Postpartum breast involution reveals regression of secretory lobules mediated by tissue-remodeling.

INTRODUCTION: A postpartum diagnosis of breast cancer is an independent predictor of metastases, however the reason is unknown. In rodents, the window of postpartum mammary gland involution promotes tumor progression, suggesting a role for breast involution in the poor prognosis of human postpartum breast cancers. Rodent mammary gland involution is characterized by the programmed elimination of the secretory lobules laid down in preparation for lactation. This tissue involution process involves massive epithelial cell death, stromal remodeling, and immune cell infiltration with similarities to microenvironments present during wound healing and tumor progression. Here, we characterize breast tissue from premenopausal women with known reproductive histories to determine the extent, duration and cellular mechanisms of postpartum lobular involution in women. METHODS: Adjacent normal breast tissues from premenopausal women (n = 183) aged 20 to 45 years, grouped by reproductive categories of nulliparous, pregnant and lactating, and by time since last delivery were evaluated histologically and by special stain for lobular area, lobular type composition, apoptosis and immune cell infiltration using computer assisted quantitative methods. RESULTS: Human nulliparous glands were composed dominantly of small (approximately 10 acini per lobule) and medium (approximately 35 acini per lobule) sized lobules. With pregnancy and lactation, a >10 fold increase in breast epithelial area was observed compared to nulliparous cases, and lactating glands were dominated by mature lobules (>100 acini per lobule) with secretory morphology. Significant losses in mammary epithelial area and mature lobule phenotypes were observed within 12 months postpartum. By 18 months postpartum, lobular area content and lobule composition were indistinguishable from nulliparous cases, data consistent with postpartum involution facilitating regression of the secretory lobules developed in preparation for lactation. Analyses of apoptosis and immune cell infiltrate confirmed that human postpartum breast involution is characterized by wound healing-like tissue remodeling programs that occur within a narrowed time frame. CONCLUSIONS: Human postpartum breast involution is a dominant tissue-remodeling process that returns the total lobular area of the gland to a level essentially indistinguishable from the nulliparous gland. Further research is warranted to determine whether the normal physiologic process of postpartum involution contributes to the poor prognosis of postpartum breast cancer.

Role of the androgen receptor in breast cancer and preclinical analysis of enzalutamide.

INTRODUCTION: The androgen receptor (AR) is widely expressed in breast cancers and has been proposed as a therapeutic target in estrogen receptor alpha (ER) negative breast cancers that retain AR. However, controversy exists regarding the role of AR, particularly in ER + tumors. Enzalutamide, an AR inhibitor that impairs nuclear localization of AR, was used to elucidate the role of AR in preclinical models of ER positive and negative breast cancer. METHODS: We examined nuclear AR to ER protein ratios in primary breast cancers in relation to response to endocrine therapy. The effects of AR inhibition with enzalutamide were examined in vitro and in preclinical models of ER positive and negative breast cancer that express AR. RESULTS: In a cohort of 192 women with ER + breast cancers, a high ratio of AR:ER (≥2.0) indicated an over four fold increased risk for failure while on tamoxifen (HR = 4.43). The AR:ER ratio had an independent effect on risk for failure above ER % staining alone. AR:ER ratio is also an independent predictor of disease-free survival (HR = 4.04, 95% CI: 1.68, 9.69; p = 0.002) and disease specific survival (HR = 2.75, 95% CI: 1.11, 6.86; p = 0.03). Both enzalutamide and bicalutamide inhibited 5-alpha-dihydrotestosterone (DHT)-mediated proliferation of breast cancer lines in vitro; however, enzalutamide uniquely inhibited estradiol (E2)-mediated proliferation of ER+/AR + breast cancer cells. In MCF7 xenografts (ER+/AR+) enzalutamide inhibited E2-driven tumor growth as effectively as tamoxifen by decreasing proliferation. Enzalutamide also inhibited DHT- driven tumor growth in both ER positive (MCF7) and negative (MDA-MB-453) xenografts, but did so by increasing apoptosis. CONCLUSIONS: AR to ER ratio may influence breast cancer response to traditional endocrine therapy. Enzalutamide elicits different effects on E2-mediated breast cancer cell proliferation than bicalutamide. This preclinical study supports the initiation of clinical studies evaluating enzalutamide for treatment of AR+ tumors regardless of ER status, since it blocks both androgen- and estrogen- mediated tumor growth.

Therapeutic targeting of erbB3 with MM-121/SAR256212 enhances antitumor activity of paclitaxel against erbB2-overexpressing breast cancer.

