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Figure S4: Cyclin E/CDK2 phosphorylation of Hbo1 does not affect the HAT activity of Hbo1.

Detailed methods of all experiments that due to limited space is included in the supplemental section.

Background: Inflammatory breast cancer (IBC) is a rare and highly aggressive form of breast cancer. Patients with IBC still have worse clinical outcomes compared to patients with non-IBC (40% versus 63% for 5 years overall survival, respectively) despite advances in breast cancer treatment. IBC remains a poorly characterized disease lacking specific therapeutic targets and prognostic biomarkers. Our group demonstrated that N-myc downstream regulated gene 1 (NDRG1) is crucial in promoting tumorigenesis and brain metastasis in mouse models of IBC. Our hypothesis is that NDRG1 is a prognostic marker associated with poor outcome in IBC patients. Methods: 64 IBC patients in a tissue microarray were evaluated by immunohistochemical staining with anti-NDRG1 primary antibody and NDRG1 levels were quantified. Using the median value, 32 patients were grouped as NDRG1-low (≤ median), and 32 as NDRG1-high (>median). Survival data were compared by Kaplan–Meier curves and log-rank test. Results: The average age of patients was 50 years. Sixty-two percent of patients had estrogen receptor (ER)-negative tumors, 83% were stage III, 80% high grade, and 67% of these patients received adjuvant radiation. The median follow-up time for the patients studied was 11.7 years, and the median overall survival (OS) time was 3.7 years. On univariate analysis, NDRG1 expression, tumor grade, disease stage, ER status, and adjuvant radiation therapy were associated with OS and disease specific survival (DSS). Patients with NDRG1-low tumors experienced better actuarial 10-year OS (p=0.0129) and DSS (p=0.0074), and showed significant higher 10-year OS and DSS rates than patients with NDRG1-high(OS, 45% vs. 19%, p=0.0278; DSS, 52% vs. 22%, p=0.0139). The median OS and DSS times were shorter for NDRG1-high patients (OS, 2.5 years; DSS, 3.1 years) than for NDRG1-low patients (OS, 5.9 years; DSS, 10.7 years). Multivariable analysis, NDRG1 was an independent predictor of OS (hazard ratio [HR]=2.449, p=0.0274) and DSS (HR=2.727, p=0.0039). ER status, disease stage and adjuvant radiation were also independent predictor for both OS and DSS. Furthermore, we observed that NDRG1-high expressing ER-negative tumors exhibit worse outcome in IBC patients (OS, p=0.0003; DSS, p=0.0003). NDRG1-high expression correlated with worse outcome in patients that received adjuvant radiation treatment (OS, p=0.0088; DSS, p=0.0093), and those with stage III (OS, p=0.0450; DSS, p=0.0239). Conclusions: Our findings demonstrated that NDRG1 is positively correlated with aggressive tumor characteristics in IBC and that it is an independent prognostic factor for OS and DSS in IBC patients, suggesting that targeting NDRG1 may provide a novel therapeutic strategy to improve outcomes for patients with IBC. Our data further suggests that NDRG1 warrants further investigation in radiation response. Citation Format: Emilly Schlee Villodre, Yun Gong, Xiaoding Hu, Lei Huo, Esther C Yoon, Naoto T Ueno, Wendy A Woodward, Debu Tripathy, Juhee Song, Bisrat G Debeb. NDRG1 expression is an independent prognostic factor in inflammatory breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-08-13.

Inflammatory breast cancer (IBC) is an aggressive form of primary breast cancer characterized by rapid onset and high risk of metastasis and poor clinical outcomes. The biological basis for the aggressiveness of IBC is still not well understood and no IBC‐specific targeted therapies exist. In this study, we report that lipocalin 2 (LCN2), a small secreted glycoprotein belonging to the lipocalin superfamily, is expressed at significantly higher levels in IBC vs non‐IBC tumors, independently of molecular subtype. LCN2 levels were also significantly higher in IBC cell lines and in their culture media than in non‐IBC cell lines. High expression was associated with poor‐prognosis features and shorter overall survival in IBC patients. Depletion of LCN2 in IBC cell lines reduced colony formation, migration, and cancer stem cell populations in vitro and inhibited tumor growth, skin invasion, and brain metastasis in mouse models of IBC. Analysis of our proteomics data showed reduced expression of proteins involved in cell cycle and DNA repair in LCN2‐silenced IBC cells. Our findings support that LCN2 promotes IBC tumor aggressiveness and offer a new potential therapeutic target for IBC., In this study, the authors demonstrate that lipocalin 2 (LCN2) is highly upregulated in inflammatory breast cancer (IBC), a rare, rapidly‐progressing and aggressive variant of primary breast cancer. Depletion of LCN2 in IBC cell lines reduced colony formation, migration, and cancer stem cell populations in vitro and inhibited tumor growth, skin invasion, and brain metastasis in mouse models of IBC.

Prophylactic cranial irradiation (PCI) can reduce the incidence of brain metastasis and improve overall survival in some patients with acute lymphoblastic leukemia or small-cell lung cancer. We examined the potential effects of PCI in a mouse model of breast cancer brain metastasis. The HER2+ inflammatory breast cancer cell line MDA-IBC3 was labeled with green fluorescent protein and injected via tail-vein into female SCID/Beige mice. Mice were then given 0 Gy or 4 Gy of whole-brain irradiation 2 days before tumor-cell injection or 5 days, 3 weeks, or 6 weeks after tumor-cell injection. Mice were sacrificed 4-weeks or 8-weeks after injection and brain tissues were examined for metastasis by fluorescent stereomicroscopy. In the unirradiated control group, brain metastases were present in 77% of mice at 4 weeks and in 90% of mice at 8 weeks; by comparison, rates for the group given PCI at 5 days after tumor-cell injection were 20% at 4 weeks (p=0.01) and 30% at 8 weeks (p=0.02). The PCI group also had fewer brain metastases per mouse at 4 weeks (p=0.03) and 8 weeks (p=0.006) versus the unirradiated control as well as a lower metastatic burden (p=0.01). Irradiation given either before tumor-cell injection or 3-6 weeks afterward had no significant effect on brain metastases compared to the unirradiated control. These results underscore the importance of timing for irradiating subclinical disease. Clinical whole brain strategies to target subclinical brain disease as safely as possible may warrant further study.

The deadly complication of brain metastasis (BM) is largely confined to a relatively narrow cross-section of systemic malignancies, suggesting a fundamental role for biological mechanisms shared across commonly brain metastatic tumor types. To identify and characterize such mechanisms, we performed genomic, transcriptional, and proteomic profiling using whole-exome sequencing, mRNA-seq, and reverse-phase protein array analysis in a cohort of the lung, breast, and renal cell carcinomas consisting of BM and patient-matched primary or extracranial metastatic tissues. While no specific genomic alterations were associated with BM, correlations with impaired cellular immunity, upregulated oxidative phosphorylation (OXPHOS), and canonical oncogenic signaling pathways including phosphoinositide 3-kinase (PI3K) signaling, were apparent across multiple tumor histologies. Multiplexed immunofluorescence analysis confirmed significant T cell depletion in BM, indicative of a fundamentally altered immune microenvironment. Moreover, functional studies using in vitro and in vivo modeling demonstrated heightened oxidative metabolism in BM along with sensitivity to OXPHOS inhibition in murine BM models and brain metastatic derivatives relative to isogenic parentals. These findings demonstrate that pathophysiological rewiring of oncogenic signaling, cellular metabolism, and immune microenvironment broadly characterizes BM. Further clarification of this biology will likely reveal promising targets for therapeutic development against BM arising from a broad variety of systemic cancers.

