conclude that RhoC remains an important clinical target with the potential to alter patient outcomes. Citation Format: Joel A. Yates, Michelle L. Wynn, ZhiFen Wu, Charles R. Evans, Charles Burant, Santiago D. Schnell, Sofia D. Merajver. Metastasis-associated oncogene RhoC as a regulator of glutamine metabolism in the inflammatory breast cancer cell line SUM149. [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 1203. doi:10.1158/1538-7445.AM2015-1203

Abstract P1-06-05: Expansion of tumor initiating cells is mediated by tumor microenvironment in breast cancer metastasis

Abstract Background: Breast cancer metastasis which ultimately results in breast cancer death, is an event believed to be initiated by the migration of tumor initiating cells (TIC) from the primary tumor to niches for micrometastatic disease. Recent data suggests the tumor microenvironment promotes TIC. The clinical relevance of secreted factors from the microenvironment on TIC surrogate, mammosphere (MS) formation and MS sensitivity to drug therapy was investigated using breast cancer patient fluids inherently conditioned by the tumor microenvironment: post-operative seromas and malignant pleural effusions. Methods: Fluids from 48 patients with breast cancer (15 seromas and 33 pleural effusions) and mesenchymal stem cells (MSC) from healthy donors were collected on IRB approved protocols. Cellular components were eliminated from patient-derived fluids using density-gradient centrifugation. MSC conditioned media (MSC-CM) was collected from 3D cultures of primary MSC. Luminex multiplex array platform was used to characterize 79 cytokine and growth factor components of all fluids. In addition, MSC-CM and patient-derived fluids were added to cultures of breast cancer cell lines: MCF-7, an estrogen receptor (ER)-positive cell line; SUM149, a triple-negative inflammatory breast cancer cell line; and SUM159, a triple-negative metaplastic breast cancer cell line and MS forming efficiency was examined. Results: Our results show that pleural effusions and seromas are enriched for factors also secreted by MSC such as MCP-1, GRO, IL-6, and VEGF-A. We found remarkable similarities regarding the cytokines and growth factors profile in pleural effusions and seromas. Both patient-derived fluids have comparable amount of Angiopoetin-2, Leptin, TNF-beta, VEGF, IL-2, IL-3, IL-4 and IL-10. EGF, TNF-alpha, IL-1, IL-6, IL-8 and IL-16 were significantly different between pleural effusions and seromas. Seroma fluid from bilateral drains in a patient with an invasive cancer and a contralateral benign mastectomy had very similar cytokine concentrations. Moreover, MSC-CM and pleural fluids from ER-positive and ER-negative patients increased the MS formation efficiency of both triple-negative cell lines while seroma fluids from ER-positive and ER-negative patients increased the MS formation efficiency of ER-positive cell line MCF-7. Finally, we evaluated the impact of a panel of drugs (simvastatin, pravastatin and erlotinib) on cell cultures grown with MSC-CM and patient-derived fluids. We found that the effect of chemotherapies on MS formation can be attenuated by patient-derived fluids. Conclusions: Seroma and pleural effusion fluids from breast cancer patients have similar cytokine profiles, change MS formation efficiency of standard breast cancer cell models, and mediate sensitivity to therapy. Here we demonstrate that host and microenvironmental factors are critical for determining resistance to therapy and may be independent of obvious tumor related factors. Future studies will investigate the prognostic implications of factors that promote TIC survival in the fluid tumor microenvironment. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-06-05.

Inflammatory breast cancer (IBC): clues for targeted therapies

Inflammatory breast cancer (IBC) is the most aggressive type of advanced breast cancer characterized by rapid proliferation, early metastatic development and poor prognosis. Since there are few preclinical models of IBC, there is a general lack of understanding of the complexity of the disease. Recently, we have developed a new model of IBC derived from the pleural effusion of a woman with metastatic secondary IBC. FC-IBC02 cells are triple negative and form clusters (mammospheres) in suspension that are strongly positive for E-cadherin, β-catenin and TSPAN24, all adhesion molecules that play an important role in cell migration and invasion. FC-IBC02 cells expressed stem cell markers and some, but not all of the characteristics of cells undergoing epithelial mesenchymal transition (EMT). Breast tumor FC-IBC02 xenografts developed quickly in SCID mice with the presence of tumor emboli and the development of lymph node and lung metastases. Remarkably, FC-IBC02 cells were able to produce brain metastasis in mice on intracardiac or intraperitoneal injections. Genomic studies of FC-IBC02 and other IBC cell lines showed that IBC cells had important amplification of 8q24 where MYC, ATAD2 and the focal adhesion kinase FAK1 are located. MYC and ATAD2 showed between 2.5 and 7 copies in IBC cells. FAK1, which plays important roles in anoikis resistance and tumor metastasis, showed 6–4 copies in IBC cells. Also, CD44 was amplified in triple-negative IBC cells (10–3 copies). Additionally, FC-IBC02 showed amplification of ALK and NOTCH3. These results indicate that MYC, ATAD2, CD44, NOTCH3, ALK and/or FAK1 may be used as potential targeted therapies against IBC.Electronic supplementary materialThe online version of this article (doi:10.1007/s10549-013-2600-4) contains supplementary material, which is available to authorized users.

