Cell Line SUM149 Research Articles

Abstract P6-01-20: Effects of local and radiation enhanced TGFβ on the invasive nature of inflammatory breast cancer cells

Abstract Inflammatory Breast Cancer (IBC) is the deadliest form of epithelial breast cancer, accounting for ~10% of breast cancer deaths in the United States. A hallmark of IBC is the formation intralymphatic emboli that are known to be chemotherapy and radiation resistant and contribute to rapid metastasis. This form of breast cancer progresses very quickly, expressing aggressive behavior. IBC and melanoma share a number of similarities in disease presentation and progression. Both spread via dermal lymphatics, form intralymphatic emboli and have a propensity to form cutaneous metastases. Melanoma can also present as “inflammatory melanoma”, which resembles IBC phenotypically. Thus, new leads for studying cutaneous metastasis can be gathered from the melanoma literature. Studies demonstrate a role for transforming growth factor beta (TGFβ) in the etiology of melanoma cutaneous metastasis. TGFβ promotes tumor cell invasion and its expression can be induced in the stroma by radiation treatment. Recently, our lab has demonstrated low expression of TGFβ in IBC patients, which we believe promotes cohesive invasion of IBC cells. Stimulation of IBC cells with 2ng/ml of TGFβ causes altered tumor cell behavior such as stimulating single cell invasion. The invasion of KPL4, SUM149 and MDA-MB231 cell lines were significantly higher in TGFβ stimulated cells compared to non-stimulated cells. As in melanoma cells we hypothesize that radiation enhanced local TGFβ production in the stroma. We radiated normal human epidermal fibroblasts cells with 0gy, 0.5gy, 1.0gy, 2.5gy and 5.0gy intensity and observed that the invasion was significantly higher in 1.0gy, 2.5gy and 5.0gy. We have also looked at the levels of TGFβ-1 in different conditioned medium. ELISA results don't show significant level of increase in TGFβ-1. Since there are two more TGFβ receptors in the TGFβ pathway (TGFβ -2 TGFβ -3), next I will look at the level of TGFβ-2 and 3 on the different intensity of radiation-conditioned medium. Our prediction is that the increase in the invasion of IBC cells is because of TGFβ, which alters the cohesive nature of IBC cells, and enhance single cell invasion. Moreover, radiation increases TGFβ levels in the stroma, which is responsible for rapid metastasis of IBC cells to the skin. Citation Format: Barkataki S, van Golen K. Effects of local and radiation enhanced TGFβ on the invasive nature of inflammatory breast cancer cells [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 P6-01-20.

Matriptase regulates c-Met mediated proliferation and invasion in inflammatory breast cancer.

The poor prognosis for patients with inflammatory breast cancer (IBC) compared to patients with other types of breast cancers emphasizes the need to better understand the molecular underpinnings of this disease with the goal of developing effective targeted therapeutics. Dysregulation of matriptase expression, an epithelial-specific member of the type II transmembrane serine protease family, has been demonstrated in many different cancer types. To date, no studies have assessed the expression and potential pro-oncogenic role of matriptase in IBC. We examined the functional relationship between matriptase and the HGF/c-MET signaling pathway in the IBC cell lines SUM149 and SUM190, and in IBC patient samples. Matriptase and c-Met proteins are localized on the surface membrane of IBC cells and their expression is strongly correlated in infiltrating cancer cells and in the cancer cells of lymphatic emboli in patient samples. Abrogation of matriptase expression by silencing with RNAi or inhibition of matriptase proteolytic activity with a synthetic inhibitor impairs the conversion of inactive pro-HGF to active HGF and subsequent c-Met-mediated signaling, leading to efficient impairment of proliferation and invasion of IBC cells. These data show the potential of matriptase inhibitors as a novel targeted therapy for IBC, and lay the groundwork for the development and testing of such drugs.

Abstract 3236: Macrophages increase the expression of RhoC in inflammatory breast cancer leading to increased migration

