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Inflammatory breast cancer (IBC), a rare form of breast cancer associated with increased angiogenesis and metastasis, is largely driven by tumor-stromal interactions with the vasculature and the extracellular matrix (ECM). However, there is currently a lack of understanding of the role these interactions play in initiation and progression of the disease. In this study, we developed the first three-dimensional, in vitro, vascularized, microfluidic IBC platform to quantify the spatial and temporal dynamics of tumor-vasculature and tumor-ECM interactions specific to IBC. Platforms consisting of collagen type 1 ECM with an endothelialized blood vessel were cultured with IBC cells, MDA-IBC3 (HER2+) or SUM149 (triple negative), and for comparison to non-IBC cells, MDA-MB-231 (triple negative). Acellular collagen platforms with endothelialized blood vessels served as controls. SUM149 and MDA-MB-231 platforms exhibited a significantly (p < .05) higher vessel permeability and decreased endothelial coverage of the vessel lumen compared to the control. Both IBC platforms, MDA-IBC3 and SUM149, expressed higher levels of vascular endothelial growth factor (p < .05) and increased collagen ECM porosity compared to non-IBCMDA-MB-231 (p < .05) and control (p < .01) platforms. Additionally, unique to the MDA-IBC3 platform, we observed progressive sprouting of the endothelium over time resulting in viable vessels with lumen. The newly sprouted vessels encircled clusters of MDA-IBC3 cells replicating a key feature of in vivo IBC. The IBC in vitro vascularized platforms introduced in this study model well-described in vivo and clinical IBC phenotypes and provide an adaptable, high throughput tool for systematically and quantitatively investigating tumor-stromal mechanisms and dynamics of tumor progression.

Introduction/Motivation: We previously reported IBC patients with limited breast-feeding history have a worse prognosis than those with greater breast feeding history. Further, we reported pregnancy related factors including multiple early pregnancies and not breast feeding as well as obesity are risk factors for triple negative and ER+ subtypes. Significant evidence suggests the microenvironment drives IBC symptoms and growth pattern. We sought to characterize the impact of purported risk factors on tumor growth, IBC-like skin symptoms and mammary gland microenvironment in animal models. Methods: We commissioned breeding of nulliparous, multiparous (x 2) force weaned, and multiparous naturally weaned (labeled nursing) mice. Mice in each of the three groups were fed either a low fat (10 Kcal %) or high fat (60 Kcal %) diet for three weeks before tumor cell injections. SUM 149 GFP Luc were injected into the left ventral #4 mammary fat pad. Mice were sacrificed, scored for the IBC-like symptom of gross skin invasion or evident skin symptoms, and tissue from the contralateral mammary gland were sectioned and stained with H and E. Analysis was performed on the 31 animals that developed primary tumors and were scored for skin invasion at the time of resection. Of these 26 mice had contralateral gland tissue assessed by H and E. Phenotypes including duct dilation, degree of adipose tissue, duct density, inflammation and necrosis were scored manually, 1-3 scale. Quadratic mixed models with random intercept were fitted to compare tumor growth. Fisher’s exact test and T-tests were performed to compare variables. Results: Tumor incidence and latency were not different by risk factor group. Tumor growth was faster in multiparous force weaned versus others, P < 0.0001, but unchanged by diet, P = 0.187. Force weaned animals on a high fat diet had a trend for increased skin invasion compared to others (force weaned high fat diet vs. other 85% vs. 42%; P = 0.08) suggesting a synergy for this IBC-like symptom. Contralateral gland ductal density (less dense) and ductal dilation (not dilated) were normal histologic variables which correlated with skin invasion on the tumor side (P =0.006 and P = 0.011, respectively). Gland density but not dilated ducts trended to correlation with force weaning, P = 0.07, but not diet. Discussion: In this effect size finding pilot study, combined risk factors were associated with a higher incidence of skin invasion of SUM149 xenografts. Changes in the microenvironment in the contralateral gland typical of involution were significantly associated with skin invasion, and gland density trends towards association with force weaning. These data provide provocative pilot results linking histologic effects in the microenvironment to tumor growth characteristics and support the hypothesis that involution induced by forced weaning may facilitate IBC-like tumor growth. These studies facilitate well-powered additional work to establish mechanism and automate normal tissue evaluation. Citation Format: Wintana Balema, Richard Larson, Renae Van Whye, Rong Ye, Jing Ning, Omar Rahal, Shane Stecklein, Savitri Krishnamurthy, Randa El-Zein, Wendy Woodward. Modeling limited breastfeeding and diet on IBC like tumor progression [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 P6-15-04.

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.

Statins, or 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, are medications that have been used for decades to lower cholesterol and to prevent or treat cardiovascular diseases. Since their approval by the US Food and Drug Administration in the 1980s, other potential uses for statins have been speculated on and explored. Basic science and clinical research suggest that statins are also effective in the management of breast cancer. Specifically, in various breast cancer cell lines, statins increase apoptosis and radiosensitivity, inhibit proliferation and invasion, and decrease the metastatic dissemination of tumors. Clinical trials in breast cancer patients support these laboratory findings by demonstrating improved local control and a mortality benefit for statin users. A role for statins in the management of aggressive breast cancers with poor outcomes - namely, inflammatory breast cancer and triple-negative breast cancer - is particularly implicated. However, data exist showing that statins may actually promote invasive breast disease after long-term use and thus should be prescribed cautiously. Furthermore, a general consensus on the type of statin that should be administered, for how long, and when in relation to time of diagnosis is lacking. Given their low toxicity profile, affordability, and ease of use, consideration of statins as a therapy for breast cancer patients is imminent. In this review, we summarize current evidence regarding statins and clinical breast cancer outcomes, as well as discuss potential future studies that could shed light on this increasingly relevant topic.

