Triple-negative Inflammatory Breast Cancer Research Articles

The combined presence of CD20 + B cells and PD-L1 + tumor-infiltrating lymphocytes in inflammatory breast cancer is prognostic of improved patient outcome.

The purpose of the study was to evaluate protein expression of PD-L1 and CD20 as prognostic biomarkers of patient outcome in inflammatory breast cancer (IBC) samples. PD-L1 and CD20 protein expression was measured by immunohistochemistry in 221 pretreatment IBC biopsies. PD-L1 was assessed in tumor cells (PD-L1+ tumor cells) and tumor stromal infiltrating lymphocytes (PD-L1+ TILs); CD20 was scored in tumor-infiltrating B cells. Kaplan-Meier curves and Cox proportional hazard models were used for survival analysis. PD-L1+ tumor cells, PD-L1+ TILs, and CD20+ TILs were found in 8%, 66%, and 62% of IBC, respectively. PD-L1+ tumor cells strongly correlated with high TILs, pathological complete response (pCR), CD20+ TILs, but marginally with breast cancer-specific survival (BCSS, P = 0.057). PD-L1+ TILs strongly correlated with high TILs, CD20+ TILs, and longer disease-free survival (DFS) in all IBC and in triple-negative (TN) IBC (P < 0.035). IBC and TN IBC patients with tumors containing both CD20+ TILs and PD-L1+ TILs (CD20+TILs/PD-L1+TILs) showed longer DFS and improved BCSS (P < 0.002) than patients lacking both, or those with either CD20+ TILs or PD-L1+ TILs alone. In multivariate analyses, CD20+TILs/PD-L1+TILs status was an independent prognostic factor for DFS in IBC (hazard ratio (HR): 0.53, 95% CI 0.37-0.77) and TN IBC (HR: 0.39 95% CI 0.17-0.88), and for BCSS in IBC (HR: 0.60 95% CI 0.43-0.85) and TN IBC (HR: 0.38 95% CI 0.17-0.83). CD20+TILs/PD-L1+TILs status represents an independent favorable prognostic factor in IBC and TN IBC, suggesting a critical role for B cells in antitumor immune responses. Anti-PD-1/PD-L1 and B cell-activating immunotherapies should be explored in these settings.

Clinically relevant inflammatory breast cancer patient-derived xenograft-derived ex vivo model for evaluation of tumor-specific therapies.

Inflammatory breast cancer (IBC) is a rare and aggressive presentation of invasive breast cancer with a 62% to 68% 5-year survival rate. It is the most lethal form of breast cancer, and early recognition and treatment is important for patient survival. Like non-inflammatory breast cancer, IBC comprises multiple subtypes, with the triple-negative subtype being overrepresented. Although the current multimodality treatment regime of anthracycline- and taxane-based neoadjuvant therapy, surgery, and radiotherapy has improved the outcome of patients with triple-negative IBC, overall survival continues to be worse than in patients with non-inflammatory locally advanced breast cancer. Translation of new therapies into the clinics to successfully treat IBC has been poor, in part because of the lack of in vitro preclinical models that can accurately predict the response of the original tumor to therapy. We report the generation of a preclinical IBC patient-derived xenograft (PDX)-derived ex vivo (PDXEx) model and show that it closely replicates the tissue architecture of the original PDX tumor harvested from mice. The gene expression profile of our IBC PDXEx model had a high degree of correlation to that of the original tumor. This suggests that the process of generating the PDXEx model did not significantly alter the molecular signature of the original tumor. We demonstrate a high degree of similarity in drug response profile between a PDX mouse model and our PDXEx model generated from the same original PDX tumor tissue and treated with the same panel of drugs, indicating that our PDXEx model had high predictive value in identifying effective tumor-specific therapies. Finally, we used our PDXEx model as a platform for a robotic-based high-throughput drug screen of a 386-drug anti-cancer compound library. The top candidates identified from this drug screen all demonstrated greater therapeutic efficacy than the standard-of-care drugs used in the clinic to treat triple-negative IBC, doxorubicin and paclitaxel. Our PDXEx model is simple, and we are confident that it can be incorporated into a PDX mouse system for use as a first-pass screening platform. This will permit the identification of effective tumor-specific therapies with high predictive value in a resource-, time-, and cost-efficient manner.

Abstract OT3-05-01: TBCRC 039: Phase II study of combination ruxolitinib (INCB018424) with preoperative chemotherapy for triple negative inflammatory breast cancer