Abstract Inflammatory breast cancer (IBC) is considered the most lethal form of breast cancer due to its ability to progress quickly and the frequent presence of metastasis at diagnosis. African Americas are disproportionally diagnosed with IBC and often have worse outcomes than Caucasians. By investigating IBC in both African American and Caucasian cell lines we seek to understand the differences in IBC progression and help address disparities by providing new anti-IBC strategies. RhoC GTPase is overexpressed in 90% of IBC tumors and is known to increase cell motility. Sites of inflammation, as seen in IBC, attract tumor associated macrophages (TAMs), which have been found to facilitate the movement and invasion of many breast cancers. We hypothesize that TAMs play a role in increasing RhoC expression in IBC cell lines, consequently leading to IBC's severe migratory and metastatic potential. A novel microfluidic device created by our team was used to measure the migratory phenotype of IBC cell lines in response to macrophage conditioned media (CM) and cytokine stimulation. IBC cell lines were treated with CM, cytokines, or pathway inhibitors then Western blotting was used to determine protein expression and phosphorylation to identify important signaling pathways. We found the expression of RhoC significantly increased in two different IBC cell lines, SUM149 (African American) and SUM190 (Caucasian), after culturing with conditioned media from the macrophage-differentiated U937 monocytic cell line. This increase was not detected in either the normal-like MCF-10A breast epithelial cell line or the non-IBC MDA-MB-231 triple negative breast cancer cell line. CM caused a significant increase in the migration distance and frequency of both SUM149 and SUM190 cell lines. Analysis of the CM determined CCL2, CCL5, and IL-8 to be the key mediators in the macrophage CM. Western blotting proposes that CCL2, CCL5, and IL-8 stimulation causes twice as much RhoC expression compared to the control. Further analysis suggests a role for the MAPK pathway in controlling RhoC expression and migration. Macrophage conditioned media causes an increase in RhoC expression in IBC cell lines and stimulates migration. Individual cytokines can lead to an increase in RhoC possibly through the MAPK pathway. Studies involving RhoC inhibitors are ongoing and could yield promising therapies for the prevention of metastasis in IBC. By understanding the specific mechanism of TAMs’ effects on IBC, we hope to learn how to control the lethal metastatic nature of IBC and improve outcomes for patients of all ethnicities. Citation Format: Julie Madden, Steve Allen, Yu-Chi Shen, Chelsea Fournier, ZhiFen Wu, Liwei Bao, Sofia Merajver. Macrophages increase the expression of RhoC in inflammatory breast cancer leading to increased migration. [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 3236.

Abstract 651: Male mouse model of canine and human inflammatory breast cancer (IBC)

Abstract Inflammatory breast cancer (IBC) is an aggressive type of breast cancer with poor survival in women. Canine IBC has been proposed as a good surrogate model for the study of the human disease. Male breast cancer is a rare disease, accounting for less than 1% of all malignancies in men. Although rarely, IBC has been also found in men. The aim was to evaluate the capacity of male SCID mice to develop IBC tumors after the injection of canine and human IBC cell lines (IPC-366 and SUM149, respectively). Eighty SCID mice: 40 male and 40 females, 6-8 weeks old were used. Suspensions of 106 IPC-366 and SUM149 cells were implanted subcutaneously into the fourth inguinal mammary gland in 20 male and 20 female mice (of each cell line), respectively. Mice were inspected twice a week for the development of tumors. Tumors were monitored by palpation and measured by calipers. Mice were sacrificed when tumors reached 1.5 cm3 and tumors and organs were removed for immunohistochemical (IHC) characterization. A portion of tumor was used for hormonal analysis (Progesterone, Estradiol, Estrone sulphate, Testosterone, Androstenedione). Results revealed that IPC-366 reproduced tumors in 90% (male) vs 100% (female) after 2 weeks p.i. SUM149 reproduced tumors in 40% (male) vs 80% (female) at 4 weeks p.i. Both cell lines produced metastasis in lungs. Higher metastatic rates found in females than in males (IPC-366: 90% vs 20%; SUM149: 80% vs 50%). EIA hormonal analysis revealed that male tumors had higher T and SO4E1 concentrations compared to female tumors (p< 0.05). IHC analysis revealed that male and female tumors shared immunophenotipic characteristics. All xenografts were highly proliferative (high Ki-67 indexes), triple negative (ER-, PR-, HER-2-), vimentin positive and contained less than 10% of cells positive to AE1-AE3 and CK-14. In conclusion, IPC-366 and SUM149 cell lines can reproduce tumors in male mice capable of metastasize. Male and female xenograft models shared immunophenotypic characteristics. Male mice model would be useful for future IBC research using a different endocrine environment that could help to better understand the androgens function in canine and human IBC. Citation Format: Sara Caceres, Laura Peña, Maria J. Illera, Gema Silvan, Paloma J. de andres, Hui Gao, Wendy A. Woodward, James M. Reuben, Juan C. Illera. Male mouse model of canine and human inflammatory breast cancer (IBC). [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 651.

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.

Abstract 4596: Regulation of epidermal growth factor receptor signaling in triple-negative breast cancer