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.

Although there is some evidence in animal studies that cholesterol signaling mediates radiation sensitivity of normal tissues, until recently, no connection had been made between cholesterol signaling and tumor radiosensitivity. Aberrant cholesterol signaling in breast cancer promotes oncogenesis and tumor progression by either altering membrane fluidity, membrane associated rafts, or as a direct activator of transcription. Cholesterol is synthesized de novo by the mevalonate pathway. A causal role for hydroxy-3-methylglutaryl-coenzyme A reductase, a rate-limiting enzyme of the mevalonate pathway, in oncogenic transformation, progression, and sensitivity to treatment offers an opportunity for drugs that inhibit this enzyme (i.e., statins) as well as other cholesterol mediating strategies as radiosensitizing treatments. This review discusses potential mechanisms by which statins alter cholesterol signaling in breast cancer and potentially enhances radiation sensitivity and outcome with a special focus on inflammatory breast cancer.

Purpose: Inflammatory breast cancer (IBC) is an aggressive variant of breast cancer characterized by visible skin symptoms on the breast at the time of presentation. We demonstrated mesenchymal stem cells (MSC) promote skin symptoms in the SUM149 xenograft model. Here, we extend this finding to IBC patient-derived xenograft (PDX) lines and performed RNA-sequencing analysis of tumors from IBC patient-derived xenograft (PDX) lines by presence of skin symptoms. We hypothesize host MSC variation may explain sporadic skin symptoms in IBC PDX tumors across generations. Methods: MSC were co-injected at the time of tumor initiation in PDX tumors (IBC-3, 5, 6, and 7) and skin symptoms assessed visually and at the time of gross resection. A priori analysis plan was to group PDX by treatment group irrespective of PDX line. IBC PDX models spontaneously but inconsistently develop skin symptoms after multiple passages. RNA sequencing was performed on IBC-PDX tumors (n=33) and non-IBC PDX tumors (n=12) across generations from IBC PDX lines 5, 6, and 7 and non-IBC PDX lines 2, 3, and 4. Four samples were discarded due to poor quality. TSNE was applied using the top differential expressed genes. Xenome analysis was performed to examine host stromal expression by skin involvement. Samples from PDX lines 5 were compared with lines 6 and 7 and the top differentially expressed genes were compared with the Molecular Signatures Databases (MSigDB). CIBERSORT was performed to deconvolute cell type composition. Results: Co-injecting MSC with PDX transplant increased skin symptoms (57% of PDX animals (cohort from IBC5, IBC6, IBC7 and non-IBC4) with MSC vs. 0% without (cohort from IBC5, IBC6, IBC7, non-IBC2 and non-IBC4) (P = 0.0007). TSNE analysis of IBC PDX split samples into three subgroups distinguished by PDX lines and skin invasion. Xenome was used to split reads from human or mouse: samples show high percentage of reads coming from human (mean±SD, 97.44%± 8.98%). Seventy-four genes show FDR ≤ 0.2 between PDX line 5 and line 6 and comparing these top 74 genes with MSigDb for interaction revealed pathways such as GPCR, signal transduction, and sensory perception. CIBERSORT revealed no enrichment for MSC in IBC PDX with skin invasion, however significant differences in CD8 expression in these immunosuppressed PDX models casts doubt on the robustness of this finding. Conclusions: Exogenous MSC enhance skin symptoms in a cohort of IBC and non-IBC PDX. Skin symptoms segregate IBC PDX tumors by expression suggesting specific biology drives this presentation. However, we are unable to demonstrate evidence for spontaneous MSC signaling in these models. Future work is warranted to link top skin symptom related signaling to stromal signals and unravel the unexpected CD8 signaling identified in these models. Citation Format: Omar Rahal, Jie Yang, Li Li, Richard Larson, Steven Van Laere, Naoto Ueno, Erik Sulman, Wendy Woodward. Transcriptome analysis of patient derived xenograft mouse models of inflammatory breast cancer to gain insights into the contribution of tumor microenvironment [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 P6-06-08.

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.