Abstract Background: Stage III triple negative(TN) inflammatory breast cancer(IBC) is associated with a poor prognosis evidenced by a 15 month(mo) median disease free survival(DFS) and overall survival(OS) of 34 mo. The substantial incidence of developing distant metastasis may be due to the prevalence of cancer cells with stem cell-like features (e.g. CD44+/CD24-) in TNIBC. The transcriptional pathway JAK2/STAT3 is associated with the survival of CD44+/CD24- cells, and preclinical data demonstrates overexpression of activated STAT3(pSTAT3) in &amp;gt; 95% of TNIBC. Preclinical studies have shown that ruxolitinib (Incyte®Corporation), an approved JAK1/JAK2 inhibitor, suppresses pSTAT3 in IBC patient derived xenograft models, and when combined with paclitaxel, results in a synergistic reduction in tumor weight. Given the lack of a known therapeutic target in TNIBC, this preoperative proof of principle study exploits the survival mechanism of CD44+/CD24- stem cells prevalent in this disease, by combining ruxolitinib(Rux) with paclitaxel(T) followed by doxorubicin/cyclophosphamide(AC). Methods: Up to 64 pts with newly diagnosed stage III TNIBC (cT4d, any N, M0) are eligible if they have adequate organ function and are willing to undergo 2 research biopsies(rbx) of the affected breast. Following baseline rbx, pts are randomized to a 7 day(d) run-in phase of Rux vs Rux(15 mg bid) + T(80mg/m2/wkx1). A 2nd rbx is obtained after the run-in phase. Pts randomized to RuxT continue to receive a total of Tx12 wks+Rux. Pts randomized to Rux alone, are re-randomized to receive Tx12 wks+Rux vs Tx12wks alone. Following T, all pts receive AC (A-60mg/m2,C-600mg/m2) every 14d x 4. Pts proceed to modified radical mastectomy (MRM) followed by chest wall/regional lymph nodes radiation therapy. Correlatives: To assess the effect of JAK inhibition with Rux on pSTAT3 and STAT3 related gene expression, molecular and genomic markers (e.g. RNA-seq, ChIPseq, FISH) will be determined in each rbx and residual tumor at MRM. The relative frequency and topology of CD44+/CD24- cell population and pSTAT3 expression by IHC will also be assessed in these tumor specimens. IL-6 and CRP plasma concentrations will be measured at baseline, prior to T and AC and prior to MRM. Statistics: The primary endpoint is change in markers of JAK/STAT inhibition. If the proportion of rbx exhibit a biologic response to Rux alone (i.e. change from pSTAT3 expression to pSTAT3 negative) is &amp;lt;10%, then Rux alone is minimally effective on JAK inhibition vs alternative hypothesis that Rux inhibits JAK if the proportion of biologic response is ≥33%. If ≥5/25 rbx treated with Rux alone have a biologic response then the hypothesis that biologic response is ≤10% is rejected with an error rate of 0.098 (target 0.10). If ≤4/25 rbx have a biologic response then the hypothesis that biologic response is ≥33% is rejected with an error rate of 0.05 (target 0.10). Biologic response of rbx with Rux alone will also be compared with the proportion of biologic response to RuxT (33% vs. 66% based upon presumed synergy with RuxT). Secondary endpoints are clinical: pathologic complete response in breast/lymph nodes, Residual Cancer Burden, DFS and OS. Clinical Trial Information: NCT02876302. Citation Format: Overmoyer B, Regan M, Polyak K, Brock J, Van Poznak C, King T, Haddad T, Stearns V, Hwang S, Winer E. TBCRC 039: Phase II study of combination ruxolitinib (INCB018424) with preoperative chemotherapy for triple negative inflammatory breast cancer [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 OT3-05-01.

Abstract OT3-06-04: A randomized phase II study of neoadjuvant panitumumab /carboplatin/paclitaxel (PaCT) versus carboplatin/paclitaxel (CT) followed by adriamycin and cyclophosphamide (AC) for newly diagnosed primary triple-negative inflammatory breast cancer (TNIBC)

Abstract Primary Objective: To determine pathological complete response (pCR) rate in patients with primary TNIBC treated with PaCT in comparison with CT, followed by AC. To explore if the pCR rate correlates with reduced nodal expression status; and with arginine methylation status of epidermal growth factor receptor (EGFR). We will identify molecular biomarkers predictive of the pCR rate by analysis of multiplexed immunohistochemical (IHC) staining, identify molecular biomarkers predictive of the pCR rate by genomic and proteomic analysis, and determine whether the inhibition of the EGFR pathway down regulates the COX-2 pathway and mesenchymal marker. Background: EGFR is overexpressed in triple negative breast cancer (TNBC) and inflammatory breast cancer (IBC). Therefore, EGFR targeted therapy may have a promising role in TNBC and IBC. A study showed that EGFR-targeted therapy may enhance the initial chemosensitivity of TNBC cells. Panitumumab blocks epidermal growth factor ligands and transforming growth factor EGFá (TGFá) binding to EGFR, inhibits tumor growth, and elicits both tumor regression and eradication of established tumors in murine xenograft tumor models. Panitumumab, a fully humanized anti-EGFR antibody, has been shown to be active in a breast cancer preclinical model using human breast cancer cell line MDA-MB-468, which has been shown to overexpress EGFR by both IHC and fluorescence in situ hybridization (FISH). Furthermore, EGFR tyrosine kinase inhibitors such as erlotinib have antitumor activity against human IBC cell lines. Thus, EGFR targeted therapy may have a promising role in TNBC and IBC. Study Design: In this open label randomized phase II trial, up to 72 patients with primary IBC, have no HER2 overexpression, and have &amp;lt;10% expression of ER and PgR, who also meet other criteria will be randomized to PaCT arm - receiving panitumumab single agent in window study and 4 cycles PaCT, or CT arm - receiving 4 cycles of CT. All patients will receive 4 cycles of AC before surgery. Statistical Considerations: A sample size of 36 patients per arm will achieve 84% power to detect a difference of 0.24 in pCR rate between 0.2 in the CT arm and 0.44 in the PaCT arm with a type I error rate of 10% using one-sided Z test. Based on historical data, we expect that the pCR rate of a PaCT regimen to achieve 24% additional efficacy compared with the CT regimen. Status of the study: Activation date: Oct. 2016. So far 6 patients have been enrolled. Enrollment continues. Sponsor: Amgen. State of Texas appropriation for rare and aggressive breast cancer research. NIH grant 1R01CA205043-01A1 Citation Format: Willey JS, Parker CA, Lim B, Valero V, Le-Petross HT, Krishnamurthy S, Woodward WA, Lucci A, Wood AL, Sun H, Babiera GV, Song J, Shen Y, Valero V, Wang X, Ueno NT. A randomized phase II study of neoadjuvant panitumumab /carboplatin/paclitaxel (PaCT) versus carboplatin/paclitaxel (CT) followed by adriamycin and cyclophosphamide (AC) for newly diagnosed primary triple-negative inflammatory breast cancer (TNIBC) [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 OT3-06-04.