Abstract Although the basal-like 1 (BL1) and BL2 subtypes of triple negative breast cancer (TNBC) share similar genetic and biological characteristics, patients with BL2 type tumors have markedly poorer prognosis. In addition, despite the overexpression of epidermal growth factor receptor (EGFR) characteristically observed in basal-like TNBC, EGFR inhibitors have performed poorly in clinical trials. In order to understand the mechanism of resistance to EGFR inhibition in these tumors, our laboratory utilized cell line models of BL2 (SUM-149 and SUM-229) and BL1 (MDA-MB-468; MM468) TNBC, all of which overexpress EGFR, are PTEN null and exhibit elevated AKT phosphorylation (p-AKT). Compared to MCF10A and MM-468 cells, AKT phosphorylation in the SUM-149 and SUM-229 cell lines was resistant to treatment with the EGFR inhibitor gefitinib, as well as to the PI3K inhibitor BKM120 (BKM) and the mTOR inhibitor KU-0063794 (KU). Even treatment with the allosteric AKT inhibitor MK2206 (MK) only partially reduced p-AKT. RT-PCR and immunoblot analysis of AKT isoform expression showed that in SUM-149 and SUM-229 cells, AKT3 is expressed at higher levels than AKT1 or AKT2 at both the protein and message level. Because AKT3 has a higher IC50 for MK than AKT1 or AKT2, increasing doses of MK were used in an effort to abrogate AKT phosphorylation. The results showed that MK treatment at higher concentrations (5 μM) decreased p-AKT. In addition, shRNA-mediated knock-down of AKT3 sensitized the cells to inhibition of AKT by BKM, KU and MK. Interestingly, MK treatment alone had little effect on colony forming efficiency, even at higher doses, but when added in combination with gefitinib, MK dramatically reduced colony formation. We then investigated the effect of PTEN re-expression on signaling in these cell lines. PTEN re-expression alone had little effect on p-AKT in SUM-149 and SUM-229 cells, but resulted in a dramatic reduction in p-AKT in MM-468 cells. Although PTEN expression alone had little effect on p-AKT in SUM-149 and 229 cells, these cells exhibited decreased p-AKT following treatment with gefitinib and were sensitive to inhibition of AKT by MK. In addition, treatment of SUM-149/PTEN cells with gefitinib markedly reduced colony-forming efficiency. Our laboratory has previously demonstrated that in SUM-149 cells, a set of genes that are essential for survival, including PLK1, are uncoupled from regulation by EGFR. Thus, we examined the effect of AKT inhibition on expression of PLK1 and found that inhibition of AKT or re-expression of PTEN was able to recouple EGFR-mediated regulation of PLK1 expression. Our results demonstrate that PI-3 kinase and mTORC2 drive phosphorylation of AKT1 but not AKT3 in SUM-149 cells, which explains their relative resistance to these targeted drugs. Citation Format: Christiana S. Kappler, Ericka L. Smith, Stephen Ethier. Regulation of epidermal growth factor receptor signaling in triple-negative breast cancer. [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 4596.

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.

Canine cell line, IPC-366, as a good model for the study of inflammatory breast cancer.

Inflammatory breast cancer (IBC) is an aggressive type of cancer with poor survival in women. Inflammatory mammary cancer (IMC) in dogs is very similar to human IBC and it has been proposed as a good surrogate model for study the human disease. The aim was to determine if IPC-366 shared characteristics with the IBC cell line SUM149. The comparison was conducted in terms of ability to grow (adherent and nonadherent conditions), stem cell markers expression using flow cytometry, protein production using western blot and tumorigenic capacity. Our results revealed that both are capable of forming long-term mammospheres with a grape-like morphology. Adherent and nonadherent cultures exhibited fast growth in vivo. Stem cell markers expressions showed that IPC-366 and SUM149 in adherent and nonadherent conditions has mesenchymal-like characteristics, E-cadherin and N-cadherin, was higher in adherent than in nonadherent cultures. Therefore, this study determines that both cell lines are similar and IPC-366 is a good model for the human and canine disease.

Sulforaphane Analogues with Heterocyclic Moieties: Syntheses and Inhibitory Activities against Cancer Cell Lines.

Recent studies have shown that sulforaphane (SFN) selectively inhibits the growth of ALDH+ breast cancer stem-like cells.Herein, a series of SFN analogues were synthesized and evaluated against breast cancer cell lines MCF-7 and SUM-159, and the leukemia stem cell-like cell line KG-1a. These SFN analogues were characterized by the replacement of the methyl group with heterocyclic moieties, and the replacement of the sulfoxide group with sulfide or sulfone. A growth inhibitory assay indicated that the tetrazole analogs 3d, 8d and 9d were significantly more potent than SFN against the three cancer cell lines. Compound 14c, the water soluble derivative of tetrazole sulfide 3d, demonstrated higher potency against KG-1a cell line than 3d. SFN, 3d and 14c significantly induced the activation of caspase-3, and reduced the ALDH+ subpopulation in the SUM159 cell line, while the marketed drug doxrubicin(DOX) increased the ALDH+ subpopulation.

The effects of CEP-37440, an inhibitor of focal adhesion kinase, in vitro and in vivo on inflammatory breast cancer cells.