Background Inflammatory breast cancer (IBC) is a clinical diagnosis. Here, we examined the association of a “classic” triad of clinical signs, swollen involved breast, nipple change, and diffuse skin change, with overall survival (OS). Method Breast medical photographs from patients enrolled on a prospective IBC registry were scored by two independent reviewers as classic (triad above), not classic, and difficult to assign. Chi‐squared test, Fisher's exact test, and Wilcoxon rank‐sum test were used to assess differences between patient groups. Kaplan–Meier estimates and the log‐rank test and Cox proportional hazard regression were used to assess the OS. Results We analyzed 245 IBC patients with median age 54 (range 26–81), M0 versus M1 status (157 and 88 patients, respectively). The classic triad was significantly associated with smoking, post‐menopausal status, and metastatic disease at presentation (p = 0.002, 0.013, and 0.035, respectively). Ten‐year actuarial OS for not classic and difficult to assign were not significantly different and were grouped for further analyses. Ten‐year OS was 29.7% among patients with the classic sign triad versus 57.2% for non‐classic (p, Inflammatory breast cancer (IBC) is a clinical diagnosis. Here, we examined the association of a “classic” triad of clinical signs, swollen involved breast, nipple change, and diffuse skin change, with overall survival (OS). Our study concluded for the first time that a triad of classic IBC signs independently predicted OS in patients diagnosed with IBC.

Brain metastases (BMs) occur in approximately 20–40% of patients with advanced cancer, and the estimated prevalence of new BMs in United States is between 200,000–300,000 per year. While the incidence of BM has increased over the past decades due to improvements in brain tumor detection technology, the prognosis is still very poor with the median overall survival times from weeks to few months. Therefore, identification of the precise molecular landscape and therapeutic targets for BMs is absolutely essential in tangible improvement of patient management. Here, we performed integrated genomic, transcriptional, and proteomic profiling in a cohort of lung, breast, and renal cell carcinomas consisting of both BMs and patient-matched primary or extracranial metastatic tissues to identify shared cellular and molecular factors driving BMs across distinct primary tumor histologies. Although the comprehensive analysis identified the unique genomic, transcriptional and proteomic landscapes according to the different histopathologies, elevated PI3K/AKT and RAS/MAPK signaling was observed as a generalizable feature across the entire specimen cohort, along with relative immunosuppression and metabolic upregulation of the electron transport chain (ETC). Interestingly, immunosuppression via T cell depletion was significantly associated with unfavorable prognosis of patients with BMs, and ETC inhibition as the prospective therapeutic target for BM patients was demonstrated using in vitro and in vivo disease models. Taken together, our findings suggest that abnormalities involving oncogenic signaling, metabolism, and the immune microenvironment are shared across multiple histologies of BMs, and may be amenable to therapeutic targeting.

Inflammatory breast cancer (IBC) is a clinically distinct and highly aggressive form of breast cancer with rapid onset and a strong propensity to metastasize. The molecular mechanisms underlying the aggressiveness and metastatic propensity of IBC are largely unknown. Herein, we report that decorin (DCN), a small leucine-rich extracellular matrix proteoglycan, is downregulated in tumors from patients with IBC. Overexpression of DCN in IBC cells markedly decreased migration, invasion, and cancer stem cells in vitro and inhibited IBC tumor growth and metastasis in vivo. Mechanistically, DCN functioned as a suppressor of invasion and tumor growth in IBC by destabilizing E-cadherin and inhibiting EGFR/ERK signaling. DCN physically binds E-cadherin in IBC cells and accelerates its degradation through an autophagy-linked lysosomal pathway. We established that DCN inhibits tumorigenesis and metastasis in IBC cells by negatively regulating the E-cadherin/EGFR/ERK axis. Our findings offer a potential therapeutic strategy for IBC, and provide a novel mechanism for IBC pathobiology.

Inflammatory breast cancer (IBC) is a clinically distinct and highly aggressive form of breast cancer with rapid onset and a strong propensity to metastasize. The molecular mechanisms underlying the aggressiveness and metastatic propensity of IBC are largely unknown. Herein, we report that decorin (DCN), a small leucine-rich extracellular matrix proteoglycan, is downregulated in tumors from patients with IBC. Overexpression of DCN in IBC cells markedly decreased migration, invasion, and cancer stem cells in vitro and inhibited tumor growth and metastasis in IBC xenograft mouse models. Mechanistically, DCN functioned as a suppressor of invasion and tumor growth in IBC by destabilizing E-cadherin and inhibiting EGFR/ERK signaling. DCN physically binds E-cadherin in IBC cells and accelerates its degradation through an autophagy-linked lysosomal pathway. We established that DCN inhibits tumorigenesis and metastasis in IBC cells by negatively regulating the E-cadherin/EGFR/ERK axis. Our findings offer a potential therapeutic strategy for IBC, and provide a novel mechanism for IBC pathobiology., Xiaoding Hu et al. find that expression of the proteoglycan decorin is decreased in patients with inflammatory breast cancer compared to normal breast tissue and some other types of breast cancer. They demonstrate that decorin acts as a tumor suppressor in cancer cells and human xenograft mouse models by destabilizing the E-cadherin-EGFR signaling axis, and their findings suggest potential therapeutic strategies for this aggressive breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive form of breast cancer characterized by metastasis, drug resistance and high rates of recurrence. With a lack or targeted therapies, TNBC is challenging to treat and carries a poor prognosis. Patients with TNBC tumors expressing high levels of ERK2 have a poorer prognosis than those with low ERK2-expressing tumors. The MAPK pathway is often found to be highly activated in TNBC, however the precise functions of the ERK isoforms (ERK1 and ERK2) in cancer progression have not been well defined. We hypothesized that ERK2, but not ERK1, promotes the cancer stem cell (CSC) phenotype and metastasis in TNBC. Stable knockdown clones of the ERK1 and ERK2 isoforms were generated in SUM149 and BT549 TNBC cells using shRNA lentiviral vectors. ERK2 knockdown significantly inhibited anchorage-independent colony formation and mammosphere formation, indicating compromised self-renewal capacity. This effect correlated with a reduction in migration and invasion. SCID-beige mice injected via the tail vein with ERK clones were employed to determine metastatic potential. SUM149 shERK2 cells had a significantly lower lung metastatic burden than control mice or mice injected with SUM149 shERK1 cells. The Affymetrix HGU133plus2 microarray platform was employed to identify gene expression changes in ERK isoform knockdown clones. Comparison of gene expression levels between SUM149 cells with ERK2 or ERK1 knockdown revealed differential and in some cases opposite effects on mRNA expression levels. Those changes associated with ERK2 knockdown predominantly altered regulation of CSCs and metastasis. Our findings indicate that ERK2 promotes metastasis and the CSC phenotype in TNBC.