Abstract 1600: Simvastatin targets breast cancer stem-like cells by inhibiting intracellular signaling pathways leading to radiosensitization of inflammatory breast cancer cells.

Abstract Background: Inflammatory breast cancer (IBC) is responsible for 10% of breast cancer deaths although contributing to a small percentage (∼1%) of total breast cancer cases each year. The hallmarks of IBC are skin involvement and a high propensity to metastasize. It is thought that a small population of stem-like cancer cells is responsible for the metastatic potential and treatment resistance to therapies such as radiation therapy in IBC. In this study we explored the radiosensitization of several breast cancer cell lines in vitro with simvastatin and the mechanism for this. Methods: In order to determine cell proliferation, breast cancer cell lines SUM149, SUM159 and MCF-7 were grown in 96 well plates with different concentrations of simvastatin for one week. In order to determine the effect of simvastatin in stem-like cancer cells, cells were seeded in serum free, growth factor enriched, anchorage independent, treated with simvastatin for 24 hours and mammosphere formation was quantified a week later. In addition, SUM149, SUM159 and MCF-7 cells were cultured in standard monolayer cultures and in stem cell enriching anchorage independent cultures with simvastatin and treated with increasing concentrations of radiation. Survival curves were generated using Sigmaplot 8.0 and t-test was used to compare surviving fraction (SF) of groups. Finally, cell lysates were obtained from cultures untreated and treated with simvastatin and radiation and analyzed through western blot. Results: Simvastatin significantly decreased cancer cell proliferation in vitro in all cell lines (p<0.002). Cell lines SUM 159 and MCF 7 grown in mammosphere enriched media showed significant decreased mammosphere formation when grown in the presence of simvastatin (p<0.05). Moreover, simvastatin radiosensitized all cell lines in both monolayer and anchorage independent clonogenic assays. The triple-negative IBC cell line SUM149 had the greatest response to combined treatment regardless of the radiation dose used in monolayer cultures (SF2: 0.417 vs 0.319, SF4: 0.136 vs 0.075, SF6: 0.026 vs 0.018, in control vs treated respectively, all p<0.0001) and in stem cell enriching cultures (SF2: 0.880 vs 0.762, SF4: 0.863 vs 0.492, SF6: 0.920 vs 0.492, in control vs treated respectively, all p<0.0001). The triple-negative non-IBC cell line SUM159 was more sensitive to combined treatment in monolayer cultures (p<0.001) than in stem cell enriching cultures. Combination of simvastatin and radiation therapy decreased protein expression of phospho-rho A, VEGF-A, and phospho-ras. Conclusions: IBC remains a difficult disease to treat with a high rate of local recurrence after radiation therapy. Statins have been shown to increase tumor free survival in breast cancer. Herein we show simvastatin targets breast cancer stem-like cells resulting in radiosensitizing of IBC in vitro. Citation Format: Adam R. Wolfe, Lara Lacerda, Richard Larson, Bisrat G. Debeb, Wei Xu, Naoto T. Ueno, Wendy A. Woodward. Simvastatin targets breast cancer stem-like cells by inhibiting intracellular signaling pathways leading to radiosensitization of inflammatory breast cancer cells. [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 1600. doi:10.1158/1538-7445.AM2013-1600

Abstract PD03-06: Simvastatin radiosensitizes differentiated and stem-like breast cancer cell lines and is associated with improved local control in inflammatory breast cancer patients treated with post-mastectomy radiation