BackgroundInflammatory breast cancer (IBC) is an aggressive type of advanced breast cancer with a poor prognosis. We recently found that focal adhesion kinase 1 (FAK1) is upregulated and phosphorylated (active) in IBC. In this study, we investigated the effect of CEP-37440, a dual inhibitor of FAK1 and anaplastic lymphoma kinase (ALK), using human IBC cell lines and preclinical models of IBC.MethodsCell proliferation assays were performed in the presence of several concentrations of CEP-37440 using IBC and triple-negative breast cancer non-IBC cell lines. In vitro, we studied the expression of total FAK1, phospho-FAK1 (Tyr 397), total ALK and phospho-ALK (Tyr 1604). In vivo, we tested CEP-37440 using FC-IBC02, SUM149, and SUM190 IBC xenograft mouse models.ResultsCEP-37440 at low concentration decreased the proliferation of the IBC cell lines FC-IBC02, SUM190, and KPL4, while not affecting the proliferation of normal breast epithelial cells. At higher concentration, CEP-37440 was also able to inhibit the proliferation of the IBC cell line MDA-IBC03 and the triple-negative non-IBC cell lines MDA-MB-231 and MDA-MB-468; the IBC cell line SUM149 showed a slight response to the drug. CEP-37440 decreased the cell proliferation of FC-IBC02, SUM190, and KPL4 by blocking the autophosphorylation kinase activity of FAK1 (Tyr 397). None of the cells evaluated expressed ALK. In vivo, after 7 weeks of CEP-37440 treatment, the SUM190, FC-IBC02, and SUM149 breast tumor xenografts were smaller in mice treated with 55 mg/kg bid CEP-37440 compared to the controls; the tumor growth inhibition (TGI) was 79.7 %, 33 %, and 23 %, respectively. None of the FC-IBC02 breast xenografts mice treated with CEP-37440 developed brain metastasis while 20 % of the mice in the control group developed brain metastasis. Expression array analyses in FC-IBC02 cells showed that CEP-37440 affects the expression of genes related to apoptosis, interferon signaling, and cytokines.ConclusionsCEP-37440 is effective against some IBC cells that express phospho-FAK1 (Tyr 397), and its antiproliferative activity is related to its ability to decrease phospho-FAK1. Our results suggest that combinational therapies could be more effective than using CEP-37440 as a single agent.Electronic supplementary materialThe online version of this article (doi:10.1186/s13058-016-0694-4) contains supplementary material, which is available to authorized users.

Profile of Steroid Receptors and Increased Aromatase Immunoexpression in Canine Inflammatory Mammary Cancer as a Potential Therapeutic Target.

Canine inflammatory mammary cancer (IMC) has been proposed as a model for the study of human inflammatory breast cancer (IBC). The aims of this study were to compare the immunohistochemical expression of aromatase (Arom) and several hormone receptors [estrogen receptor α (ERα), estrogen receptor β (ERβ), progesterone receptor (PR) and androgen receptor (AR)], in 21 IMC cases vs 19 non-IMC; and to study the possible effect of letrozole on canine IMC and human inflammatory breast cancer (IBC) in vitro using IPC-366 and SUM-149 cell lines. Significant elevations of the means of Arom Total Score (TS), ERβ TS and PR TS were found in the IMC group (p = 0.025, p = 0.038 and p = 0.037, respectively). Secondary IMC tumours expressed higher levels of Arom than primary IMC (p = 0.029). Non-IMC PR- tumours contained higher levels of Arom than non-IMC PR+ tumours (p = 0.007). After the addition of letrozole, the number of IMC and IBC cells dropped drastically. The overexpression of Arom found and the results obtained in vitro further support canine IMC as a model for the study of IBC and future approaches to the treatment of dogs with mammary cancer, and especially IMC, using Arom inhibitors.

Abstract P3-04-04: Estrogen receptor β5 increases aggressiveness of the triple negative breast cancer cell line SUM159

Abstract Recent clinical studies are indicating that the estrogen receptor β variant β5 (ERβ5) expression correlates to worse prognosis. We wanted to know if expression of ERβ5 is changing the growth behavior of the triple negative cell line SUM159. Estrogen receptor β5 is highly similar to estrogen receptor β1 except for a truncated C-terminus making the remaining ligand binding domain incapable of binding to estrogen. In addition a 4 amino acid unique peptide is added to the C-terminal end. Stably expressing ERβ5 using a transposon integrated tetracycline regulated expression system we find that expression of ERβ5 increases proliferation of the triple negative SUM159 cells especially in reduced serum condition compared to control cells. Since SUM159 have been shown to depend on autocrine stimulation for growth we are suggesting that expression of ERβ5 is affecting the production of autocrine growth factors. Citation Format: Faria M, Tin-U C, Dey P, Gustafsson J-A, Strom AM. Estrogen receptor β5 increases aggressiveness of the triple negative breast cancer cell line SUM159. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-04-04.

Abstract P1-03-01: Lipoproteins regulate the effects of macrophages and mesenchymal stem cells on radiation response of inflammatory breast cancer cells