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 &amp;lt; 0.05), and in vivo (P &amp;lt; 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 &amp;lt; 0.05) and in vivo (P &amp;lt; 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 &amp;lt; 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 &amp;lt; 0.05), cell membrane rigidity (P &amp;lt; 0.05), and induction of apoptosis (P &amp;lt; 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 OT1-01-03: A phase II multi-center study of BGB324 in combination with pembrolizumab in patients with previously treated, locally advanced and unresectable or metastatic triple negative breast cancer (TNBC) or triple negative inflammatory breast cancer (TN-IBC)

Abstract Background. The AXL receptor tyrosine kinase is associated with poor overall survival in breast cancer. AXL signaling is an important regulator of tumor plasticity related to epithelial-to-mesenchymal transition (EMT) and stem cell traits that drive metastasis and drug resistance. Upregulation of AXL has been associated with reduced response to anti-PD-1 therapy. Signaling via AXL is also a key suppressor of the anti-tumor innate immune response, and AXL is expressed on several cells associated with the tumor immune microenvironment. Hence AXL signaling contributes uniquely to both tumor cell intrinsic and microenvironmental anti-tumor immune suppression mechanisms. We show that AXL is required for tumor immune evasion in the 4T1/Balb/C mammary adenocarcinoma model and that blocking AXL signaling with BGB324, a selective clinical-stage small molecule AXL kinase inhibitor, enhanced the effect of immune checkpoint blockade. BGB324 + anti-CTLA-4/anti-PD-1 treated tumors displayed enhanced infiltration of cytotoxic T lymphocytes and Natural Killer cells. Importantly, responding animals rejected orthotopic 4T1 tumor cell re-challenge, demonstrating sustained tumor immunity. These data provided a translational rationale for combining AXL targeted therapy with immune checkpoint inhibitors to enhance anti-cancer immune response. Study Design. BGBC007 (NCT03184558) is an open-label, single arm, multi-center phase II study designed to assess the anti-tumor activity of BGB324 in combination with pembrolizumab in patients with previously treated, locally advanced and unresectable, or metastatic TNBC or TN-IBC. Secondary objectives include safety and pharmacokinetic profile of BGB324 and pembrolizumab in combination. A single arm, extension of Simon's 2-stage design is employed with an interim and final analysis. Up to 56 evaluable patients will be enrolled. Recruitment will be halted once 28 evaluable patients have been entered to determine the Objective Response Rate (ORR, complete response and partial response). If 5 or fewer responses are observed in up to 28 patients, the trial will be terminated in favor of the null for futility. If 11 or more responses are observed, then the trial will be stopped in favor of the alternative for demonstration of activity. If 6 to 10 patients have an observed response then a further 28 patients may be evaluated. This design provides an overall power of 80.6% to test the stated null and alternative hypothesis. BGB324 will be administered orally, once daily, in a fasted state. Days 1, 2 and 3 of BGB324 administration consists of a 'loading' dose of 400 mg followed by a dose of 200 mg daily. A fixed dose of 200 mg pembrolizumab will be given by intravenous infusion over 30 minutes every 3 weeks. BGB324 and pembrolizumab will be given until disease progression, unacceptable dose toxicity, or until 106 weeks (35 cycles). Efficacy endpoints including ORR, Duration of Response, Progression Free Survival are based on tumor imaging evaluation by RECIST 1.1. Tumor specimens will be taken to assess AXL and PD-L1 expression. Citation Format: Yule M, Wnuk-Lipinska K, Davidsen K, Blø M, Hodneland L, Engelsen A, Kang J, Lie M, Bougnaud S, Aguilera K, Ahmed L, Rybicka A, Milde Nævdal E, Deyna P, Boniecka A, Straume O, Thiery J-P, Chouaib S, Brekken RA, Gausdal G, Lorens JB. A phase II multi-center study of BGB324 in combination with pembrolizumab in patients with previously treated, locally advanced and unresectable or metastatic triple negative breast cancer (TNBC) or triple negative inflammatory breast cancer (TN-IBC) [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 OT1-01-03.

Interleukin‐18 (IL‐18) is equally expressed in inflammatory breast cancer and noninflammatory locally advanced breast cancer: A possible association with chemotherapy response

Inflammatory breast cancer (IBC) is the most aggressive form of locally advanced breast cancer. The signs of inflammation such as hyperemia and hyperthermia might suggest the possible participation of inflammatory mediators. This study investigates stromal and tumor expression of nuclear factor-kappa B (NF-κB) and interleukin-18 (IL-18) in samples obtained from IBC and noninflammatory locally advanced breast cancer (LABC) and the influence of these markers on patients' prognosis. Demographic data, tumor molecular characteristics and overall survival in both groups were also assessed. Furthermore, in this study, we evaluated the expression of IL-18 and p50 nuclear fraction of NF-κB by immunohistochemistry in specimens from IBC and LABC (T4b). We observed that 24.6% of women were diagnosed with IBC up to age 40. In addition, the patients with IBC showed a lower overall survival when compared to LABC. In regard to molecular markers, ER+ , C-erbB2- or triple negative IBC patients showed a significantly reduced overall survival. In addition, a higher IL-18 immunostaining in stroma of IBC and LABC was observed in comparison with tumor cells, but stromal immunoexpression was similar between IBC and LABC. Besides, IL-18 positivity seemed be related with a better clinical response to neoadjuvant chemotherapy. However, NF-κB expression was identical in both groups. The IL-18 is present in tumor stroma of IBC and LABC and seems to be associated with the complete response to neoadjuvant chemotherapy.