Inflammatory breast cancer (IBC) is the most pro‐metastatic form of breast cancer. Better understanding of its pathophysiology and identification of actionable genetic alterations (AGAs) are crucial to improve systemic treatment. We aimed to define the DNA profiles of IBC vs noninflammatory breast cancer (non‐IBC) clinical samples in terms of copy number alterations (CNAs), mutations, and AGAs. We applied targeted next‐generation sequencing (tNGS) and array‐comparative genomic hybridization (aCGH) to 57 IBC and 50 non‐IBC samples and pooled these data with four public datasets profiled using NGS and aCGH, leading to a total of 101 IBC and 2351 non‐IBC untreated primary tumors. The respective percentages of each molecular subtype [hormone receptor‐positive (HR+)/HER2−, HER2+, and triple‐negative] were 68%, 15%, and 17% in non‐IBC vs 25%, 35%, and 40% in IBC. The comparisons were adjusted for both the molecular subtypes and the American Joint Committee on Cancer (AJCC) stage. The 10 most frequently altered genes in IBCs were TP53 (63%), HER2/ERBB2 (30%), MYC (27%), PIK3CA (21%), BRCA2 (14%), CCND1 (13%), GATA3 (13%), NOTCH1 (12%), FGFR1 (11%), and ARID1A (10%). The tumor mutational burden was higher in IBC than in non‐IBC. We identified 96 genes with an alteration frequency (p, We compared the DNA mutational and copy number profiles and mRNA expression profiles of a large series of IBC and noninflammatory breast cancer (non‐IBC) untreated primary tumors. Compared to non‐IBC samples, IBC samples displayed more frequent alterations in the NOTCH and DNA repair pathways independently form the unbalance in term of molecular subtypes and American Joint Committee on Cancer stages.

Advanced-stage breast cancer patients comprise a smaller proportion of breast cancer patients than do early stage patients and are more likely to experience a poor outcome. Understanding the underlying molecular mechanisms and identifying new biomarkers for treatment in this subgroup of patients is paramount. With the aim of identifying microRNAs that are regulated in advanced-stage breast cancer, we found lower expression of miR-26b, a member of the miR-26 family, in inflammatory breast cancer and noninflammatory locally advanced breast cancer tissue than in normal breast tissue, by quantitative real-time polymerase chain reaction and in situ hybridization. Quantitative real-time polymerase chain reaction (but not in situ hybridization) also revealed lower miR-26b expression in inflammatory breast cancer than in noninflammatory locally advanced breast cancer. Furthermore, lower expression of miR-26b was correlated with shorter distant metastasis-free survival and overall survival in univariate analysis, and with shorter overall survival in multivariate analysis. The expression of miRNA-26b was inversely associated with EZH2 protein expression in several breast cancer cell lines, and overexpression and knockdown of miR-26b caused corresponding changes in EZH2 expression. Our study shows that miR-26b may regulate EZH2 expression in breast cancer and may be useful as a therapeutic target for inflammatory breast cancer and noninflammatory locally advanced breast cancer.

Background Inflammatory breast cancer (IBC) is associated with a poor prognosis and high risk of central nervous system (CNS) metastases. Methods We retrospectively reviewed stage III-IBC patients compared with noninflammatory invasive ductal carcinoma (NI-IDC) patients treated between January 1, 1984, and December 31, 2011, who began primary treatment within 1 year of diagnosis and had been followed up for at least 1 year before the development of CNS metastasis or death. Cumulative CNS metastasis incidence and post-CNS metastasis overall survival (OS) estimates were computed. Multivariable Cox proportional hazard models explored factors for post-CNS metastasis survival. Results A total of 2323 patients were identified (589-IBC/1734-NI-IDC). Eighty-one IBC patients developed CNS metastasis, versus 154 NI-IDC patients. The 2-, 5-, and 10-year cumulative CNS metastasis incidence rates in IBC and NI-IDC were 9.8%, 15.8%, 17.4% and 6.5%, 10.1%, and 12.7%, respectively. This was significantly different between IBC and NI-IDC patients (P = .0037). Multicovariate competing risk regression models in IBC and NI-IDC patients showed no statistically significant associations with the risk of developing CNS metastasis, except neoadjuvant taxane use in NI-IDC patients (hazard ratio, 0.45; 95% confidence interval, 0.24-0.83; P = .011). The median follow-up was 7.2 years, and the median post-CNS metastasis OS was not significantly different between IBC (7.6 months) and NI-IDC (5.6 months) patients. One hundred ninety patients with CNS metastasis died. HER2-positive patients had better OS, with a median 14.1 versus 4.3 months (P 50 years (P = .012) but not IBC status was a significant predictor of post-CNS metastasis survival. Conclusion IBC patients demonstrated higher CNS metastasis incidence rates but OS following CNS metastases is similar in both groups. HER2 status and age may play prognostic roles. Cancer 2018;124:2299-305. © 2018 American Cancer Society.

Purpose: Breastfeeding alters the breast microenvironment, and several lines of evidence suggest the breast microenvironment contributes to the clinical phenotype of inflammatory breast cancer. We investigated breastfeeding history as a modifier of locoregional recurrence (LRR), distant metastasis (DM), disease-free survival (DFS), and overall survival (OS) in parous women with inflammatory breast cancer. Methods: Parous women with inflammatory breast cancer were identified from a prospective registry at The University of Texas MD Anderson Cancer Center. We compared patient and tumor characteristics, LRR, DM, DFS, and OS patients with (BF+) and without (BF-) a history of breastfeeding. Results: Eighty-two patients were included. At a median follow-up of 50 months, BF+ patients had significantly lower risk of LRR (9.0% vs. 23.6%; p=0.01), a lower risk of DM (26.8% vs. 53.8%; p=0.008), and better DFS (73.1% vs. 48.1%; p=0.006) than BF- patients. On multivariate analysis, BF+ history was associated with significantly lower risk of DM (hazard ratio 0.38, 95% confidence interval 0.15-0.97; p=0.04) and better DFS (hazard ratio 0.37, 95% confidence interval 0.15-0.93; p=0.04) after adjusting for established predictive and prognostic variables. The prognostic significance of breastfeeding may be most pronounced in women with triple-negative IBC. Conclusion: A lack of breastfeeding history in parous women with inflammatory breast cancer may predict worse prognosis. We speculate that breastfeeding-induced alterations in the breast microenvironment may alter the aggressiveness of inflammatory breast cancer.

International audience; In 2006, a remarkable collaboration between University of Texas MD Anderson Cancer Center clinicians andTexas and New Mexico State legislators led to the formation of a dedicated IBC Research Program and Clinicat MD Anderson. This initiative provided funding and infrastructure to foster coordination of an IBC WorldConsortium of national and international experts, and launch the first ever IBC international conference in2008, which brought together experts from around the world to facilitate collaborations and accelerateprogress. Indeed great progress has been made since then. National and international experts in IBCconvened at the 10th Anniversary Conference of the MD Anderson IBC Clinic and Research Program andpresented the most extensive sequencing analysis to date comparing IBC to non-IBC, gene- andprotein-based immunoprofiling of IBC versus non-IBC patients, and converging lines of evidence on thespecific role of the microenvironment in IBC. Novel models, unique metabolic mechanisms, and prominentsurvival pathways have been identified and were presented. Multiple clinical trials based on the work of thelast decade are in progress or in development. The important challenges ahead were discussed. This progressand a coordinated summary of these works are presented herein.