Abstract Inflammatory breast cancer (IBC) represents the most aggressive manifestation of breast cancer and results in up to 10% of all breast cancer-related deaths. Lack of IBC-specific targetable drivers suggests involvement of other cell types besides breast tumor epithelial cells. The multidisciplinary treatment of IBC consists of pre-operative neoadjuvant chemotherapy (with targeted agents for Her2- and ER-positive cases), radical mastectomy, followed by radiotherapy. Given that: 1) radiation therapy is a main component in treatment of IBC, 2) we have previously shown that high-density lipoproteins (HDL) mediates outcomes after radiation for IBC, and 3) the possible involvement of tumor micro-environment, it is critically important to understand the role of microenvironment such as tumor-associated macrophages and mesenchymal stem cells (MSC) on radiation response of IBC cells and how this stroma-epithelial crosstalk is regulated by lipoproteins. Here, we used in vitro co-culture system and 2D clonogenic assays of radiation resistance to examine the impact of both polarized human THP1 macrophages and MSCs on radiation response of human IBC cell lines. Further, we determined the effect of MSC-educated THP1 and THP1-educated MSCs, when co-cultured with IBC cells, on radiation response. We also determined the effect of HDL on radiation response of IBC cells co-cultured with M2-polarized (TLR3) MSCs. Our findings demonstrate that while LPS-treated, M1-polarized MSC (TLR4) co-cultured with IBC cell lines SUM149 and IBC3 leads to radio-sensitization, co-culture of IBC cells with Poly (I:C)-treated, M2-polarized MSC (TLR3) leads to radio-resistance of IBC cells. In a similar manner, co-culture of IBC cells with THP1 macrophages polarized to either M1 phenotype (LPS and IFN-γ treated) or M2 phenotype (IL4 and IL13 treated), mediate radio-sensitivity and radio-resistance, respectively, of SUM149 IBC cells. In order to provide a more comprehensive model of macrophage-MSC-IBC crosstalk, we co-cultured IBC cells with THP1 macrophages that have been previously co-cultured (i.e. "educated") with MSC and compared the effect of MSC-educated THP1 versus non-educated on radiation response of IBC cells. Our data show that MSC-education of THP1 enhances radioresistance of IBC SUM149 and KPL4 cells co-cultured with THP1 cells. In a recent publication, we showed that HDL radiosensitize IBC cells and decrease their self-renewal potential. To expand our recently published findings, here we tested whether HDL co-treatment has any effect on MSC-M2 (TLR3)-mediated radioresistance of IBC cells. Our current findings, show that co-treatment of HDL inhibits MSC-M2-mediated radioresistance of SUM149 IBC cells. In sum, these data suggest that cells within tumor micro-environment such as macrophages and MSCs regulate radiation response of IBC cells and this can be altered by lipoproteins. Citation Format: Rahal OM, Wolfe AR, Larson RA, Ueno NT, Reuben JM, Woodward WW. Lipoproteins regulate the effects of macrophages and mesenchymal stem cells on radiation response of inflammatory breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-03-01.

Abstract B12: Co-targeting PTEN-defined TNBC with p110beta-isoform specific inhibitor and PARP inhibitor BMN 673: A predictive context to sensitize PARP inhibitors in TNBC

Abstract Background: TNBC is a highly aggressive form of BRCA-associated BC subtype for which chemotherapy still remains a major form of treatment to which patients initially respond yet a majority of them inevitably relapse. PARP1 has been identified as a target in BRCA-defined cancers and inhibitors of PARP1 has been recently approved by FDA as targeted agents in BRCA-defined cancers. The loss of PTEN is the most common “first event” associated with basal-like subtype (Martins et al., 2012) and this PI3K-pathway activating event (deletion/mutation/loss of PTEN) occurs more frequently (35%) than PIK3CA mutations in this subtype of BC (Ellis & Perou, 2013). In the cytoplasm, PTEN-loss mediated upregulation of PI3K/AKT signal is known to depend on the PI3Kbeta-isoform. In contrast nuclear PTEN controls DNA repair (Bassi et al., 2013). A failure to repair the damaged DNA upon inhibition of PARP causes accumulation of DNA double-strand breaks and leads to apoptosis of cancer cells. Thus we hypothesized that a combination of p110beta-isoform specific inhibitor and PARP inhibitor will sensitize PARP inhibitors in PTEN-null TNBC model. Methods: Five PARP inhibitors, Talazoparib (BMN673, B), Niraparib (N), Olaparib (O), Rucaparib (R) and Veliparib (V) were tested in four PTEN-null TNBC cell lines, MDA-MB468, HCC70 (p.F90fs*9), BT549 (p.V275fs*1) and SUM149 cell lines in combination with p110beta specific inhibitor, AZD6482. Proliferative, apoptotic and PARylation signals following drug combinations were demonstrated by WB in a dose and time dependent manner. Pro-apoptotic and anti-proliferative effects were verified using complementary 3D ON-TOP assay, real time proliferation (Incucyte), AnnexinV and cl-caspase3 analyses. In order to understand the initial effects of isoform-specific inhibitors of PI3K we also compared anti-proliferative signals of GDC-0032 (p110 beta sparing inhibitor) and GDC-0941 (pan PI3K inhibitor) with that of AZD6482 at 3/6 hours in MDA-MB468 and SUM149 cells. Results: In PTEN null TNBC cells 500 nM BMN673 alone was effective in slowing cell proliferation and inducing apoptosis while AZD6482 (both 5-10 uM) did not have anti-proliferative or pro-apoptotic effects at 48 and 72 hours. Combinations of different PARPi (100nM of B, 1uM of N, 10uM of O, 10uM of R and 10uM of V) with AZD6482 blocked 3D growth of PTEN null BT549 and MDA-MB468 TNBT cells in a time dependent manner (96 hours & 7 days). PARP inhibitors abrogated PAR signals either alone or in combination with carboplatin in both BRCA1/2 WT and mutated cells. The most pronounced anti-tumor effect was observed with the combination of B and AZD6482 which was mechanistically explained by the robust increase of AnnexinV positive cells following a single 500nM dose of B at both 48 and 72 hours. In both MDA-MB468 and SUM149 cells although panPI3K inhibitor, GDC-0941 downregulated pAKT (S473, T308), pP70S6K (T389), pS6RP and p4EBP1 (T37/46) levels, p110 alpha-specific inhibitor GDC-0032 failed to perturb these signals while AZD6482 blocked them all. In summary, we demonstrate a remarkable sensitivity of tumor cells to PARP inhibition in PTEN-defined TNBC models and identify that PTEN-nullness as a potential predictive context for a possible co-targeting of PI3K pathway to further sensitize TNBC to PARP inhibitors. Significance: Our findings squarely place PTEN expression at the nexus of oncogenic signals in TNBC and indicate that TNBC patients with no PTEN activity in their tumors might benefit from combined p110beta-isoform specific inhibitor and PARP inhibitor. Citation Format: Nandini Dey, Jennifer H. Carlson, Pradip De, Brian Leyland-Jones. Co-targeting PTEN-defined TNBC with p110beta-isoform specific inhibitor and PARP inhibitor BMN 673: A predictive context to sensitize PARP inhibitors in TNBC. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B12.