Abstract 1235: EPHA2-targeted therapy enhances the cytotoxicity of eicosapentaenoic acid against triple-negative inflammatory breast cancer

Abstract Background: Inflammatory breast cancer (IBC) is the most aggressive form of breast cancer. We have previously reported that mediators of inflammation, such as COX-2, promote the growth of Triple-Negative receptor (TN) IBC xenografts; therefore, inflammation in TN-IBC has a unique opportunity as a therapeutic strategy. Eicosapentaenoic acid (EPA), a non-toxic omega-3 fatty acid with anti-inflammatory properties, has partially reduced tumor growth in pre-clinical models of TN-IBC. Therefore, our goal is to develop a novel non-toxic approach that enhances EPA efficacy against TN-IBC in combination with targeted therapy. Methods and Results: Using a high-throughput, siRNA screen (939 genes) in the TN-IBC cell line SUM149PT, we identified Ephrin type-A receptor 2 (EPHA2), an oncogenic cell-surface receptor tyrosine kinase, as a target that modulates the sensitivity of TN-IBC cells to EPA treatment. To determine the clinical relevance of EPHA2, we interrogated a meta-analysis of breast cancer mRNA expression data sets, and found that high EPHA2 tumor expression was significantly correlated with poor overall survival in TN-IBC patients, compared to low EPHA2 expressing tumors (P = 0.01). We observed no significant correlations to other breast cancer subtypes. Similar findings were observed in vitro were EPHA2 expression predominantly occurred in the TN-IBC subtypes (19 of 30) among 49 breast cancer cell lines. Gain/loss-of-expression studies were performed to functionally validate EPHA2 as a synergistic combinational target with EPA in two EPHA2-expressing TN-IBC models, SUM149PT and BCX010, using proliferation and apoptosis assays in vitro and established tumor xenografts in vivo. EPHA2 gene silencing significantly reduced cell growth and induced apoptosis in combination with EPA when compared with untreated control and monotherapy in vitro (P &amp;lt; 0.05) and in vivo (P &amp;lt; 0.001), while vector-induced EPHA2 expression reversed cell growth reduction and apoptosis induction following combination treatment with EPA in vitro (P &amp;lt; 0.05). To translate our findings to the clinic, we validated that dasatinib, a small molecule inhibitor of EPHA2, in combination with EPA significantly enhanced cell death of SUM149PT and BCX010 cells in vitro when compared to non-treated and monotherapy (P &amp;lt; 0.05). Finally, using membrane fluidity assessment and reverse-phase protein array (300 antibodies), we determined that combination treatment efficacy depended on EPA/EPHA2 inhibition-mediated increase in cell membrane rigidity (P &amp;lt; 0.001, compared to monotherapy), which subsequently inhibited receptor tyrosine kinase signaling activity, potentially resulting in induction of apoptosis. Conclusions: Our preclinical findings provide a rationale for the development of a phase 1 clinical trial investigating combination EPA and EPHA2-inhibitors in patients with EPHA2-positive TN-IBC. Citation Format: Angie M. Torres-Adorno, Heidi Vitrac, Yuan Qi, Yiwen Yang, Peiying Yang, Bedrich L. Eckhardt, Naoto T. Ueno. EPHA2-targeted therapy enhances the cytotoxicity of eicosapentaenoic acid against triple-negative inflammatory breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1235. doi:10.1158/1538-7445.AM2017-1235

Abstract 433: Glycogen accumulation in aggressive breast cancers during hypoxic exposure

Abstract Isolation of specific metabolic alterations in aggressive triple negative and inflammatory breast cancers represents a compelling avenue for the development of treatments for these diseases. The ability of cancer to utilize diverse metabolic pathways to modulate increased survival and proliferation is well established. Indeed, we have previously described a series of metabolic adaptations in the triple negative inflammatory breast cancer cell line SUM149 and demonstrated a role for the small GTPase RhoC in the metabolic phenotype of the cell line. In this work we seek to describe a survival mechanism for cancer cells that are subjected to hypoxic environments. Hypoxic survival or growth is often necessitated at the center of a growing tumor mass or if the tumor is growing in the lymphatic system, as is often the case for inflammatory breast cancers. Here we investigate the regulation of glycogen levels in breast cancer and normal-like cell lines in atmospheric and 1% oxygen environments. Reserves of glucose are typically stored in liver and muscle cells as the polysaccharide glycogen. Elevated levels of glycogen have previously been observed in various cancers, and recent studies have implicated the importance of glycogen metabolism in promoting cancer cell survival. We hypothesize that aggressive breast cancers utilize modifications of the glycogen synthesis and degradation pathways to provide nutritional flexibility as they proliferate and invade into diverse environments. MCF10A, SUM149, and SUM190 cell lines demonstrate a greater than 10-fold increase in glycogen accumulation when subjected to hypoxic growth conditions. MDA-MB-231 cells also increase glycogen storage, though at levels on the order of 2-3 times higher in hypoxia when compared to normoxic growth. Conditioned media for all cell lines in normoxic and hypoxic conditions were analyzed for glucose and lactate levels to determine changes in metabolite consumption. The data presented indicate an unexpected accumulation of glycogen in response to hypoxic conditions. Studies to determine the mechanisms through which these changes occur and why they are important for continued survival and growth of the cancer cells are ongoing and will be presented. Citation Format: Megan Altemus, Joel Yates, ZhiFen Wu, LiWei Bao, Sofia Merajver. Glycogen accumulation in aggressive breast cancers during hypoxic exposure [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 433. doi:10.1158/1538-7445.AM2017-433