// Adam R. Wolfe 1, 2 , Nicholaus J Trenton 5 , Bisrat G. Debeb 1, 2 , Richard Larson 1, 2 , Brian Ruffell 6 , Khoi Chu 4 , Walter Hittelman 4 , Michael Diehl 5 , Jim M Reuben 1 , Naoto T. Ueno 1, 3 , Wendy A. Woodward 1, 2 1 MD Anderson Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 2 Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 3 Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 4 Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA 5 Department of Bioengineering, Rice University, Houston, TX, USA 6 Department of Immunology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA Correspondence to: Wendy A. Woodward, email: wwoodward@mdanderson.org Keywords: inflammatory breast cancer, macrophages, mesenchymal stem cells, IL-6, statins Received: August 23, 2016 Accepted: October 06, 2016 Published: October 15, 2016 ABSTRACT Inflammatory breast cancer (IBC) is a unique and deadly disease with unknown drivers. We hypothesized the inflammatory environment contributes to the IBC phenotype. We used an in vitro co-culture system to investigate interactions between normal and polarized macrophages (RAW 264.7 cell line), bone-marrow derived mesenchymal stem cells (MSCs), and IBC cells (SUM 149 and MDA-IBC3). We used an in vivo model that reproduces the IBC phenotype by co-injecting IBC cells with MSCs into the mammary fat pad. Mice were then treated with a macrophage recruitment inhibitor, anti-CSF1. MSC and macrophages grown in co-culture produced higher levels of pro-tumor properties such as enhanced migration and elevated IL-6 secretion. IBC cells co-cultured with educated MSCs also displayed enhanced invasion and mammosphere formation and blocked by anti-IL-6 and statin treatment. The treatment of mice co-injected with IBC cells and MSCs with anti-CSF1 inhibited tumor associated macrophages and inhibited pSTAT3 expression in tumor cells. Anti-CSF1 treated mice also exhibited reduced tumor growth, skin invasion, and local recurrence. Herein we demonstrate reciprocal tumor interactions through IL-6 with cells found in the IBC microenvironment. Our results suggest IL-6 is a mediator of these tumor promoting influences and is important for the IBC induced migration of MSCs.

NDRG1 is widely described as a metastasis suppressor in breast cancer. However, we found that NDRG1 is critical in promoting tumorigenesis and brain metastasis in mouse models of inflammatory breast cancer (IBC), a rare but highly aggressive form of breast cancer. We hypothesized that NDRG1 is a prognostic marker associated with poor outcome in patients with IBC. NDRG1 levels in tissue microarrays from 64 IBC patients were evaluated by immunohistochemical staining with NDRG1 (32 NDRG1-low (&le, median), 32 NDRG1-high (&gt, median)). Overall and disease-free survival (OS and DSS) were analyzed with Kaplan&ndash, Meier curves and log-rank test. Univariate analysis showed NDRG1 expression, tumor grade, disease stage, estrogen receptor (ER) status, and receipt of adjuvant radiation to be associated with OS and DSS. NDRG1-high patients had poorer 10-year OS and DSS than NDRG1-low patients (OS, 19% vs. 45%, p = 0.0278, DSS, 22% vs. 52%, p = 0.0139). On multivariable analysis, NDRG1 independently predicted OS (hazard ratio (HR)=2.034, p = 0.0274) and DSS (HR = 2.287, p = 0.0174). NDRG1-high ER-negative tumors had worse outcomes OS, p = 0.0003, DSS, p = 0.0003, and NDRG1-high tumors that received adjuvant radiation treatment had poor outcomes (OS, p = 0.0088, DSS, p = 0.0093). NDRG1 was a significant independent prognostic factor for OS and DSS in IBC patients. Targeting NDRG1 may represent a novel strategy for improving clinical outcomes for patients with IBC.

Purpose We previously showed that high-density lipoprotein (HDL) radiosensitizes inflammatory breast cancer (IBC) cells in vitro 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 in vitro. Methods and Materials MiR-33a expression was analyzed by reverse transcriptase–polymerase chain reaction in 4 cell 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 in vitro 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. Results 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 24 hours 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 cell lines 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). Conclusions Our results reveal miR-33a negatively regulates HDL-induced radiation sensitivity in 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.

// Bisrat G. Debeb 1, 5 , Lara Lacerda 1, 5 , Richard Larson 1, 5 , Adam R. Wolfe 1, 5 , Savitri Krishnamurthy 2, 5 , James M. Reuben 3, 5 , Naoto T. Ueno 4, 5 , Michael Gilcrease 2 , Wendy A. Woodward 1, 5 1 Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 2 Department of Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 3 Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 4 Department of Breast Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA 5 MD Anderson Morgan Welch Inflammatory Breast Cancer Research Program and Clinic, Houston, TX 77030, USA Correspondence to: Wendy A. Woodward, email: wwoodward@mdanderson.org Bisrat G. Debeb, email: bgdebeb@mdanderson.org Keywords: HDAC inhibitors, cancer stem cells, pentose phosphate pathway, G6PD Received: September 24, 2015 Accepted: March 16, 2016 Published: April 07, 2016 ABSTRACT Purpose: We recently demonstrated that histone deacetylase (HDAC) inhibitors can “reprogram” differentiated triple-negative breast cancer cells to become quiescent stem-like cancer cells. We hypothesized that the metabolic state of such cells differs from that of their differentiated progeny. Results: In untreated cells, glucose uptake was higher in ALDH + cells than in ALDH – cells ( p = 0.01) but lactate production was not different; treating ALDH – or ALDH + cells with VA or SAHA similarly increased glucose uptake without changing lactate production but upregulated G6PD, a rate-limiting enzyme in pentose phosphate pathway metabolism. NADPH production was higher in HDAC inhibitor-treated stem-like cells than in vehicle-treated cells ( p < 0.05). Two G6PD inhibitors, 6-aminonicotinamide and dehydroepiandrosterone, decreased mammosphere formation efficiency and ALDH activity and 6-aminonicotinamide reduced the VA-induced increase in ALDH + cells. Finally, patients expressing high G6PD mRNA had significantly worse overall survival ( p < 0.001), and patients with high G6PD protein showed a similar trend towards worse disease-specific survival ( p = 0.06). Methods: Glucose consumption, lactate and NADPH production, and reactive oxygen species generation were compared in aldehyde dehydrogenase (ALDH)-positive and –negative cells in the presence or absence of the HDAC inhibitors valproic acid (VA) or suberoylanilide hydroxamic acid (SAHA). Glucose-6-phosphate dehydrogenase (G6PD) expression was evaluated in a tissue microarray from 94 patients with node-positive invasive breast carcinoma and in two publically available databases and correlated with overall survival. Conclusions: Energy metabolism in HDAC inhibitor-induced stem-like cancer cells differed sharply from that of differentiated cell types. HDAC inhibitor-induced dedifferentiation promoted metabolic reprogramming into the pentose phosphate pathway, which is targeted effectively by G6PD inhibition. These findings highlight a potential dual-therapy approach to targeting bulk differentiated cells with HDAC inhibitors and CSCs with G6PD inhibitors.

Background and purpose There is mounting evidence that histone deacetylase (HDAC) inhibitors, e.g. valproic acid (VPA), synergize with radiation to improve outcomes in several cancers. This study was conducted to ascertain whether VPA affected outcomes in breast cancer patients with brain metastases treated with whole brain radiotherapy (WBRT). Materials and methods Records from 253 breast cancer patients with brain metastases treated with WBRT were reviewed. Data regarding use of all antiepileptic drugs (AEDs) were extracted. Kaplan–Meier survival times were calculated using the date of brain involvement as time zero. Cox proportional hazard models were used to determine the association between patient and tumor characteristics and overall survival (OS). Results Median OS for the entire patient cohort was 6months. Patients receiving VPA ( n =20) had a median OS of 11months versus 5months for those not receiving VPA ( p =0.028). Median OS was 9months for patients taking any AED ( n =101) versus 4months for those not taking AEDs ( p =0.0003). On multivariate analysis both VPA and AED use were associated with improved OS (HR 0.61, p =0.0419; HR 0.59, p =0.0002, respectively). Conclusions This study suggests the use of AEDs, including VPA, is associated with improved OS in breast cancer patients with brain metastases following WBRT.