Introduction: Inflammatory breast cancer (IBC) is an aggressive and rare disease with poor prognosis, accounting for 10% of breast cancer mortality [1]. A primary factor contributing to the bleak prognosis is the lack of IBC specific treatments. There are currently no IBC specific therapies due to a lack of IBC specific diagnostic and targeting markers. Efforts focused on identifying driver mutations and tumor targets have implicated tumor stroma including stromal cells such as macrophages in mediating IBC-like symptoms. This highlights the significance of understanding the interactions of tumor cells with the tumor stroma in greater detail and the knowledge would enable determination of targetable biology from these interactions which would facilitate development of IBC specific treatments and therapeutics. What is needed is a model to capture the complexity of IBC, identify critical spatial hetero-cellular interactions and target them successfully in a physiologically relevant and high-throughput manner. Approach: To address this need, we developed a 3D IBC microfluidic platform, unique in its simultaneous integration of functional blood vessels, tumor cells, macrophages, and type I collagen whose density, stiffness, and porosity mimics cancerous breast stroma. The platform will be used to study the influence of macrophage-tumor-endothelial interactions on 2 key critical features of IBC: vascular sprouting and formation of IBC emboli surrounded by vascular sprouts. Results: The 3D IBC microfluidic platform composed of MDA-IBC3 cells and a functional endothelial blood vessel demonstrated both vascular sprouting and emboli formation, key features of IBC tumors seen in IBC patient derived xenograft (PDX) models. Additionally, we observed vascular nesting of MDA-IBC3 emboli, recreating a characteristic IBC phenomenon observed in Mary-X PDX models. Incorporation of macrophages significantly increased the number of new vascular sprouts, sprouting rate and resulted in sprouts forming at earlier time points. Additionally, the presence of macrophages resulted in the formation of a significantly more porous collagen matrix (p Conclusion: IBC is an aggressive and invasive breast cancer with a poor prognosis linked to tumor-stroma interactions. Current preclinical to study IBC consist primarily of PDX models where determining the influence of specific signaling pathways and microenvironmental stimuli on tumor progression is challenging. Here we present a novel 3D microfluidic IBC platform to study tumor stromal interactions in a controlled manner. The MDA-IBC3 breast tumor platform demonstrated both vascular sprouting and emboli formation, key features of IBC seen in PDX models and the presence of macrophages increased both angiogenic sprouting and remodeling of the collagen matrix. The stark differences in the tumor platform response associated with macrophage presence strengthens the hypothesis of tumor stroma as a key player driving the aggressive nature of IBC and reveals a potential target for IBC therapeutics. [1] Fernandez, S.V., et al., Breast cancer research and treatment, 140(1): p. 23-33, 2013 Citation Format: Manasa Gadde, Caleb Phillips, Omar Rahal, Wendy Woodward, Marissa Rylander, Thomas Yankeelov. An in vitro microfluidic tumor platform for modeling and investigating tumor stromal interactions in inflammatory breast cancer [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 P6-06-02.

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

Purpose To determine the role of macrophage polarization on the response of inflammatory breast cancer (IBC) cells to radiation and whether modulation of macrophage plasticity can alter radiation response. Methods and Materials The human THP-1 monocyte cell line and primary human monocytes isolated from peripheral blood mononuclear cells were differentiated into macrophages and polarized to either an “antitumor” (M1) or a “protumor” (M2) phenotype. These polarized macrophages were co-cultured with IBC cells (SUM149, KPL4, MDA-IBC3, or SUM190) without direct contact for 24 hours, then subjected to irradiation (0, 2, 4, or 6 Gy). Interleukin (IL)4/IL13-induced activation of STAT6 signaling was measured by Western blotting of phospho-STAT6 (Tyr641), and expression of M2 polarization gene markers (CD206, fibronectin, and CCL22) was measured by quantitative polymerase chain reaction. Results Expression of M2 polarization markers was higher in M2-polarized macrophages after IL4/IL13 treatment than in control (M0) or M1-polarized macrophages. Co-culture of IBC cell lines with M1-polarized THP-1 macrophages mediated radiosensitivity of IBC cells, whereas co-culture with M2-polarized macrophages mediated radioresistance. Phosphopeptide mimetic PM37, targeting the SH2 domain of STAT6, prevented and reversed IL4/IL13-mediated STAT6 phosphorylation (Tyr641) and decreased the expression of M2 polarization markers. Pretreatment of M2-THP1 macrophages with PM37 reduced the radioresistance they induced in IBC cells after co-culture. Targeted proteomics analysis of IBC KPL4 cells using a kinase antibody array revealed induction of protein kinase C zeta (PRKCZ) in these cells only after co-culture with M2-THP1 macrophages, which was prevented by PM37 pretreatment. KPL4 cells with stable short hairpin RNA knockdown of PRKCZ exhibited lower radioresistance after M2-THP1 co-culture. Conclusions These data suggest that inhibition of M2 polarization of macrophages by PM37 can prevent radioresistance of IBC by down-regulating PRKCZ.

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.

Despite the well-accepted notion that early maternal influences persist beyond fetal life and may underlie many adult diseases, the risks imposed by the maternal environment on breast cancer development and underlying biological mechanisms remain poorly understood. In this study, we investigated whether early exposure to blueberry (BB) via maternal diet alters oncogene Wnt1-induced mammary tumorigenesis in offspring. Wnt1-transgenic female mice were exposed to maternal Casein (CAS, control) or blueberry-supplemented (CAS + 3%BB) diets throughout pregnancy and lactation. Offspring were weaned to CAS and mammary tumor development was followed until age 8 months. Tumor incidence and latency were similar for both groups; however, tumor weight at killing and tumor volume within 2 weeks of initial detection were lower (by 50 and 60%, respectively) in offspring of BB- versus control-fed dams. Dietary BB exposure beginning at weaning did not alter mammary tumor parameters. Tumors from maternal BB-exposed offspring showed higher tumor suppressor (Pten and Cdh1) and lower proproliferative (Ccnd1), anti-apoptotic (Bcl2) and proangiogenic (Figf, Flt1 and Ephb4) transcript levels, and displayed attenuated microvessel density. Expression of Pten and Cdh1 genes was also higher in mammary tissues of maternal BB-exposed offspring. Mammary tissues and tumors of maternal BB-exposed offspring showed increased chromatin-modifying enzyme Dnmt1 and Ezh2 transcript levels. Body weight, serum insulin and serum leptin/adiponectin ratio were lower for maternal BB-exposed than control tumor-bearing offspring. Tumor weights and serum insulin were positively correlated. Results suggest that dietary influences on the maternal environment contribute to key developmental programs in the mammary gland to modify breast cancer outcome in adult progeny.