Abstract 1137: ADAM8 drives aggressive phenotype of triple-negative inflammatory breast cancer &amp; constitutes a novel therapeutic target

Abstract Inflammatory Breast Cancer (IBC) is a rare, highly aggressive form of cancer that is frequently locally advanced or metastasized at the time of diagnosis. The Triple-Negative subtype of IBC (TN-IBC), in particular, is characterized by very poor overall survival. TN-IBC lacks targeted therapies and is primarily treated with radiation or chemotherapy, which are inefficient. Recently, we identified the cell surface transmembrane ADAM8 (A Disintegrin and Metalloproteinase) protein as a driver of Triple-Negative Breast Cancer (TNBC) growth and metastasis via its Metalloproteinase (MP) and Disintegrin (DI) domains, respectively. In in vivo proof-of-concept experiments with a prototype reagent, we demonstrated that simultaneous, antibody-based, targeting of the ADAM8 MP and DI domains is an effective therapeutic approach for TNBC (Romagnoli et al., EMBO Mol. Med. 6:278, 2014). The aggressive behavior of IBC cells has been attributed to a stem-like cancer cell compartment with high ALDH activity (ALDH+). Our studies of the green tea compound epigallocatechin-3 gallate (EGCG) revealed that it reduced growth of tumors derived from ALDH+ SUM-149 TN-IBC cells. We now report that EGCG concurrently decreases tumor levels of ADAM8 mRNA. This led us to assess the role of ADAM8 in TN-IBC. Tumor biopsies from 15 patients taken at the time of diagnosis and/or after neo-adjuvant treatment with chemotherapy, as well as paired lymph node and skin samples (when available) were analyzed by immunohistochemistry for ADAM8 expression. We report that 45.5% of primary TN-IBC patient tumors and 50.0% of metastases within the axillary lymph nodes express high levels of ADAM8 and its expression is largely unaffected by chemotherapy. In addition, ADAM8 expression was higher in the aggressive ALDH+ stem-like SUM-149 cell compartment. Consistently, knockdown of ADAM8 dramatically reduced the ability of SUM-149 cells to grow in an anchorage independent fashion and to migrate through Matrigel. A newly prepared anti-human ADAM8 mouse monoclonal antibody (ADP13) inhibited the MP and DI domains of ADAM8 on SUM-149 cells. ADP13 reduced orthotopic growth of tumors derived from SUM-149 cells by 40% in mice treated by i.p. injection 2 days a week with a dose of 4.5 mg/kg (n=7). Dose-response curves and survival experiments are in progress. Conclusions: ADAM8 expression is present in almost half of TN-IBC patient tumors and their metastases, and promotes aggressive phenotype of TN-IBC cells in in vitro 3D-assays. A pre-clinical mouse model of TN-IBC validated ADAM8 as an accessible and promising new target for therapeutic intervention against this highly aggressive disease. Citation Format: Mathilde Romagnoli, Stefania Pianetti, Sonia G. Das, Delphine Loussouarn, Carole Gourmelon, Mario Campone, Sophie Barillé-Nion, Giang T. Nguyen, Srimathi Srinivasan, Gail E. Sonenshein, Nora D. Mineva. ADAM8 drives aggressive phenotype of triple-negative inflammatory breast cancer &amp; constitutes a novel therapeutic target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1137. doi:10.1158/1538-7445.AM2017-1137

Syndecan-1 is a novel molecular marker for triple negative inflammatory breast cancer and modulates the cancer stem cell phenotype via the IL-6/STAT3, Notch and EGFR signaling pathways

BackgroundInflammatory breast cancer (IBC), a particularly aggressive form of breast cancer, is characterized by cancer stem cell (CSC) phenotype. Due to a lack of targeted therapies, the identification of molecular markers of IBC is of major importance. The heparan sulfate proteoglycan Syndecan-1 acts as a coreceptor for growth factors and chemokines, modulating inflammation, tumor progression, and cancer stemness, thus it may emerge as a molecular marker for IBC.MethodsWe characterized expression of Syndecan-1 and the CSC marker CD44, Notch-1 & -3 and EGFR in carcinoma tissues of triple negative IBC (n = 13) and non-IBC (n = 17) patients using qPCR and immunohistochemistry. Impact of siRNA-mediated Syndecan-1 knockdown on the CSC phenotype of the human triple negative IBC cell line SUM-149 and HER-2-overexpressing non-IBC SKBR3 cells employing qPCR, flow cytometry, Western blotting, secretome profiling and Notch pharmacological inhibition experiments. Data were statistically analyzed using Student’s t-test/Mann-Whitney U-test or one-way ANOVA followed by Tukey’s multiple comparison tests.ResultsOur data indicate upregulation and a significant positive correlation of Syndecan-1 with CD44 protein, and Notch-1 & -3 and EGFR mRNA in IBC vs non-IBC. ALDH1 activity and the CD44(+)CD24(-/low) subset as readout of a CSC phenotype were reduced upon Syndecan-1 knockdown. Functionally, Syndecan-1 silencing significantly reduced 3D spheroid and colony formation. Intriguingly, qPCR results indicate downregulation of the IL-6, IL-8, CCL20, gp130 and EGFR mRNA upon Syndecan-1 suppression in both cell lines. Moreover, Syndecan-1 silencing significantly downregulated Notch-1, -3, -4 and Hey-1 in SUM-149 cells, and downregulated only Notch-3 and Gli-1 mRNA in SKBR3 cells. Secretome profiling unveiled reduced IL-6, IL-8, GRO-alpha and GRO a/b/g cytokines in conditioned media of Syndecan-1 knockdown SUM-149 cells compared to controls. The constitutively activated STAT3 and NFκB, and expression of gp130, Notch-1 & -2, and EGFR proteins were suppressed upon Syndecan-1 ablation. Mechanistically, gamma-secretase inhibition experiments suggested that Syndecan-1 may regulate the expression of IL-6, IL-8, gp130, Hey-1, EGFR and p-Akt via Notch signaling.ConclusionsSyndecan-1 acts as a novel tissue biomarker and a modulator of CSC phenotype of triple negative IBC via the IL-6/STAT3, Notch and EGFR signaling pathways, thus emerging as a promising therapeutic target for IBC.