Background: Inflammatory breast cancer (IBC) is a clinically distinct and highly aggressive form of primary breast cancer. Although considered rare, IBC accounts for 10% of breast cancer-related deaths owing to its rapid proliferation and strong propensity to metastasize. The molecular mechanisms underlying the aggressiveness and metastatic propensity of IBC remain elusive. Through transcriptome profiling we identified Decorin (DCN) as being significantly altered in metastatic IBC cell sublines. DCN is a secreted, small leucine-rich proteoglycan known to function as a tumor/metastasis suppressor by inhibiting several signaling pathways including EGFR, TGFβ, and c-MET. However, whether or how DCN regulates IBC tumorigenesis or metastasis is unknown. The aim of this study was to investigate the function and mechanism of DCN in IBC tumorigenesis and metastasis. Methods: Three IBC cell lines [ER-/HER2+ (MDA-IBC3, SUM190) and ER-/HER2- (SUM149)] were used. DCN gene expression in clinical samples was analyzed from publically available datasets and the IBC Consortium dataset. DCN was stably expressed in IBC cell lines by using lentiviral vectors. For in vivo studies, DCN-overexpressing stable cell lines were injected into cleared mammary fat pads of SCID/Beige mice and tumor growth was monitored via caliper measurements. Tumor-skin involvement was assessed visually during primary tumor growth and tumor excision. Reverse phase protein array analysis was used for proteomic profiling. Protein-protein interactions were analyzed by reciprocal immunoprecipitation of exogenous or endogenous proteins. Results: DCN expression was significantly lower in breast cancer samples than in normal breast (p Conclusions: We found that DCN inhibited tumorigenesis and skin invasion in IBC via its direct interaction with and stabilization of E-cadherin and its suppression of EGFR signaling. Our findings provide new insights and a novel mechanism for IBC pathobiology that may be therapeutically targetable. Future studies will determine the role of DCN in IBC metastasis and the detailed mechanism of DCN-mediated suppression of tumorigenesis and metastasis in IBC. Citation Format: Xiaoding Hu, Emilly Schlee Villodre, Richard Larson, Omar M. Rahal, Xiaoping Wang, Savitri Krishnamurthy, Debu Tripathy, Naoto T Ueno, Wendy A Woodward, Bisrat Godefay Debeb. Decorin-mediated suppression of tumorigenesis and skin invasion in inflammatory breast cancer via inhibition of the E-cadherin/EGFR axis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-06.

Background: Inflammatory breast cancer (IBC) is a rare (1-2%) but lethal form of primary breast cancer responsible for 10% of breast cancer-related deaths in the United States. IBC tumors grow aggressively and have a high risk of metastasis, particularly to the brain. Unraveling the pathways involved in IBC aggressiveness and metastasis could reveal new therapeutic targets. We recently identified NDRG1 (N-Myc downstream regulated gene1) in a newly isolated subline of MDA-IBC3 cells as a top candidate protein associated with enhanced propensity for brain metastasis. Although the metastasis suppressor properties of NDRG1 are widely described, its function in the pathobiology of IBC is unknown. Here we investigated the function of NDRG1 in tumorigenesis and metastatic progression to brain in IBC. Methods: NDRG1 gene expression was analyzed in large publically available breast cancer clinical datasets and the IBC Consortium dataset, and median expression level was used to stratify patients into NDRG1-high and NDRG1-low groups. Tissue microarrays of IBC patient samples were immunostained with anti-NDRG1 antibody. Stable knockdown (KD) of NDRG1 in IBC cell lines (HER2+: MDA-IBC3; TNBC: SUM149, BCX010) was obtained by lentiviral gene transfer. For in vivo studies, control and NDRG1 KD IBC cell lines were transplanted into cleared mammary fat pads of SCID/Beige mice and tumor growth monitored via caliper measurements. Reverse phase protein array analysis was used for proteomic profiling. Results: In several independent cohorts of patients with breast cancer (including The Cancer Genome Atlas), NDRG1 mRNA expression was significantly higher in ER-negative and high-grade breast tumors (p Conclusions: We showed that high NDRG1 was associated with worse clinical outcomes in patients with IBC and that NDRG1 drives tumor progression in IBC potentially via activation of the EGFR-STAT3 signaling pathway. Targeting the NDRG1-EGFR axis may be a novel therapeutic approach in IBC and other aggressive breast cancers. Additional investigations are underway to determine the detailed mechanisms of NDRG1-mediated IBC tumorigenesis and brain metastasis Citation Format: Emilly Schlee Villodre, Xiaoding Hu, Richard Larson, Bedrich L Eckhardt, Yun Gong, Lei Huo, Juhee Song, Savitri Krishnamurthy, Nuhad Ibrahim, Naoto T Ueno, Debu Tripathy, Wendy A Woodward, Bisrat G Debeb. Ndrg1-egfr axis in inflammatory breast cancer tumorigenesis and brain metastasis [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-01-10.

Introduction We hypothesized that breast tissue not involved by tumor in inflammatory breast cancer (IBC) patients contains intrinsic differences, including increased mammary stem cells and macrophage infiltration, which may promote the IBC phenotype. Materials and methods Normal breast parenchyma ae 5 cm away from primary tumors was obtained from mastectomy specimens. This included an initial cohort of 8 IBC patients and 60 non-IBC patients followed by a validation cohort of 19 IBC patients and 25 non-IBC patients. Samples were immunostained for either CD44(+)CD49f(+)CD133/2(+) mammary stem cell markers or the CD68 macrophage marker and correlated with IBC status. Quantitation of positive cells was determined using inForm software from PerkinElmer. We also examined the association between IBC status and previously published tumorigenic stem cell and IBC tumor signatures in the validation cohort samples. Results 8 of 8 IBC samples expressed isolated CD44(+)CD49f(+)CD133/2(+) stem cell marked cells in the initial cohort as opposed to 0/60 non-IBC samples (p = 0.001). Similarly, the median number of CD44(+)CD49f(+)CD133/2(+) cells was significantly higher in the IBC validation cohort as opposed to the non-IBC validation cohort (25.7 vs. 14.2, p = 0.007). 7 of 8 IBC samples expressed CD68 + histologically confirmed macrophages in initial cohort as opposed to 12/48 non-IBC samples (p = 0.001). In the validation cohort, the median number of CD68 + cells in IBC was 3.7 versus 1.0 in the non-IBC cohort (p = 0.06). IBC normal tissue was positively associated with a tumorigenic stem cell signature (p = 0.02) and with a 79-gene IBC signature (p < 0.001). Conclusions Normal tissue from IBC patients is enriched for both mammary stem cells and macrophages and has higher association with both a tumorigenic stem cell signature and IBC-specific tumor signature. Collectively, these data suggest that IBC normal tissue differs from non-IBC tissue. Whether these changes occur before the tumor develops or is induced by tumor warrants further investigation.