MiR-33a Decreases High-Density Lipoprotein-Induced Radiation Sensitivity in Breast Cancer

We previously showed that high-density lipoprotein (HDL) radiosensitizes inflammatory breast cancer (IBC) cells invitro and is associated with better local control after radiation therapy in IBC patients. The microRNA miR-33 family negatively regulates the adenosine triphosphate binding cassette transporter subfamily A member 1. We hypothesized that variations in miR-33a expression in IBC cancer cells versus non-IBC cells would correlate with radiation sensitivity following exposure to HDL invitro. MiR-33a expression was analyzed by reverse transcriptase-polymerase chain reaction in 4cell lines representing common clinical breast cancer subtypes. Overexpression and knockdown of miR-33a was demonstrated via transfection of an miR-33a mimic or an anti-miR-33a construct in high- and low-expressing miR-33a cell lines. Clonogenic survival invitro in these cells was quantified at baseline and following HDL treatment. MiR-33a expression on distant relapse-free survival (DRFS) of 210 cases downloaded from the Oxford breast cancer dataset was determined. Expression levels of miR-33a were lower in IBC cell lines and IBC tumor samples than in non-IBC cell lines and normal breast tissue. Cholesterol concentrations in the cell membranes were higher in IBC cells than in non-IBC cells. Clonogenic survival following 24hours of HDL treatment was decreased in response to irradiation in the low-miR-33a-expressing cell lines SUM149 and KPL4, but survival following HDL treatment decreased in the high-miR-33a-expressing celllines MDA-MB-231 and SUM159. In the high-miR-33a-expressing cell lines, anti-miR-33a transfection decreased radiation resistance in clonogenic assays. Conversely, in the low-miR-33a-expressing cell lines, the miR-33a mimic reversed the HDL-induced radiation sensitization. Breast cancer patients in the top quartile based on miR-33a expression had markedly lower rates of DRFS than the bottom quartile (P=.0228, log-rank test). For breast cancer patients treated with radiation, high miR-33a expression predicted worse overall survival (P=.06). Our results reveal miR-33a negatively regulates HDL-induced radiation sensitivity in breast cancer.

Abstract 430: MCP-1/CCL2 and IL-8 regulate proteolytic activity of triple negative inflammatory breast cancer via cathepsin B, ERK1/2, JAK1 and Src signaling pathways

Abstract Background Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. Recently, we have shown that macrophages isolated from the tumor microenvironment of IBC patients secrete monocyte chemoattractant protein-1/CC-chemokine ligand 2 (MCP-1/CCL2) and IL-8 that augment dissemination and metastasis of IB carcinoma cells. However, the precise molecular mechanism by which these cytokines exert their effect is still unclear. Methods In the present study, the triple negative breast cancer (TNBC) cell lines Sum149 (IBC) and HCC70 (non-IBC) cells were employed to analyze the effect of IL-8 and MCP-1/CCL2 on the proteolytic activity using live cell imaging assay, expression of cathepsin B and the activation status of STAT3, AKT, JAK1 and Src. In addition, we enrolled TNBC patients sub-grouped into IBC (n = 15), non-IBC (n = 19) patients. Results Our results revealed that upon stimulation with MCP-1/CCL2 and IL-8, Sum149 cells and HCC70 exhibited an increase in DQ-collagen degradation-mediated proteolytic activity. Mechanistically, MCP-1/CCL2 and IL-8 increase collagen degradation via enhanced expression of cathepsin B single chain mature enzyme (31 kDa) and the heavy chain of double chain mature enzyme (25/26 kDa). Moreover, MCP-1/CCL2 and IL-8 enhance activation of STAT3, ERK1/2 and AKT in both Sum149 and HCC70 cells. Interestingly, we detected over-expression of cathepsin B in the carcinoma tissues of TNBC-IBC patients compared to non-IBC patients. Over expression of cathepsin B found to be associated with activation of Src and ERK1/2, in IBC as compared to non-IBC tissues. Conclusions Our data indicate that MCP-1/CCL2 and IL-8 stimulate proteolytic activity, cathepsin B expression and Src-ERK1/2 pathway in IBC tissues versus non-IBC. Targeting MCP-1/CCL2 and IL-8 in triple negative IBC patients represents a promising therapeutic strategy. Citation Format: Sherif A. Ibrahim, Eslam A. Elghonaimy, Mohamed El-Shinawi, Medhat El-Halawany, Mohamed A. Nouh, Tahani El-Mamlouk, Bonnie F. Sloane, Mona Mostafa Mohamed. MCP-1/CCL2 and IL-8 regulate proteolytic activity of triple negative inflammatory breast cancer via cathepsin B, ERK1/2, JAK1 and Src signaling pathways. [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 430. doi:10.1158/1538-7445.AM2015-430