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 P3-13-21: Primary surgery in inflammatory breast cancer

Abstract Background: Inflammatory Breast Cancer (IBC) is a rare but very aggressive variant of breast cancer accounting for around 1-5% of all breast cancer cases. Current standard of care is multimodality therapy with preoperative chemotherapy followed by modified radical mastectomy and adjuvant radiotherapy. Method and Material: We searched the Surveillance Epidemiology and End results Registry to identify female patients diagnosed with Stage III IBC between 1988-2013.We performed descriptive and univariate analyses of the patients with IBC. Variables assessed included patient age, race/ethnicity, histologic subtype, tumor grade, hormone receptor status, surgery type. Results: We identified 11604 women with IBC from SEER database diagnosed between 1988-2013.The median age was 57 yrs., 80 % ( 9273) of patients were white,14.4 % ( 1667) blacks, and around 73% had nodal involvement. 32.4 % (3756) patients were diagnosed with IBC between 1988-2000, while 67.6 % ( 7848) between 2001-2013.We excluded patients with missing primary surgery information with 7586 patients remaining for analysis. The median age in this final cohort was 56yrs, 80.4 % ( 6097) patients were white, and 13.7 % (1044) were black. The predominant tumor type was invasive ductal carcinoma, majority of the patients had grade 3 tumor 59.4 % ( 4504), grade2 in 23.2 % ( 1757),47.1% were estrogen receptor (ER)positive,43% were ER negative,53% were progesterone receptor negative, and 7% had HER 2 positive tumor. In this cohort 6.4 % (487) patients underwent partial mastectomy and 93.6 % (7099) underwent mastectomy. In the cohort undergoing breast conservation, 15% were during 1988-2000 and 84.4% during years 2001-2013 (p value=0.06). Surgery in non-metastatic inflammatory Breast CancerType of Surgery1988-20002001-2013p-valuePartial Mastectomy76 (6.7%)1140(93.3%)0.06Mastectomy411 (6.9%)5959(93.1%) The median survival was 46 months among patients diagnosed between 1988-2000 and 61 months for 2001-2013 time period(p&amp;lt;0.0001).The median survival in patients undergoing partial mastectomy was 42 months versus 48 months among patients undergoing mastectomy (p=0.0001). Discussion: IBC continues to be an aggressive breast cancer subtype, but survival has improved over last decade. The utility of partial mastectomy has increased in the last decade compared to 1988-2000. Mastectomy still remains the standard surgical option, but with more effective systemic therapeutic agents, better tumor responses are noted especially in Her 2 positive and triple negative IBC. Partial Mastectomy may be a viable option for patients achieving complete clinical response. Citation Format: Muzaffar M, Vohra N, Wong J. Primary surgery in inflammatory breast cancer [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 P3-13-21.

Abstract OT2-01-20: Phase IIB study of neoadjuvant panitumumab combined with carboplatin and paclitaxel (PaCT) for anthracycline-resistant triple-negative breast cancer (TNBC)

Abstract BACKGROUND: Approximately 50% of patients with TNBC treatedwithstandardtaxane/anthracycline-based neoadjuvant chemotherapy (NACT)have chemo-insensitive disease (CID), i.e., residual disease burden (RCB)-II/III at the time of surgery, and 40-80% of patients develop recurrence within 3 years. Recent developments in molecular profiling have identified subsets of TNBC with distinct, targetable molecular features. We developed a clinical trial to identify and characterize CID (ARTEMIS: A Randomized, TNBC-Enrolling trial to confirm Molecular profiling Improves Survival). In ARTEMIS, patients with localized TNBC will undergo a pretreatment biopsy, then begin anthracycline-based NACT. During NACT, we use molecular profiling and response assessment to identify CID and allocate patients to alternative therapies to overcome CID. Epidermal growth factor receptor (EGFR) is overexpressed in 25-30% of TNBC. In preclinical studies, suppression of EGFR signaling has shown efficacy in controlling cancers through suppression of the stem cell population, enhanced apoptosis via MAPK/PI3K signaling, and modulation of epithelial-mesenchymal transition (EMT). Moreover, in a phase II trial of triple negative inflammatory breast cancer, neoadjuvant PaCT yielded significantly higher pathologic complete response (pCR) rates than historic control. Taken together, we hypothesize that using PaCT to suppress EGFR in TNBC will enhance the pCR rate. OBJECTIVES: Primary objective: determine pCR and RCB-0/I rates in TNBC patients with CID given PaCT. Secondary objective: determine the benefit of using baseline genomic signatures to develop an alternative second phase of NACT. TRIAL DESIGN AND STATISTICAL METHODS: Patients with &amp;gt;10% volume reduction for non-CID or &amp;lt;80% for CID will enroll in a biomarker-guided, experimental, nonrandomized phase II study and be given PaCT (panitumumab 2.5 mg/kg, carboplatin AUC 5, paclitaxel 80 mg/m2). Because pCR rates in pts with CID with additional cycles of taxane-based therapy are low (∼5%), a 20% response rate (RCB-0 or RCB-I) will be considered clinically meaningful. A two-stage Gehan-type design will be employed. If at least 1 of 14 patients responds, 23 more patients will be added, for a total of 37 patients. This design has a 49% chance of terminating after the first stage if the true response rate is 0.05, 23% if the rate is 0.10, 10% if the rate is 0.15, and 4% if the rate is 0.20. If accrual continues to the second stage and 37 patients are enrolled, the 95% confidence interval for a 0.20 response rate will be 0.10 to 0.35. BRIEF ELIGIBILITY CRITERIA: Inclusion: localized TNBC; enrolled in ARTEMIS trial; adequate organ, bone marrow, and cardiac parameters; Exclusion: pregnant or lactating, known or suspected metastasis. CORRELATIVE SCIENCE: Circulating tumor cells (CTCs) and cell free (cf) DNA in baseline and subsequent blood samples, EGFR expression (immunohistochemistry), stem cell/EMT/apoptosis marker changes in tissue and CTCs, PD-L1 glycosylation for EGFR sensitivity. Citation Format: Lim B, Helgason T, Hess KR, Piwnica-Worms H, Yang W, Adrada BE, Rauch GM, Gilcrease M, Symmans FW, Huo L, Mittendorf EA, Thompson A, Stacy M-TL, Debu T, Ueno NT. Phase IIB study of neoadjuvant panitumumab combined with carboplatin and paclitaxel (PaCT) for anthracycline-resistant triple-negative breast cancer (TNBC) [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 OT2-01-20.