Purpose: Brain metastasis poses a major treatment challenge and remains an unmet clinical need. Finding novel therapies to prevent and treat brain metastases requires an understanding of the biology and molecular basis of the process, which currently is constrained by a dearth of experimental models and specific therapeutic targets. The purpose of this study was to develop preclinical models and identify molecular mediators of brain metastasis from breast cancer. Methods: We used MDA-MB-IBC3 (ER-/HER2+), SUM149 (ER-/HER2-), MCF7 (ER+/HER2-), SUM159 (ER-/HER2-) and MDA-MB-231 (ER-/HER2-) cell lines for this study. GFP-labeled cells were injected via tail vein into SCID/beige mice and metastatic colonization to the brain and lung evaluated by fluorescent stereomicroscope and histology 8-weeks after injection. miRNA microarray was performed with miRNA 3.0 Array. Stable knockdown of miR-141 was achieved with the lentiviral miRZip system. MiR-141 serum levels in 105 breast cancer patients were measured using quantitative PCR. Results: We developed novel brain metastasis models in which tail-vein injection of both triple-negative and a HER2-overexpressing inflammatory breast cancer lines led to a high rate of brain metastases (67%) in SCID/Beige mice (SUM149, 6 of 9 mice; MDA-MB-IBC3, 10 of 15 mice). Sub-lines derived from lung or brain metastases in these models were morphologically and molecularly distinct. The brain metastasis-derived sublines showed epithelial morphology and overexpressed epithelial markers and miR-141 while sublines from lung metastases showed mesenchymal morphology and overexpressed mesenchymal markers. Knockdown of miR-141 significantly inhibited metastatic colonization to the brain compared to controls (miR-141 knockdown vs. control: SUM149, 0 of 8 mice vs. 6 of 9 mice, p=0.009; MDA-MB-IBC3, 2 of 14 mice vs. 10 of 15 mice, p=0.007) but it did not affect colonization to the lung. Importantly, ectopic expression of miR-141 in non-expressing MDA-MB-231 significantly enhanced brain metastatic colonization (5 of 9 mice vs. 0 of 10 mice, P=0.02). On multivariate analyses high serum level of miR-141 was an independent predictor of progression free survival [HR 4.8 (95%CI, 2.6-8.7), P Conclusion: We demonstrated high rates of brain metastases from a heterogeneous group of cell lines that have not previously been associated with brain metastases, demonstrated miR-141 as a key regulator of brain metastasis and provided clinical evidence supporting the prognostic relevance of miR-141. We propose that miR-141 should be examined as a biomarker and potential target in the prevention and treatment of brain metastases from breast cancer. Citation Format: Bisrat G Debeb, Lara Lacerda, Simone Anfossi, Parmeswaran Diagaradjane, Khoi Chu, Lei Huo, Caimiao Wei, Richard A Larson, Adam R Wolfe, Wei Xu, Daniel L Smith, Li Li, Cristina Ivan, Pamela K Allen, Xiang H Zhang, George A Calin, Savitri Krishnamurthy, Naoto T Ueno, Thomas A Buchholz, James M Reuben, Wendy A Woodward. The microRNA miR-141 is a key regulator of brain metastasis from breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-16-01.

In 2006, a remarkable collaboration between University of Texas MD Anderson Cancer Center clinicians and Texas and New Mexico State legislators led to the formation of a dedicated IBC Research Program and Clinic at MD Anderson. This initiative provided funding and infrastructure to foster coordination of an IBC World Consortium of national and international experts, and launch the first ever IBC international conference in 2008, which brought together experts from around the world to facilitate collaborations and accelerate progress. Indeed great progress has been made since then. National and international experts in IBC convened at the 10th Anniversary Conference of the MD Anderson IBC Clinic and Research Program and presented the most extensive sequencing analysis to date comparing IBC to non-IBC, gene- and protein-based immunoprofiling of IBC versus non-IBC patients, and converging lines of evidence on the specific role of the microenvironment in IBC. Novel models, unique metabolic mechanisms, and prominent survival pathways have been identified and were presented. Multiple clinical trials based on the work of the last decade are in progress or in development. The important challenges ahead were discussed. This progress and a coordinated summary of these works are presented herein.

Inflammatory breast cancer (IBC) is associated with a poor prognosis and high risk of central nervous system (CNS) metastases.We retrospectively reviewed stage III-IBC patients compared with noninflammatory invasive ductal carcinoma (NI-IDC) patients treated between January 1, 1984, and December 31, 2011, who began primary treatment within 1 year of diagnosis and had been followed up for at least 1 year before the development of CNS metastasis or death. Cumulative CNS metastasis incidence and post-CNS metastasis overall survival (OS) estimates were computed. Multivariable Cox proportional hazard models explored factors for post-CNS metastasis survival.A total of 2323 patients were identified (589-IBC/1734-NI-IDC). Eighty-one IBC patients developed CNS metastasis, versus 154 NI-IDC patients. The 2-, 5-, and 10-year cumulative CNS metastasis incidence rates in IBC and NI-IDC were 9.8%, 15.8%, 17.4% and 6.5%, 10.1%, and 12.7%, respectively. This was significantly different between IBC and NI-IDC patients (P = .0037). Multicovariate competing risk regression models in IBC and NI-IDC patients showed no statistically significant associations with the risk of developing CNS metastasis, except neoadjuvant taxane use in NI-IDC patients (hazard ratio, 0.45; 95% confidence interval, 0.24-0.83; P = .011). The median follow-up was 7.2 years, and the median post-CNS metastasis OS was not significantly different between IBC (7.6 months) and NI-IDC (5.6 months) patients. One hundred ninety patients with CNS metastasis died. HER2-positive patients had better OS, with a median 14.1 versus 4.3 months (P.0001). Age50 years (P = .012) but not IBC status was a significant predictor of post-CNS metastasis survival.IBC patients demonstrated higher CNS metastasis incidence rates but OS following CNS metastases is similar in both groups. HER2 status and age may play prognostic roles. Cancer 2018;124:2299-305. © 2018 American Cancer Society.

Radiation and chemotherapy are effective treatments for cancer, but are also toxic to healthy cells. Little is known about whether prior exposure to these treatments is related to markers of cellular aging years later in breast cancer survivors. We examined whether past exposure to chemotherapy and/or radiation treatment was associated with DNA damage, telomerase activity, and telomere length 3–6 years after completion of primary treatments in breast cancer survivors (stage 0–IIIA breast cancer at diagnosis). We also examined the relationship of these cellular aging markers with plasma levels of Interleukin (IL)-6, soluble TNF-receptor-II (sTNF-RII), and C-reactive protein (CRP). Ninety-four women (36.4–69.5 years; 80% white) were evaluated. Analyses adjusting for age, race, BMI, and years from last treatment found that women who had prior exposure to chemotherapy and/or radiation compared to women who had previously received surgery alone were more likely to have higher levels of DNA damage (P = .02) and lower telomerase activity (P = .02), but did not have differences in telomere length. More DNA damage and lower telomerase were each associated with higher levels of sTNF-RII (P’s

Epithelial-mesenchymal transition (EMT) is associated with characteristics of breast cancer stem cells, including chemoresistance and radioresistance. However, it is unclear whether EMT itself or specific EMT regulators play causal roles in these properties. Here we identify an EMT-inducing transcription factor, zinc finger E-box binding homeobox 1 (ZEB1), as a regulator of radiosensitivity and DNA damage response (DDR). Radioresistant subpopulations of breast cancer cells derived from ionizing radiation exhibit hyperactivation of ATM and upregulation of ZEB1, and ZEB1 promotes tumor cell radioresistance in vitro and in vivo. Mechanistically, ATM kinase phosphorylates and stabilizes ZEB1 in response to DNA damage, and ZEB1 in turn directly interacts with USP7 and enhances its ability to deubiquitinate and stabilize CHK1, thereby promoting homologous recombination-dependent DNA repair and resistance to radiation. These findings identify ZEB1 as an ATM substrate linking ATM to CHK1 and as the mechanism underlying the association between EMT and radioresistance.