Abstract 4454: Targeting the gp130 receptor in preclinical models of triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer (BC) with poor clinical outcomes and limited therapeutic options. To date, efforts to implement targetted therapy in TNBC have had limited success. Cumulative evidence suggest that cancer stem cells (CSCs) drive resistance to therapy, tumor recurrence, and may play a determinant role in the aggressive biology of TNBC. Recent studies indicate that CSCs are in reciprocal interaction with tumor microenvironment through cytokine-mediated signaling pathways. IL-6 is emerging as an important mediator of tumor growth and CSC survival. IL-6 is the most studied ligand of the gp130 receptor, which is a potent activator of the JAK/STAT3 signaling pathway important in survival and proliferation. The gp130 receptor is emerging as a promising target for cancer therapy. SC144 is a novel first-in-class gp130 inhibitor that has shown preclinical efficacy in ovarian cancer models. This study explores the effect of gp130 inhibition with SC144 on TNBC preclinical models. Methods: SC144 was provided by Dr. Neamati's laboratory currently at the University of Michigan. We conducted in vitro studies using well-characterized human BC cell lines with a triple-negative phenotype. The effect of the novel compound SC144 on TNBC cell lines was evaluated using MTT assays for growth inhibition, ALDEFLUOR assay for identification of CSCs, ELISA tests for cytokine levels, and qRT-PCR for gene expression analysis. Results: SC144 inhibits cell growth in the TNBC cell lines SUM159, SUM149, MDAMB231, and HCC38 with IC50 values in a submicromolar range (0.87+0.17 μmol/L, 0.9+0.1 μmol/L, 0.5 μmol/L, 0.4 μmol/L, respectively). In an in vitro culture system of the TNBC cell line SUM159, docetaxel treatment induces a three-fold increase in IL-6 levels by ELISA and increases the CSC population measured by ALDEFLUOR. Pretreatment of SUM159 cells with SC144 significantly sensitized cells to docetaxel. Treatment of SUM159 cells with SC144 in addition to docetaxel significantly reduced the ALDH+ population measured by ALDEFLUOR compared to docetaxel alone (7.79+1% vs 22.23+5%). Investigating the potential effect of SC144 on the CSC population, we found a higher expression level of the IL-6 receptor in ALDH+ cells compared to ALDH- and the bulk population. Furthermore, in SUM159 cells treated with SC144 vs. control for 24 hours we found a significant decrease on expression of genes with documented relevance in CSC maintenance including CD44 (0.08 vs. 0.13), ALDH1a1 (0.001 vs. 0.006), STAT1 (0.004 vs. 0.024) and STAT3 (0.003 vs. 0.014). The in vivo study to test the efficacy of SC144 in a SUM159 xenograft model is undergoing. Conclusions: SC144, a novel gp130 inhibitor, has antitumor activity in human BC cell lines with the triple-negative phenotype. SC144 sensitizes cells to docetaxel therapy, and demonstrates an inhibitory effect on the CSC subpopulation. These findings identify a potential therapeutic target for TNBC. Citation Format: Leonel F. Hernandez-Aya, Yajing Liu, Kelsey Kerr, Shuzo Tamura, Nouri Neamati, Max S. Wicha, Monika L. Burness. Targeting the gp130 receptor in preclinical models of triple-negative breast 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 4454. doi:10.1158/1538-7445.AM2015-4454

Abstract 106: Characterization of polycomb repressive complex 2 (PRC2) subunits retinoblastoma-binding protein 4 and 7 (RbBP4/7) in triple-negative breast cancer