Breast cancer is the leading cause of cancer deaths in women. Diet and lifestyle are major contributing factors to increased breast cancer risk. While mechanisms underlying dietary protection of mammary tumor formation are increasingly elucidated, there remains a dearth of knowledge on the nature and precise actions of specific bioactive components present in foods with purported health effects. The 43-amino acid peptide lunasin (LUN) is found in soybeans, is bioavailable similar to the isoflavone genistein (GEN), and thus may mediate the beneficial effects of soy food consumption. Here, we evaluated whether LUN displays common and distinct actions from those of GEN in non-malignant (mouse HC11) and malignant (human MCF-7) mammary epithelial cells. In MCF-7 cells, LUN up-regulated tumor suppressor phosphatase and tensin homolog deleted in chromosome ten (PTEN) promoter activity, increased PTEN transcript and protein levels and enhanced nuclear PTEN localization, similar to that shown for GEN in mammary epithelial cells. LUN-induced cellular apoptosis, akin to GEN, was mediated by PTEN, but unlike that for GEN, was p53-independent. LUN promoted E-cadherin and β-catenin non-nuclear localization similar to GEN, but unlike GEN, did not influence the proliferative effects of oncogene Wnt1 on HC11 cells. Further, LUN did not recapitulate GEN inhibitory effects on expansion of the cancer stem-like/progenitor population in MCF-7 cells. Results suggest the concerted actions of GEN and LUN on cellular apoptosis for potential mammary tumor preventive effects and highlight whole food consumption rather than intake of specific dietary supplements with limited biological effects for greater health benefits.

Mammary stem cells are undifferentiated epithelial cells, which initiate mammary tumors and render them resistant to anticancer therapies, when deregulated. Diets rich in fruits and vegetables are implicated in breast cancer risk reduction, yet underlying mechanisms are poorly understood. Here, we addressed whether dietary factors selectively target mammary epithelial cells that display stem-like/progenitor subpopulations with previously recognized tumor-initiating potential. Using estrogen receptor-positive MCF-7 and estrogen receptor-negative MDA-MB-231 human breast cancer cell lines and freshly isolated epithelial cells from MMTV-Wnt-1 transgenic mouse mammary tumors, we demonstrate that sera of adult mice consuming soy isoflavone genistein (GEN) or blueberry (BB) polyphenol-containing diets alter the population of stem-like/progenitor cells, as measured by their functional ability to self-renew and form anchorage-independent spheroid cultures in vitro at low frequency (1-2%). Serum effects on mammosphere formation were dose-dependently replicated by GEN (40 nM >2 μM) and targeted the basal stem-like CD44+/CD24-/ESA+ and the luminal progenitor CD24+ subpopulations in MDA-MB-231 and MCF-7 cells. GEN inhibition of mammosphere formation was mimicked by the Akt inhibitor perifosine and was associated with enhanced tumor suppressor phosphatase and tensin homologue deleted on chromosome ten (PTEN) expression. In contrast, a selected mixture of BB phenolic acids was only active in MDA-MD-231 cells and its CD44+/CD24-/ESA+ subpopulation, and this activity was independent of induction of PTEN expression. These findings delineate a novel and selective function of distinct dietary factors in targeting stem/progenitor cell populations in estrogen receptor-dependent and -independent breast cancers.

The mammary gland is composed of two major cellular compartments: a highly dynamic epithelium that undergoes cycles of proliferation, differentiation and apoptosis in response to local and endocrine signals and the underlying stroma comprised of fibroblasts, endothelial cells and adipocytes, which collectively form the mammary fat pad. Breast cancer originates from subversions of normal growth regulatory pathways in mammary epithelial cells due to genetic mutations and epigenetic modifications in tumor suppressors, oncogenes and DNA repair genes. Diet is considered a highly modifiable determinant of breast cancer risk; thus, considerable efforts are focused on understanding how certain dietary factors may promote resistance of mammary epithelial cells to growth dysregulation. The recent indications that stromal cells contribute to the maintenance of the mammary epithelial 'niche' and the increasing appreciation for adipose tissue as an endocrine organ with a complex secretome have led to the novel paradigm that the mammary stromal compartment is itself a relevant target of bioactive dietary factors. In this review, we address the potential influence of dietary factors on mammary epithelial-stromal bidirectional signaling to provide mechanistic insights into how dietary factors may promote early mammary epithelial differentiation to decrease adult breast cancer risk.

Mammary stromal adipocytes constitute an active site for the synthesis of the adipokine, adiponectin (APN) that may influence the mammary epithelial microenvironment. The relationship between “local,” mammary tissue-derived APN and breast cancer risk is poorly understood. Here, we identify a novel mechanism of APN-mediated signaling that influences mammary epithelial cell proliferation, differentiation, and apoptosis to modify breast cancer risk. We demonstrate that early dietary exposure to soy protein isolate induced mammary tissue APN production without corresponding effects on systemic APN levels. In estrogen receptor (ER)-negative MCF-10A cells, recombinant APN promoted lobuloalveolar differentiation by inhibiting oncogenic signal transducer and activator of transcription 3 activity. In ER-positive HC11 cells, recombinant APN increased ERβ expression, inhibited cell proliferation, and induced apoptosis. Using the estrogen-responsive 4X-estrogen response element promoter-reporter construct to assess ER transactivation and small interfering RNA targeting of ERα and ERβ, we show that APN synergized with the soy phytoestrogen genistein to promote ERβ signaling in the presence of estrogen (17β-estradiol) and ERβ-specific agonist 2,3-bis(4-hydroxyphenyl)-propionitrile and to oppose ERα signaling in the presence of the ERα-specific agonist 4,4′,4′-(4-propyl-(1H)-pyrazole-1,3,5-triyl)trisphenol. The enhancement of ERβ signaling with APN + genistein cotreatments was associated with induction of apoptosis, increased expression of proapoptotic/prodifferentiation genes (Bad, p53, and Pten), and decreased antiapoptotic (Bcl2 and survivin) transcript levels. Our results suggest that mammary-derived APN can influence adjacent epithelial function by ER-dependent and ER-independent mechanisms that are consistent with reduction of breast cancer risk and suggest local APN induction by dietary factors as a targeted approach for promotion of breast health.