Background: Inflammatory breast cancer (IBC) is the most lethal form of primary breast cancer and accounts for a significant 10 % of breast cancer deaths in the USA owing to its aggressive proliferation and metastasis, and a lack of effective therapeutic options. Unraveling the underlying mechanisms of growth and metastasis of this aggressive disease could lead to effective therapeutic strategies for an improved outcome in IBC patients. We recently generated in vitro and in vivo IBC models for brain metastasis studies [Debeb et al. JNCI, 2016] and observed an upregulation of Lipocalin 2 (LCN2), a small, secreted iron-trafficking protein which plays a significant role in immune and inflammatory responses and the promotion of malignant progression. The purpose of this study was to investigate the function of LCN2 in IBC tumorigenesis and metastasis. Methods: Stable knockdown (KD) of LCN2 in IBC cell lines was achieved with lentiviral vectors. Proteomic and gene expression profiling were performed using RPPA and Affymetrix Clariom D microarray. For in vivo studies, control and LCN2 KD IBC cells were transplanted into the cleared mammary fat pad of SCID/Beige mice. Tumor-skin involvement was assessed visually during primary tumor growth and tumor excision. LCN2 gene expression levels in clinical samples were analyzed from the IBC Consortium as well as public data sets. LCN2 serum levels in IBC patients were measured using ELISA and were correlated with clinicopathological variables and outcome data. Results: LCN2 gene expression is higher in IBC versus non-IBC patients (p=0.00036), independently of the molecular subtypes, and higher in more aggressive (TNBC and HER2+) than hormone receptor-positive subtypes (p Conclusions: Our findings suggest that LCN2 may drive IBC tumor progression and skin invasion/recurrence potentially via the EGFR signaling pathway.Future studies will determine the role of LCN2 in metastasis and pinpoint the detailed mechanisms of LCN2-mediated IBC tumorigenesis and recurrence. Citation Format: Villodre ES, Larson R, Hu X, Stecklein SR, Gomez K, Finetti P, Krishnamurthy S, Ivan C, Su X, Ueno NT, Van Laere S, Bertucci F, Tripathy D, Vivas-Mejía P, A Woodward W, Debeb BG. Lipocalin 2 promotes inflammatory breast cancer tumorigenesis and skin invasion [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-03.

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

Background: Inflammatory breast cancer (IBC) is a rare but highly aggressive form of breast cancer, responsible for 8%–10% of breast cancer–related deaths. To date, a unique molecular signature for IBC able to explain the dramatic differences in survival and tumor aggressiveness compared with non-IBC has not been identified. Triple-negative (TN) subtype is associated with worse clinical outcome in IBC patients. Tumor-associated dendritic cells (DCs) are the central regulators of immune responses in the tumor microenvironment, where the crosstalk with IBC cells may induce proinflammatory immune responses responsible of the development of highly aggressive tumor cells: cancer stem cells with epithelial mesenchymal transition (CSCs/EMT) phenotype. The two miR-19a target genes PTEN and SOCS1 regulate DC activation and maturation by inhibiting toll-like receptor (TLR) and CD40 signaling. As we found that the TN-IBC SUM149 cells expressed and secreted higher levels of miR-19a compared with the TN non-IBC SUM159 cells, we hypothesized that miR-19a secreted by SUM149 cells can be taken up by DCs and downregulate PTEN and SOCS1, leading to increased maturation of DC and production of proinflammatory cytokines upon TLR-mediated activation. Methods: DCs were generated from healthy donor-derived monocytes cultured for 5 days in RPMI 10% FBS supplemented with IL-4 and GM-CSF 1000 U/ml. To track miRNA transfer to DCs, SUM149 cells were transfected with either Dy547-labeled non-targeting miRNA or miR-19a-mimic, then co-cultured with DCs in 1.0 mm pore size transwell chambers for 24 h. MiRNA uptake by DCs was assessed by confocal fluorescence microscopy and qRT-PCR. The effect of miR-19a uptake by DCs were assessed by measuring: the levels of PTEN and SOCS1 mRNA after 24 h of DC co-culture with miR-19a-transfected SUM149; the expression levels of the costimulatory-activation markers CD80, CD86, CD40, CD83, HLA-DR (by FACS); and the production of IL-6, TNF-α (by ELISA) after DC activation by TLR4/LPS (100 ng for 18 h). As IL-6 induces STAT3, a transcription factor for miR-19a, we also evaluated the effect of IL-6 on miR-19a expression in SUM149 cells. Results: Fluorescent Dy547-labeled miRNA and miR-19a were transferred from SUM149 to DCs. The uptake of miR-19a induced the downregulation of both PTEN and SOCS1 mRNA in DCs, leading to increased expression of costimulatory-activation markers and production of IL-6 and TNF-α after DC activation by TLR4/LPS vs control. Stimulation of SUM149 cells with IL-6 upregulated miR-19a expression that was associated with an increased expression of CSC markers (CD44+CD24−Aldefluor+) and upregulation of EMT-regulating transcription factors. Conclusion: DCs can uptake miR-19a secreted by SUM149 cells, leading to increased production of IL-6 and TNF-α upon DC activation by TLR stimulation. IL-6 upregulated miR-19a expression in SUM149 cells. Therefore, miR-19a/IL-6 self-sustaining loop may represent a novel way by which TN-IBC cells maintain a proinflammatory tumor microenvironment able to induce the development of tumor cells with CSCs/EMT phenotype responsible of the poor prognosis of patients with TN-IBC. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-12-12.

Spontaneous canine inflammatory mammary cancer (IMC) shares epidemiologic, histopathological and clinical characteristics with the disease in humans and has been proposed as a natural model for human inflammatory breast cancer (IBC). Few cell lines are available to study IBC, such us SUM 149, SUM 190 and MDA-IBC3. The aim of this study was to characterize a new cell line from IMC (IPC-366) for the comparative study of both IMC and IBC. Tumors cells from a female dog with clinical IMC were collected. The pathological diagnosis of IMC was confirmed at the Veterinary Pathology Service of UCM- Veterinary Clinical Hospital. The cells were grown under adherent conditions in DMEM/F12 with 5% fetal bovine serum and 1% antibiotics (streptomycin and penicillin). The growth and mammospheres production capability, and cytological, ultrastructural and immunohistochemical (IHC) characteristics of IPC-366 were evaluated. Tumorigenicity and metastatic potential of IPC-366 were also assessed by inoculating the cells on the mammary fat pad of 18 female Balb/SCID mice and the development of tumor was monitored by imaging and luciferase assay. Microscopic examination of tumor revealed an epithelial morphology with marked anysocytosis. Doubling time of the tumor cells was approximately 24 h. Under non-adherent conditions, IPC-366 cells formed mammospheres in approximately 3 days. Cytological and histological examination of smears and ultrathin sections by electron microscopy revealed that IPC-366 is formed by highly malignant large round or polygonal cells characterized by marked atypia and prominent nucleoli and frequent multinucleated cells. Some cells had cytoplasmic empty spaces covered by cytoplasmic membrane resembling capillary endothelial cells, a phenomenon described as vasculogenic mimicry. IHC characterization of IPC-366 was basal-like: epithelial cells (AE1/AE3+, CK14+, vimentin+, actin-, p63-, ER-, PR-, HER-2 – (DAKO, HER2 P4 ), overexpressed COX-2 and high Ki-67 proliferation index (87.15 %). Imaging and luciferase assay revealed that at 3 weeks after inoculating the IPC-366 cells, a tumor mass was found in 50 % of mice. At 8 weeks after inoculation metastases in lung, liver and lymph nodes were found. Xenograph tumors maintained the original IHC characteristics of the female dog tumor. In summary, the cell line IPC-366 is a fast growing malignant triple negative cell line model of inflammatory mammary carcinoma that can be used for the comparative study of both IMC and IBC. Citation Format: Sara Caceres, Laura Peña, Lara C Alvarez de la Cerda, Maria J Illera, Richard Larson, Hui Gao, Bisrat G Debeb, Wendy A Woodward, James M Reuben, Juan C Illera. Characterization of a new canine inflammatory mammary cancer (IMC) cell line (IPC-366) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-07-04.