A Case Series of Non-Hodgkin's Lymphoma Recurrence in Lower Extremities Mimicking Deep Vein Thrombosis

Background: While uncommon, non-Hodgkin's Lymphoma (NHL) can relapse in extranodal sites in the lower extremities. It is well established that venous thomboembolism occurs in higher incidence in patients with underlying malignancy. We describe 3 cases of NHL in which relapsed disease was discovered after lower extremity swelling prompted evaluation for deep vein thrombosis (DVT), and revealed recurrent NHL. CASE 1: A 61 YO F with history of Stage IEA Marginal Zone Lymphoma (MZL) of the dura diagnosed in 2012 received treatment with whole-brain and local brain radiation. In 2016 she developed recurrence in cervical lymph nodes. Six months after receiving 4 cycles of bendamustine and rituximab she developed left leg pain and calf swelling. PET/CT showed a hypermetabolic lesion in left tibia. MRI confirmed large lesion in mid-shaft of tibia extending down the length of the bone. CT-guided bone biopsy confirmed MZL. CASE 2: A 67 YO F with history of triple-negative inflammatory breast cancer treated in 2000 with neoadjuvant chemotherapy and mastectomy. PET/CT in 2008 showed 2 left femoral lesions. She underwent CT-guided bone biopsy of femoral lesion which confirmed follicular lymphoma (FL). Interim PET/CT in 2010 showed decreased FDG uptake in left femur. She was followed with watchful waiting. Then in 2014 patient developed leg swelling and pain, thought to be a DVT. Duplex ultrasound of the lower extremity was negative. PET/CT showed a left inguinal mass and left calf mass. MRI confirmed large mass within the left lower leg centered on the left fibular diaphysis with a very large associated soft tissue component with extensive muscular and neurovascular involvement. Biopsy of left inguinal lymph node confirmed diffuse large B-Cell lymphoma (DLBCL). CASE 3: A 61 YO M with history Stage IV FL in 1988 and initially treated with 4 cycles of PROMACE-CytaBOM. He relapsed nodally in 1995 and was treated on the IDEC investigational protocol with rituximab which led to a complete remission. In 2001 he developed a subcutaneous nodule on his back, biopsy showed FL, grade 1. A staging CT scan of neck, chest, abdomen, and pelvis was unremarkable, but a PET scan showed left axillary disease. He was re-treated with rituximab and local radiation which led to a complete response. Patient presented in 2008 with right calf swelling. Ultrasound at that time showed no DVT but confirmed swelling and suggested right inguinal adenopathy. PET/CT showed FDG uptake in multiple areas in groin and pelvis plus extensive uptake in thickened calf muscles extending from the right knee to the toe. Biopsy of right inguinal lymph node confirmed FL. Conclusion: Among NHLs, relapse patterns can vary significantly. Timing and frequency of relapse is largely dependent on whether NHL is classified as indolent or aggressive. However, location of relapsed disease in NHL is less well established. NHL relapse can occur in a fashion mimicking DVT with lower extremity swelling. When patients with history of NHL present with lower extremity swelling, relapsed disease should be included in the differential diagnosis. Table Patient characteristics Table. Patient characteristics Disclosures Cohen: Takeda Oncology: Honoraria; Pharmacyclics: Honoraria; Seattle Genetics: Honoraria; Amgen: Honoraria. Broome:True North Therapeutics: Honoraria; Alexion Pharmaceuricals: Honoraria.