The tumor suppressors phosphatase and tensin homologue deleted on chromosome ten (PTEN) and p53 are closely related to the pathogenesis of breast cancer, yet pathway-specific mechanisms underlying their participation in mediating the protective actions of dietary bioactive components on breast cancer risk are poorly understood. We recently showed that dietary exposure to the soy isoflavone genistein (GEN) induced PTEN expression in mammary epithelial cells in vivo and in vitro, consistent with the breast cancer preventive effects of soy food consumption. Here, we evaluated PTEN and p53 functional interactions in the nuclear compartment of mammary epithelial cells as a mechanism for mammary tumor protection by GEN. Using the non-tumorigenic human mammary epithelial cells MCF10-A, we demonstrate that GEN increased PTEN expression and nuclear localization. We show that increased nuclear PTEN levels initiated an autoregulatory loop involving PTEN-dependent increases in p53 nuclear localization, PTEN-p53 physical association, PTEN-p53 co-recruitment to the PTEN promoter region and p53 transactivation of PTEN promoter activity. The PTEN-p53 cross talk induced by GEN resulted in increased cell cycle arrest; decreased pro-proliferative cyclin D1 and pleiotrophin gene expression and the early formation of mammary acini, indicative of GEN promotion of lobuloalveolar differentiation. Our findings provide support to GEN-induced PTEN as both a target and regulator of p53 action and offer a mechanistic basis for PTEN pathway activation to underlie the antitumor properties of dietary factors, with important implications for reducing breast cancer risk.

Cells within the tumor microenvironment, including but not limited to macrophages and mesenchymal stem cells (MSCs), can promote the phenotype and aggressiveness of inflammatory breast cancer (IBC). For example, co-injection of MSCs with SUM149 IBC cells significantly increased the clinical features of IBC such as skin invasion and metastasis. Our preliminary work showed that MSCs can be educated by co-culture with M1 polarized (anti-tumor) or M2 polarized (pro-tumor) mouse Raw macrophages. Such education of MSCs by M2 Raw macrophages leads to increased IL6 secretion by MSCs, relative to M1-educated or uneducated MSCs. M2-educated MSCs also have increased migration toward IBC cell lines SUM149 and IBC3, effects that can be blocked by an anti-IL6 antibody. Co-culture with M2-educated MSCs also enhances migration and mammosphere formation of IBC cells. Radiation response of IBC cells upon interactions with cells from the tumor microenvironment was also analyzed. Preliminary work shows that co-culture of IBC cells (SUM149 and KPL4) with M2-polarized human THP1 macrophages, prior to ionizing radiation, mediates radiation resistance of IBC cells, and this effect can be decreased by either adding HDL lipoproteins during co-culture period or by STAT6 inhibitors that block IL4/IL13-mediated phosphorylation of STAT6 and M2-polarization in THP1 macrophages. MSCs can also be polarized into either a MSC1 phenotype or a MSC2 phenotype by exposure to toll-like receptor (TLR) ligands TLR4 or TLR3, respectively. Indoleamine-pyrrole 2,3-dioxygenase (IDO) expression in MSCs is a marker of MSC2 polarization that is induced after exposure with TLR3 ligand (PolyIC) relative to MSC1 (TLR4 stimulated; LPS-treated) or parental MSCs. Similar to macrophage polarization, while MSC1 mediates anti-tumor effects, MSC2 are immunosuppressive and thus contribute to tumor growth. Preliminary work also shows that co-culture of IBC cells with MSC2 mediates radioresistance and this can be decreased as well by exposure to HDL during co-culture period prior to radiation. HDL protective effects, in part, can be explained by decreased expression of TLR3-induced IDO mRNA levels in MSC2. In the present work, we extended the above mentioned observations regarding the crosstalk between mouse Raw macrophages and MSCs by analyzing the effect of co-culture of human THP1 macrophages (parental designated as M0, M1- or M2-polarized) with MSCs on the IDO mRNA expression in MSCs, a marker of MSC2 polarization. Surprisingly, co-culture of M1-polarized THP1 with MSCs resulted in a robust increased expression of IDO mRNA in MSC relative to parental MSC (uneducated) or MSCs co-cultured with M2-THP1. Further studies are needed to determine the effects of increased IDO expression in MSC, upon M1-THP1 co-culture, on the aggressive behavior of IBC cells and whether this could be altered with IDO inhibitors. Our results suggest that there could be inter-species differences between mouse and human macrophages on the education of human MSCs. Based on our findings we propose testing a combination of STAT6 inhibitors that reverse M2-polarization of macrophages and IDO inhibitors that can decrease MSC2 phenotype mediated by TLR3 exposure and/or M1-THP1 education. Citation Format: Rahal OM, Wolfe AR, Mandal PK, Larson R, Tin S, Reuben JM, McMurray JS, Woodward WA. Understanding the complexity of macrophage and mesenchymal stem cell interactions to improve treatment outcome for IBC patients [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 PD6-06.