Expression of cyclin E proteolytic cleavage products, low-molecular weight cyclin E (LMW-E), is associated with poor clinical outcome in patients with breast cancer and it enhances tumorigenecity in mouse models. Here we report that LMW-E expression in human mammary epithelial cells induces an epithelial-to-mesenchymal transition phenotype, increases the CD44hi/CD24lo population, enhances mammosphere formation, and upregulates aldehyde dehydrogenase expression and activity. We also report that breast tumors expressing LMW-E have a higher proportion of CD44hi/CD24lo tumor cells as compared with tumors expressing only full-length cyclin E. In order to explore how LMW-E enriches cancer stem cells in breast tumors, we conducted a protein microarray analysis that identified the histone acetyltransferase (HAT) Hbo1 as a novel cyclin E/CDK2 substrate. The LMW-E/CDK2 complex phosphorylated Hbo1 at T88 without affecting its HAT activity. When coexpressed with LMW-E/CDK2, wild-type Hbo1 promoted enrichment of cancer stem-like cells (CSC), whereas the T88 Hbo1 mutant reversed the CSC phenotype. Finally, doxorubicin and salinomycin (a CSC-selective cytotoxic agent) synergized to kill cells expressing LMW-E, but not full-length cyclin E. Collectively, our results suggest that the heightened oncogenecity of LMW-E relates to its ability to promote CSC properties, supporting the design of therapeutic strategies to target this unique function. Cancer Res; 73(17); 5556–68. ©2013 AACR.

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

Recent studies have shown that differentiated cancer cells can dedifferentiate into cancer stem cells (CSCs) although to date no studies have reported whether this transition is influenced by systemic anti-cancer agents. Valproic acid (VA) is a histone deacetylase (HDAC) inhibitor that promotes self-renewal and expansion of hematopoietic stem cells and facilitates the generation of induced pluripotent stem cells from somatic cells and is currently being investigated in breast cancer clinical trials. We hypothesized that HDAC inhibitors reprogram differentiated cancer cells toward the more resistant stem cell-like state. Two highly aggressive breast cancer cell lines, SUM159 and MDA-231, were sorted based on aldehyde dehydrogenase (ALDH) activity and subsequently ALDH-negative and ALDH-positive cells were treated with one of two known HDAC inhibitors, VA or suberoylanilide hydroxamic acid. In addition, primary tumor cells from patients with metastatic breast cancer were evaluated for ALDH activity following treatment with HDAC inhibitors. We demonstrate that single-cell-sorted ALDH-negative cells spontaneously generated ALDH-positive cells in vitro. Treatment of ALDH-negative cells with HDAC inhibitors promoted the expansion of ALDH-positive cells and increased mammosphere-forming efficiency. Most importantly, it significantly increased the tumor-initiating capacity of ALDH-negative cells in limiting dilution outgrowth assays. Moreover, while HDAC inhibitors upregulated β-catenin expression and significantly increased WNT reporter activity, a TCF4 dominant negative construct abolished HDAC-inhibitor-induced expansion of CSCs. These results demonstrate that HDAC inhibitors promote the expansion of breast CSCs through dedifferentiation and have important clinical implications for the use of HDAC inhibitors in the treatment of cancer.

Purpose Prophylactic cranial irradiation (PCI) involves giving radiation to the entire brain with the goals of reducing the incidence of brain metastasis and improving overall survival. Experimentally, we have demonstrated that PCI prevents brain metastases in a breast cancer mouse model. We developed a computational model to expand on and aid in the interpretation of our experimental results. Methods and Materials MATLAB was used to develop a computational model of brain metastasis and PCI in mice. Model input parameters were optimized such that the model output would match the experimental number of metastases per mouse from the unirradiated group. An independent in vivo–limiting dilution experiment was performed to validate the model. The effect of whole brain irradiation at different measurement points after tumor cells were injected was evaluated in terms of the incidence, number of metastases, and tumor burden and was then compared with the corresponding experimental data. Results In the optimized model, the correlation between the number of metastases per mouse and the experimental fits was >95. Our attempt to validate the model with a limiting dilution assay produced 99.9% correlation with respect to the incidence of metastases. The model accurately predicted the effect of whole-brain irradiation given 3 weeks after cell injection but substantially underestimated its effect when delivered 5 days after cell injection. The model further demonstrated that delaying whole-brain irradiation until the development of gross disease introduces a dose threshold that must be reached before a reduction in incidence can be realized. Conclusions Our computational model of mouse brain metastasis and PCI correlated strongly with our experiments with unirradiated mice. The results further suggest that early treatment of subclinical disease is more effective than irradiating established disease.

We previously reported using statins was correlated with improved metastasis-free survival in aggressive breast cancer. The purpose of this study was to examine the effect of statins on metastatic colonization by triple-negative breast cancer (TNBC) cells. TNBC cell lines were treated with simvastatin and then studied for cell cycle progression and proliferation in vitro, and metastasis formation in vivo, following injection of statin-treated cells. Reverse-phase protein assay (RPPA) analysis was performed on statin-treated and control breast cancer cells. RNA interference targeting FOXO3a was used to measure the impact of simvastatin on FOXO3a-expressing cells. The prognostic value of FOXO3a mRNA expression was examined in eight public breast cancer gene expression datasets including 1479 patients. Simvastatin increased G1/S-phase arrest of the cell cycle and inhibited both proliferation and migration of TNBC cells in vitro. In vitro pre-treatment and in vivo treatment with simvastatin reduced metastases. Phosphorylated FOXO3a was downregulated after simvastatin treatment in (RPPA) analysis. Ectopic expression of FOXO3a enhanced mammosphere formation and migratory capacity in vitro. Knockdown of FOXO3a attenuated the effect of simvastatin on mammosphere formation and migration. Analysis of public gene expression data demonstrates FOXO3a mRNA downregulation was independently associated with shorter metastasis-free survival in all breast cancers, as well as in TNBC breast cancers. Simvastatin inhibits in vitro endpoints associated with metastasis through a FOXO3a mechanism and reduced metastasis formation in vivo. FOXO3a expression is prognostic for metastasis formation in patient data. Further investigation of simvastatin as a cancer therapy is warranted.