Abstract Around 15% of all breast cancers are classified as ER−/PR−/HER2−, or triple negative (TNBC). This subtype of breast cancer does not respond to traditional endocrine therapies or targeted agents against HER2 and is particularly invasive with a high rate of recurrence due to resistance of cancer stem cells against therapy. Currently no efficient treatments are available to target cancer stem cells in TNBC. With this unmet need, it is a necessity to determine the molecular mechanisms underlying stem cell biology in TNBC to develop better targeted therapies of this disease. Recent studies have shown that the subunits of the polycomb repressive complex 2 (PRC2), such as EZH2, have been linked to invasiveness, metastasis, and chemoresistance in TNBC. PRC2 consists of four core members: EZH2, SUZ12, EED and RbBP4/7 which together mediate H3K27 trimethylation to repress gene transcription. In addition to its role in gene regulation, PRC2 has been linked to the regulation of breast cancer stem cells. However, the function of RbBP4/7 in breast cancer and breast cancer stem cells is unknown. The purpose of this study is to examine the role of RbBP4/7 in PRC2 in maintenance of the H3K27me3 mark, regulation of cancer cell stemness, and breast tumorigenesis. Analyzing the expression of RbBP4/7 in publicly available data sets through ONCOMINE, we found that both RbBP4 and RbBP7 were overexpressed in breast cancers, especially TNBC, compared with normal breast tissues. Similar to EZH2, higher expression of RbBP4 and RbBP7 was correlated to higher incidence of metastasis and poor prognosis. Using RNA interference, we successfully knocked down RbBP4 and RbBP7 in TNBC cell lines SUM149 and MDA-MB-231. Our results show that knockdown of both RbBP4 and RbBP7 significantly decreases cell growth by 70%, reduces H3K27me3 levels and drastically inhibits the mammosphere forming capability of these breast cancer cell lines by 80%. In addition, there was an increase in apoptotic cells as assessed by Annexin V staining from 12% in untreated cells to 28% in the RbBP4/7 knockdown cells. Knockdown of either RbBP4 or RbBP7, on the other hand, only has a moderate effect on cell proliferation, H3K27me3 levels and mammosphere formation. Our studies indicate that together RbBP4 and RbBP7 help maintain PRC2 function and breast cancer stem cell phenotype and could be exploited as a novel therapeutic target for treating TNBC. The effects of RbBP4 and RbBP7 knockdown on target gene regulation and breast tumor initiation and progression using xenograft models are currently under investigation. Citation Format: Rebecca A. Reed, Miao-Chia Lo, Duxin Sun. Characterization of polycomb repressive complex 2 (PRC2) subunits retinoblastoma-binding protein 4 and 7 (RbBP4/7) in triple-negative breast 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 106. doi:10.1158/1538-7445.AM2015-106

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 4226: Histone deacetylase inhibitors sensitize cancer stem cells to PARP inhibitors in triple-negative breast cancer

Abstract Background: Triple negative breast cancers (TNBCs) have a poor prognosis and are a therapeutic challenge due to resistance to multiple chemotherapy drugs. Growing evidence suggests that cancer stem cells (CSCs) may be promising therapeutic targets for treating TNBCs. Poly ADP ribose polymerase (PARP) inhibitors have shown striking activity in preclinical models of BRCA-deficient breast carcinomas. However they are less efficacious in tumors without germline BRCA mutations, which account for more than 80% of all TNBCs. Our data demonstrates that treatment of TNBC with PARP inhibitor increases CSCs, likely due to an elevated homologous recombination (HR) repair mechanism. Several small studies have suggested that histone deacetylase (HDAC) inhibitors, which decrease HR repair proteins, may sensitize non-BRCA-mutated TNBC to PARP inhibitors. Currently, little is known whether this synergistic effect can reverse the increase in CSCs caused by PARP inhibition. In this study, our aim was to investigate if HDAC inhibitors could sensitize CSCs to PARP inhibitors in TNBCs. Results: Using four TNBC cell lines, we confirmed the synthetic lethality of HDAC and PARP inhibitors (Vorinostat and Olaparib respectively) in vitro. CompuSyn analysis revealed Combination Indices of 0.66, 0.44, 0.47 and 0.16 in SUM159, MDAMB231, SUM149 and HCC1937 cells respectively at ED50. An ALDEFLUOR assay revealed ∼1.9 fold increase in the CSC population after PARP inhibition in BRCA-mutant SUM149 and HCC1937 cells, while the absolute CSC number, (total cell number times percentage of ALDEFLUOR-positive cells) remained unchanged. Whereas in BRCA-wild type TNBC cell lines SUM159 and MDAMB231, there was no change in CSCs. Addition of the HDAC inhibitor decreased absolute CSC number by 85%, 78% and 40% in SUM149, SUM159 and HCC1937 cells respectively after 7 days of PARP inhibition. Although the mechanism is not fully understood at present, we demonstrated that Rad51, a key player in HR repair, mediates the sensitivity of TNBCs to PARP inhibition, and HDAC inhibitor prevents the formation of Rad51 foci at DNA damage site. This may indicate that HDAC inhibition sensitizes the CSCs of TNBCs to PARP inhibition via suppressing the HR pathway. Conclusion: Our data suggests that PARP inhibition only targets the bulk cancer cells in BRCA-mutant TNBC cells while sparing CSCs and has no effect on BRCA WT cells. HDAC inhibition sensitized CSCs of TNBC to PARP inhibition regardless of BRCA status, possibly via interfering with Rad51 function. The inhibitors we used in the present study are either FDA-approved or at Phase III clinical trials, and the combination is predicted to have low toxicity. Thus, our findings are promising and could be rapidly translated into clinical use. Citation Format: Yajing Liu, Rachel Martin-Trevino, Li Shang, April Davis, Max Wicha, Suling Liu, Monika Burness. Histone deacetylase inhibitors sensitize cancer stem cells to PARP inhibitors in triple-negative breast 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 4226. doi:10.1158/1538-7445.AM2015-4226