Early developmental events influence the fine tuning of later susceptibility to adult diseases. Diet is a determinant of breast cancer risk, and our previous studies showed that diet-mediated changes in transcriptional programs promote early mammary gland differentiation. Although consumption of fruits is considered to elicit multiple health benefits, little is known on whether associated bioactive components modify the early differentiation program in developing mammary glands. Here, we evaluated the hypothesis that early exposure (in utero and lactational) to blueberry through maternal diet enhances mammary epithelial differentiation in female offspring. Pregnant Sprague-Dawley rats beginning at gestation day 4 were fed American Institute of Nutrition-based diets containing casein and whole blueberry powders added to casein at 2.5%, 5.0%, and 10% weight/weight. Female pups at weaning were evaluated for growth and mammary tissue parameters. Blueberry at 5% dose increased body and adipose fat weights, relative to the other diets. Mammary branch density and terminal end bud size were highest for the 5% blueberry group, whereas terminal end bud numbers were not affected by all diets. Mammary ductal epithelial cells of the 5% blueberry group had lower nuclear phosphorylated histone 3 and higher nuclear tumor suppressor phosphatase and tensin homolog deleted in chromosome 10 (PTEN) levels than the casein group. Although sera of both diet groups had similar antioxidant capacity, 5% blueberry sera elicited higher nuclear PTEN accumulation in human MCF-10A mammary epithelial cells. Our studies identify developing mammary glands as early targets of blueberry-associated bioactive components, possibly through systemic effects on epithelial PTEN signaling.

Exposure to mid range ultrat violet radiations (UVBs) has been shown to produce systemic inflammation and hyperalgesia in mice [Saade, N.E., Nasr, I.W., Massaad, C.A., Safieh-Garabedian, B., Jabbur, S.J., Kanaan, S.A., 2000. Modulation of ultraviolet-induced hyperalgesia and cytokine upregulation by interleukins 10 and 13. Br. J. Pharmacol. 131, 1317–1324]. Our aim was to characterize a new rat model of localized exposure to UVB and to determine the role of skin innervation in the observed hyperalgesia and cytokine upregulation. In several groups of rats one hindpaw was exposed to UVB (250–350 mJ/cm 2 ) and this was followed by the application, to the plantar area of the paw, of either Von Frey hairs or a few acetone drops to measure tactile and cold allodynia, respectively. Thermal hyperalgesia was assessed by the paw withdrawal latency and duration. Cytokine levels were determined, by ELISA, in processed samples of skin tissue isolated from the exposed and non-exposed paws. UVB induced a biphasic thermal hyperalgesia and cold and tactile allodynia with an early phase that peaked at 3–6 h and disappeared at 24 h and a late phase with a peak at 48 h and recovery at 72-h post-exposure. Tumor necrosis factor, interleukins 1β, 6, 8, 10 and NGF levels were significantly increased following the same biphasic temporal pattern. Chemical ablation of capsaicin sensitive afferents and guanethidine injection produced significant alteration of the hyperalgesia and allodynia. The increase in cytokine levels by UVB was also altered by both treatments. The present study describes a new animal model for localized UVB-induced inflammatory hyperalgesia and provides evidence about the involvement of neurogenic mechanisms in the observed hyperalgesia and upregulation of proinflammatory mediators.

Our previous study showed that specifically delivering BikDD, a constitutive active mutant of pro-apoptotic protein Bik, to breast cancer cell xenografts in immunocompromised mice has a potent activity against tumor initiating cells (TICs), and that the combination between tyrosine kinase inhibitors (TKI) and BikDD gene therapy yielded synergistic effect on EGFR and HER2 positive breast cancer cells in immunodeficient nude mice. Those encouraging results have allowed us to propose a clinical trial using the liposome-complexing plasmid DNA expressing BikDD gene which has been approved by the NIH RAC Advisory committee. However, it is imperative to test whether systemic delivery of BikDD-expressing plasmid DNAs with liposomes into immunocompetent mice has therapeutic efficacy and tolerable side effects as what we observed in the nude mice model. In this study, we investigated the effects of BikDD gene-therapy on the primary mammary tumors, especially on tumor initiating cells (TICs), of a genetically engineered immunocompetent mouse harboring normal microenvironment and immune response. The effects on TIC population in tumors were determined by FACS analysis with different sets of murine specific TIC markers, CD49f(high)CD61(high) and CD24(+)Jagged1(-). First we showed in vitro that ectopic expression of BikDD in murine N202 cells derived from MMTV-HER2/Neu transgenic mouse tumors induced apoptosis and decreased the number of TICs. Consistently, systemic delivery of VISA-Claudin4-BikDD by liposome complexes significantly inhibited mammary tumor growth and slowed down residual tumor growth post cessation of therapy in MMTV-HER2/Neu transgenic mice compared to the controls. In addition, the anti-tumor effects of BikDD in vivo were consistent with decreased TIC population assessed by FACS analysis and in vitro tumorsphere formation assay of freshly isolated tumor cells. Importantly, systemic administration of BikDD did not cause significant cytotoxic response in standard toxicity assays or body weight changes. Taken together, our findings validated that selective expression of BikDD in the primary mammary tumors in immunocompetent hosts significantly reduced tumor burden and inhibited the residual tumor growth at off-therapy stage by eliminating TICs. Hence, the VISA-Claudin4-BikDD-mediated gene therapy is worthy of further investigation in breast cancer clinical trials.