Abstract Among women with non-inflammatory triple-negative and triple-negative inflammatory breast cancer (IBC), the 5-yr actuarial rates of local failure after radiation are 11%–35% and 45%, respectively, in part influenced by the contribution of radioresistant cancer stem cells to these cancers. Herein we explored the radiosensitization of breast cancer stem-like cells in vitro and examined the influence on local control after post-mastectomy radiation (PMRT) among IBC patients taking statins. SUM149, SUM159 and MCF-7 cells were cultured in standard monolayer cultures and stem cell enriching anchorage independent clonogenic cultures with simvastatin and treated with increasing concentrations of radiation. Survival curves were generated and t-test was used to compare surviving fraction (SF) of groups. p < 0.05 was considered significant. Clinical information was extracted from the charts of 534 Stage III IBC patients treated with neoadjuvant systemic therapy, mastectomy and PMRT from 1970–2011. Clinical characteristics between statin and no statin cohorts were compared using the chi-square statistic. Local regional recurrence-free survival (LRFS) was determined using the Kaplan Meier method and compared with log rank. Simvastatin radiosensitized all cell lines in both types of clonogenic culture assays. The triple-negative IBC cell line SUM149 had the greatest response to combined treatment regardless of the radiation dose used in monolayer cultures (SF2: 0.417 vs 0.319, SF4: 0.136 vs 0.075, SF6: 0.026 vs 0.018, in control vs treated respectively, all p < 0.0001) and in anchorage independent cultures (SF2: 0.880 vs 0.762, SF4: 0.863 vs 0.492, SF6: 0.920 vs 0.492, in control vs treated respectively, all p < 0.0001). The triple-negative non-IBC cell line SUM159 was more sensitive to combined treatment in monolayer cultures (p < 0.001) than in stem cell enriching cultures. In a retrospective clinical study of 534 IBC patients, 84 patients used a statin. Median follow-up time for the entire cohort was 2.6 yrs. As expected, statin users were older (80% vs 44% over 50, p < 0.001) and were more often obese than non-statin users (59% vs 42%, p < 0.001). Receptor status was balanced between these groups. Statin users were more often pathologically node-negative (p < 0.01) and had fewer grade III tumors (p < 0.04). On univariate analysis actuarial 3-yr LRFS was higher among statin users, 89% vs 75%, p = 0.009. On multivariate analyisis TNBC (HR = 0.369, p < 0.0001), lymphatic invasion (HR = 0.430, p = 0.0007), neoadjuvant pathological response (HR = 0.242, p = 0.006) and statin use (HR = 0.422, p = 0.03) were independently associated with higher LRFS. Patients with IBC and triple negative non-IBC breast cancer have the highest rates of local failure and no available known radiosensitizers. Here we report significant improvement in local control after PMRT among statin users with IBC and significant radiosensitization across triple-negative and IBC cell lines of multiple subtypes using simvastatin. Clinical value in patients without hypercholesterolemia remains to be established. These encouraging data suggest simvastatin may be an appropriate radiosensitizing agent for clinical trials. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr PD03-06.

Abstract 5210: Impact of tumor microenvironment on expansion of tumor initiating cells in breast cancer metastasis

Abstract Breast cancer metastasis which ultimately results in breast cancer death, is an event believed to be initiated by the migration of tumor initiating cells (TIC) from the primary tumor to niches for micrometastatic disease. Recent data suggests the tumor microenvironment promotes TIC. The clinical relevance of secreted factors from the microenvironment on TIC surrogate, mammosphere (MS) formation and MS sensitivity to drug therapy was investigated using breast cancer patient fluids inherently conditioned by the tumor microenvironment: post-operative seromas and malignant pleural effusions. Fluids from 20 patients with breast cancer (8 seromas and 13 pleural effusions) and mesenchymal stem cells (MSC) from healthy donors were collected on IRB approved protocols. Cellular components were eliminated from patient-derived fluids using density-gradient centrifugation. MSC conditioned media (MSC-CM) was collected from 3D cultures of primary MSC. Luminex multiplex array platform was used to characterize 79 cytokine and growth factor components of all fluids. In addition, MSC-CM and patient-derived fluids were added to cultures of breast cancer cell lines: MCF-7, an estrogen receptor (ER)-positive cell line; SUM149, a triple-negative inflammatory breast cancer cell line; and SUM159, a triple-negative metaplastic breast cancer cell line and MS forming efficiency was examined. Our results show that pleural effusions and seromas are enriched for factors also secreted by MSC such as MCP-1, GRO, IL-6 and IL-8. We found remarkable similarities regarding the cytokines and growth factors profile in pleural effusions and seromas. Both patient-derived fluids have comparable amount of IL-2, IL-3, IL-4 and IL-10, as well as, VEGF, EGF and FGF-b. IL-1β and IL-16 were significantly different between pleural effusions and seromas. Seroma fluid from bilateral drains in a patient with an invasive cancer and a contralateral benign mastectomy had very similar cytokine concentrations. Moreover, MSC-CM and pleural fluids from ER+ and ER- patients increased the MS formation efficiency of both triple-negative cell lines while seroma fluids from ER+ and ER- patients increased the MS formation efficiency of ER-positive cell line MCF-7. Finally, we evaluated the impact of a panel of drugs on cell cultures grown with MSC-CM and patient-derived fluids. We found that the effect of chemotherapies on MS formation can be attenuated by patient-derived fluids. Seroma and pleural effusion fluids from breast cancer patients have similar cytokine profiles, change MS formation efficiency of standard breast cancer cell models, and mediate sensitivity to therapy. Here we demonstrate that host and microenvironmental factors are critical for determining resistance to therapy. Future studies will investigate the prognostic implications of factors that promote TIC survival in the fluid tumor microenvironment. 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 5210. doi:1538-7445.AM2012-5210