Abstract 1900: MiR-222 accelerates the progression of inflammatory breast cancer by targeting tumor suppressor gene p27kip1 (CDKN1B)

Abstract MicroRNAs (miRNAs) are small non-coding RNAs about 18-22 nucleotides in length, whose main role is to regulate gene expression by altering mRNA stability and translation. MicroRNAs have been shown to play a role in many types of cancer, including breast cancer by targeting tumor suppressor genes such as p53, p27, PTEN, BRCA1, BRCA2 and many others. The goal of this study was to investigate the role of miRNAs in inflammatory breast cancer (IBC). IBC is the most aggressive and metastatic form of breast cancer, that is characterized by very rapid progression, poor prognosis, with a 5 year survival outcome of 30-40%. The mechanism that underlies the rapid progression of IBC is currently under intense investigation, and novel approaches are needed to better understand this disease. To understand the role that microRNAs might play in IBC, we conducted next generation sequencing of the miRNAs from two IBC cell line, SUM149 which is a triple negative IBC (ER-, PR-, HER2-), and SUM190 which is HER2 overexpressing, but ER and PR negative (ER-, PR-, HER2+). We found that 463 miRNAs were differentially expressed in the two cell lines, with 243 miRNAs being overexpressed in SUM149 compared to SUM190, while 220 miRNAs were overexpressed in SUM190. Notably, our qPCR analysis confirmed the raw read counts, showing that miR-155, miR-221, and miR-222 were overexpressed in SUM149 as compared to SUM190 cells by 15, 10, and 3 fold respectively. Furthermore, the inhibition of miR-222 in SUM149 resulted in significant reduction in cell proliferation, tumorsphere formation, and migration in these cells as compared to SUM190 cells. Our flow cytometry data showed that there was cell cycle arrest at the G1 phase in SUM149 cells due to miR-222 inhibition, while SUM190 cells showed little or no change. Additionally, Western blot and immunofluorescence analyses showed that the protein levels of p27, which is one of the targets of miR-222, significantly increased in SUM149 cells, but not in SUM190 cells when miR-222 inhibitor was used in these cells. These results suggest that miR-222 may accelerate the progression of some IBCs by targeting tumor suppressor genes, resulting in rapid cell proliferation, and may be a potential therapeutic target for triple negative inflammatory breast cancers. Citation Format: Joshua Were Ogony, Erin Hayes, Jennifer Knapp, Joan Lewis-Wambi. MiR-222 accelerates the progression of inflammatory breast cancer by targeting tumor suppressor gene p27kip1 (CDKN1B). [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 1900.

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 (&amp;gt;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.

Impact of hormone receptor (HR) and human epidermal growth factor receptor 2 (HER2) status on survival in inflammatory breast cancer: Analysis of Surveillance, Epidemiology and End Results (SEER) database.

e12548Background: Inflammatory breast cancer (IBC) is associated with a poor prognosis compared to other subtypes of breast cancer. Very little is known about how HR and HER2 status influence prognosis in IBC. Methods: Patients diagnosed with IBC between 2010 and 2011 were identified from the SEER database. Overall survival (OS) and breast cancer specific survival (BCSS) were analyzed using Kaplan-Meier method and Cox proportional hazards model. Results: Among 348 patients with IBC, 56 (16.1%) were HR+/HER2+, 63 (18.1%) were HR-/HER2+, 107 (30.8%) were HR+/HER2-, and 76 (21.8%) were triple negative (TN). Receptor status was not available for 46 (13.2%) patients. Majority of the population was > 50 years of age (75%), Caucasian (61%), had stage IIIB disease at diagnosis (46%), and underwent mastectomies (62%). TN status was significantly associated with poor OS and BCSS after adjustment for other clinically relevant factors (Table). 2-year OS and BCSS for TN IBC were 46% and 53%, while for non-TN IBC it wa...

The role of the aryl hydrocarbon receptor in the development of cells with the molecular and functional characteristics of cancer stem-like cells.

BackgroundSelf-renewing, chemoresistant breast cancer stem cells are believed to contribute significantly to cancer invasion, migration and patient relapse. Therefore, the identification of signaling pathways that regulate the acquisition of stem-like qualities is an important step towards understanding why patients relapse and towards development of novel therapeutics that specifically target cancer stem cell vulnerabilities. Recent studies identified a role for the aryl hydrocarbon receptor (AHR), an environmental carcinogen receptor implicated in cancer initiation, in normal tissue-specific stem cell self-renewal. These studies inspired the hypothesis that the AHR plays a role in the acquisition of cancer stem cell-like qualities.ResultsTo test this hypothesis, AHR activity in Hs578T triple negative and SUM149 inflammatory breast cancer cells were modulated with AHR ligands, shRNA or AHR-specific inhibitors, and phenotypic, genomic and functional stem cell-associated characteristics were evaluated. The data demonstrate that (1) ALDHhigh cells express elevated levels of Ahr and Cyp1b1 and Cyp1a1, AHR-driven genes, (2) AHR knockdown reduces ALDH activity by 80 %, (3) AHR hyper-activation with several ligands, including environmental ligands, significantly increases ALDH1 activity, expression of stem cell- and invasion/migration-associated genes, and accelerates cell migration, (4) a significant correlation between Ahr or Cyp1b1 expression (as a surrogate marker for AHR activity) and expression of stem cell- and invasion/migration-associated gene sets is seen with genomic data obtained from 79 human breast cancer cell lines and over 1,850 primary human breast cancers, (5) the AHR interacts directly with Sox2, a master regulator of self-renewal; AHR ligands increase this interaction and nuclear SOX2 translocation, (6) AHR knockdown inhibits tumorsphere formation in low adherence conditions, (7) AHR inhibition blocks the rapid migration of ALDHhigh cells and reduces ALDHhigh cell chemoresistance, (8) ALDHhigh cells are highly efficient at initiating tumors in orthotopic xenografts, and (9) AHR knockdown inhibits tumor initiation and reduces tumor Aldh1a1, Sox2, and Cyp1b1 expression in vivo.ConclusionsThese data suggest that the AHR plays an important role in development of cells with cancer stem cell-like qualities and that environmental AHR ligands may exacerbate breast cancer by enhancing expression of these properties.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0240-y) contains supplementary material, which is available to authorized users.