Mammary adipose tissue may contribute to breast cancer development and progression by altering neighboring epithelial cell behavior and phenotype through paracrine signaling. Dietary exposure to soy foods is associated with lower mammary tumor risk and reduced body weight and adiposity in humans and in rodent breast cancer models. Despite the suggested linkage between obesity and breast cancer, the local influence of bioactive dietary components on mammary adiposity for antitumor effects remains unknown. Herein, we report that post-weaning dietary exposure to soy protein isolate and its bioactive isoflavone genistein (GEN) lowered mammary adiposity and increased mammary tumor suppressor PTEN and E-cadherin expression in female mice, relative to control casein diet. To ascertain GEN's role in mammary adipose deposition that may affect underlying epithelial cell phenotype, we evaluated GEN's effects on SV40-immortalized mouse mammary stromal fibroblast-like (MSF) cells during differentiation into adipocytes. MSF cells cultured in a differentiation medium with 40 nM GEN showed reductions in mature adipocyte numbers, triglyceride accumulation, andPparγ(Pparg) and fatty acid synthase transcript levels. GEN inhibition of adipose differentiation was accompanied by increased estrogen receptor β (Erβ(Esr2)) gene expression and was modestly recapitulated by ERβ-selective agonist 2,3-bis-(4-hydroxyphenyl)-propionitrile (DPN). Reduction ofErβexpression by siRNA targeting increasedPparγtranscript levels and stromal fibroblast differentiation into mature adipocytes; the latter was reversed by GEN but not by DPN. Conditioned medium from GEN-treated adipocytes diminished anchorage-independent mammosphere formation of human MCF-7 breast cancer cells. Our results suggest a mechanistic pathway to support direct regulation of mammary adiposity by GEN for breast cancer prevention.

The increasing cognizance that diet (and lifestyle) can modify breast cancer risk and progression has motivated many breast cancer patients to take increasing personal control of the direction of their therapies after diagnosis and surgery. While this has certain advantages, including higher compliance to prescribed drugs and improvements in emotional and mental well-being, it predicates the need for increased understanding of the benefits of particular diets and dietary regimen to the treatment programs and for improved translation of data obtained from studies with animal models into clinical settings. Epidemiological studies have linked high consumption of soy-rich foods to the lower incidence of breast cancer in Asia relative to that in Western countries. The potential of soy-rich foods as breast cancer protective when dietary exposure occurs early in life, has resulted in driving the use of soy and its associated bioactive components, specifically the isoflavone genistein, as chemopreventive agents or as adjuvants to conventional drug therapies. Bioactive components in soy foods may affect hormone and non-hormone-mediated mechanisms. However, their overall biological outcomes remain not well-understood and at times, contradictory, due to distinct physiological contexts and doses of exposure, multiple targets, and inconsistent measures of relevant endpoints. Here we provide an argument in support of the potential use of soy foods for breast cancer patients based on the review of the current literature as well as raise caveats that must be addressed for its successful application as standard-of-care treatment.

Krüppel-like factors (KLFs), of which there are currently 17 known protein members, belong to the specificity protein (Sp) family of transcription factors and are characterized by the presence of Cys2/His2 zinc finger motifs in their carboxy-terminal domains that confer preferential binding to GC/GT-rich sequences in gene promoter and enhancer regions. While previously regarded to simply function as silencers of Sp1 transactivity, many KLFs are now shown to be relevant to human cancers by their newly identified abilities to mediate crosstalk with signaling pathways involved in the control of cell proliferation, apoptosis, migration, and differentiation. Several KLFs act as tumor suppressors and/or oncogenes under distinct cellular contexts, underscoring their prognostic potential for cancer survival and outcome. Recent studies suggest that a number of KLFs can influence steroid hormone signaling through transcriptional networks involving steroid hormone receptors and members of the nuclear receptor family of transcription factors. Since inappropriate sensitivity or resistance to steroid hormone actions underlies endocrine-related malignancies, we consider the intriguing possibility that dysregulation of expression and/or activity of KLF members is linked to the pathogenesis of endometrial and breast cancers. In this review, we focus on recently described mechanisms of actions of several KLFs (KLF4, KLF5, KLF6, and KLF9) in cancers of the mammary gland and uterus. We suggest that understanding the mode of actions of KLFs and their functional networks may lead to the development of novel therapeutics to improve current prospects for cancer prevention and cure.

The maintenance of optimal metal levels is an essential aspect of cell homoeostasis. However, in many types of cancer these metal levels especially iron, zinc and copper diverge from normal levels. We have recently shown that the zinc chelator TPEN increases the generation of reactive oxygen species (ROS) which selectively kills colon cancer cells. We have also provided evidence that the redox cycling of copper is responsible for TPEN anticancer effects. In this study, we aimed to further decipher the mechanism of TPEN-induced cell death in colon cancer cells through studying its effect on DNA damage. HCT116 p53+/+ human colon cancer cells were seeded were treated with 5μM TPEN at 50% confluence The inhibition of cell growth was measured by MTT, while ROS production was measured by the DCFH Assay using flow cytometry. siRNAs against DNApk and Chk2 was used to investigate the involvement of these DNA damage sensors in TPEN activity. DNA damage was assessed by the comet assay. Phosphorylation of ATM, ATR, Chk1, Chk2 and H2AX by TPEN were detected immunocytochemically by multiparameter cytometry. Expression levels of Chk1/2, ATR and DNApK were determined by western blotting. We show that cell death by TPEN is associated with significant DNA damage, an effect that was dependent on ROS generation and on the redox cycling of copper, as evidenced by reversal of DNA damage in the presence of antioxidants (NAC, CAT) or the copper chelator neocuproine (Neo). DNA damage was associated with increased expression of p-H2AX and a significant activation of ATM/ATR signaling molecules, specifically p-ATM, p-ATR and p-Chk1. Interestingly, silencing DNApk and Chk2 reversed DNA damage caused by TPEN, suggesting the involvement of DNApk and ATM/ATR pathways in TPEN-mediated effects. This study shows for the first time the involvement of DNApk and Chk2 in TPEN-induced DNA damage and confirms our previous findings that the redox cycling of copper is the main mechanism by which TPEN induces cell death in human colon cancer cells. Citation Format: Hala Gali-Muhtasib, Omar Rahal, Maamoun Fatfat, Carla Hankache, Bassam Osman, Hala Khalife, Khaled Machaca. The anticancer molecule TPEN induces DNA damage in human colon cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2564. doi:10.1158/1538-7445.AM2015-2564

Breast cancer risk is highly modified by environmental factors including diet. Previously, we showed that dietary intake of soy protein isolate (SPI) decreased mammary tumor incidence and increased mammary tumor latency in rats relative to those fed a control casein (CAS) diet, when exposed to the chemical carcinogen NMU. Mammary tumor preventive effects by SPI were associated with up-regulation of the tumor suppressor PTEN and down-regulation of the oncogenic Wnt-signaling components in mammary epithelial cells (MECs) leading to enhanced differentiation. Given that breast cancer is considered to be initiated by stem cells (SCs) with tumorigenic potential, termed cancer stem cells (CSCs), and mammary over-expression of Wnt-1 in mice causes spontaneous breast tumors due to the expansion of mammary CSCs, we hypothesized that diet may alter the mammary SC population to effect mammary tumor prevention. Here, we investigated SPI effects relative to CAS, on mammary tumor development in MMTV-Wnt 1-Transgenic (Tg) female mice and on the mammary SC population in virgin wildtype (WT) and pre-neoplastic Tg female mice. Tumor incidence at 8 months of age of Tg mice fed SPI (n=30) post-weaning was lower than in those fed CAS (48.3% vs.73.5%; P Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr PD02-03.

Abstract #4069 Breast cancer is the second leading cause of death in the Western hemisphere affecting one of eight women in their lifetime. In addition to chromosomal and genetic alterations, nutrition is considered a risk determinant for breast cancer. Prevailing evidence suggests a negative correlation between intake of soy-rich foods and breast cancer incidence. Epidemiological and case control studies have shown a two-to eight fold lower occurrence of the disease in Asian women whose early intake of soy products is 20-25 times higher than their American counterparts. Previous work from our group has shown diet-induced protection against DMBA- and NMU-induced mammary tumors in vivo, with decreased tumor incidence and increased tumor latency in rats exposed to dietary soy protein isolate (SPI) or casein (CAS) supplemented with the major soy isoflavone genistein (GEN) when compared to those fed the control casein (CAS) diet. We also have shown that GEN enhanced apoptosis in mammary epithelial cells in vivo and in vitro (MCF-7 cells), attendant with increased expression of the tumor suppressor PTEN and several pro-apoptotic genes Bax, Bok, and p21. Here, we test the hypothesis that GEN induction of PTEN expression and nuclear localization in mammary epithelial cells result in decreased cellular proliferation and enhanced cellular differentiation, to confer protection from mammary tumorigenesis. Using the non-tumor human mammary epithelial cell line MCF-10A, we show that GEN at physiologically relevant concentrations (40 nM, 2 µM) increased PTEN mRNA and protein levels and enhanced nuclear accumulation of active (non-phosphorylated) PTEN. GEN induction of PTEN transcript levels was lost with the inclusion of the transcription inhibitor actinomycin D, suggesting PTEN is a direct GEN target. Further, cells treated with GEN for 6 days had lower cell viability when compared to control cells, concomitant with decreased expression of cyclin D1 and of the PTEN regulated gene pleiotrophin. Interestingly, GEN up-regulation of PTEN expression was accompanied by similar effects on another tumor suppressor p53, which co-localized with PTEN in the nuclei of GEN-treated cells. PTEN siRNA targeting, which decreased basal PTEN expression by 60%, significantly increased cyclin D1 but not p53 expression. Our findings provide support to the hypothesis that the protective effects of GEN in the mammary epithelium may be mediated by PTEN to enhance differentiation and inhibit cellular proliferation, and suggest the participation of another key tumor suppressor p53. Funding by USDA-CRIS 6251-5100002-06S. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4069.

Diet is highly linked to breast cancer risk, yet little is known about its influence on mammary epithelial populations with distinct regenerative and hence, tumorigenic potential. To investigate this, we evaluated the relative frequency of lineage-negative CD29(hi)CD24(+), CD29(lo)CD24(+) and CD29(hi)Thy1(+)CD24(+) epithelial subpopulations in pre-neoplastic mammary tissue of adult virgin MMTV-Wnt1-transgenic mice fed either control (Casein) or soy-based diets. We found that mammary epithelial cells exposed to soy diet exhibited a lower percentage of CD29(hi)CD24(+)Lin(-) population, decreased ability to form mammospheres in culture, lower mammary outgrowth potential when transplanted into cleared fat pads, and reduced appearance of tumor-initiating CD29(hi)Thy1(+)CD24(+) cells, than in those of control diet-fed mice. Diet had no comparable influence on the percentage of the CD29(lo)CD24(+)Lin(-) population. Global gene expression profiling of the CD29(hi)CD24(+)subpopulation revealed markedly altered expression of genes important to inflammation, cytokine and chemokine signaling, and proliferation. Soy-fed relative to casein-fed mice showed lower mammary tumor incidence, shorter tumor latency, and reduced systemic levels of estradiol 17-β, progesterone and interleukin-6. Our results provide evidence for the functional impact of diet on specific epithelial subpopulations that may relate to breast cancer risk and suggest that diet-regulated cues can be further explored for breast cancer risk assessment and prevention.