INTRODUCTION: Elevated expression of erbB3 rendered erbB2-overexpressing breast cancer cells resistant to paclitaxel via PI-3 K/Akt-dependent upregulation of Survivin. It is unclear whether an erbB3-targeted therapy may abrogate erbB2-mediated paclitaxel resistance in breast cancer. Here, we study the antitumor activity of an anti-erbB3 antibody MM-121/SAR256212 in combination with paclitaxel against erbB2-overexpressing breast cancer. METHODS: Cell growth assays were used to determine cell viability. Cells undergoing apoptosis were quantified by a specific apoptotic ELISA. Western blot analyses were performed to assess the protein expression and activation. Lentiviral vector containing shRNA was used to specifically knockdown Survivin. Tumor xenografts were established by inoculation of BT474-HR20 cells into nude mice. The tumor-bearing mice were treated with paclitaxel and/or MM-121/SAR256212 to determine whether the antibody (Ab) enhances paclitaxel's antitumor activity. Immunohistochemistry was carried out to study the combinatorial effects on tumor cell proliferation and induction of apoptosis in vivo. RESULTS: MM-121 significantly facilitated paclitaxel-mediated anti-proliferative/anti-survival effects on SKBR3 cells transfected with a control vector or erbB3 cDNA. It specifically downregulated Survivin associated with inactivation of erbB2, erbB3, and Akt. MM-121 enhances paclitaxel-induced poly(ADP-ribose) polymerase (PARP) cleavage, activation of caspase-8 and −3, and apoptosis in both paclitaxel-sensitive and -resistant cells. Specific knockdown of Survivin in the trastuzumab-resistant BT474-HR20 cells dramatically enhanced paclitaxel-induced apoptosis, suggesting that increased Survivin caused a cross-resistance to paclitaxel. Furthermore, the studies using a tumor xenograft model-established from BT474-HR20 cells revealed that either MM-121 (10 mg/kg) or low-dose (7.5 mg/kg) paclitaxel had no effect on tumor growth, their combinations significantly inhibited tumor growth in vivo. Immunohistochemical analysis showed that the combinations of MM-121 and paclitaxel significantly reduced the cells with positive staining for Ki-67 and Survivin, and increased the cells with cleaved caspase-3. CONCLUSIONS: The combinations of MM-121 and paclitaxel not only inhibit tumor cell proliferation, but also promote erbB2-overexpressing breast cancer cells to undergo apoptosis via downregulation of Survivin in vitro and in vivo, suggesting that inactivation of erbB3 with MM-121 enhances paclitaxel-mediated antitumor activity against erbB2-overexpressing breast cancers. Our data supports further exploration of the combinatorial regimens consisting of MM-121 and paclitaxel in breast cancer patients with erbB2-overexpressing tumors, particularly those resistant to paclitaxel.

Thyroid hormone enhances estrogen-mediated proliferation and cell cycle regulatory pathways in steroid receptor-positive breast Cancer.

Estrogen receptor (ER) α expression and associated signaling is a major driver of over two-thirds of all breast cancers (BC). ER targeting strategies are typically used as a first-line therapy in patients with steroid receptor positive (SR+) disease. Secondary resistance to anti-estrogenic agents may occur with clonal expansion and disease progression. Mechanisms underlying hormone resistance are an expanding field of significant translational importance. Cross-talk with other nuclear hormones, receptors, and signaling pathways, including thyroid hormones (TH) and their receptors (THRs), have been shown to promote endocrine therapy resistance in some studies. We have shown that TH replacement therapy (THRT) was independently and significantly associated with higher rates of relapse and mortality in SR positive (+), node-negative (LN-) BC patients, whereas it showed no association with outcomes in SR negative (-) patients. LN-, SR+ patients receiving THRT and tamoxifen had the worst outcomes, suggesting a pro-carcinogenic interaction that significantly and independently shortened survival and increased mortality. Using in vivo and in vitro models, we previously showed hormonal cross-talk, altered gene signaling, target gene activation, and resistance to tamoxifen in the presence of TH. In this report, we show TH ± E2 ± tamoxifen inhibits cell cycle control signaling, reduces apoptosis, and enhances cell proliferation, tumor growth, tamoxifen resistance, and clonal expansion. Mechanistically these changes involve numerous genes and pathways, including critical cell cycle regulatory proteins and genes identified using various molecular methods. These studies facilitate a greater mechanistic understanding of the biological and molecular impact of TH on SR+ BC.

Patient-derived luminal breast cancer xenografts retain hormone receptor heterogeneity and help define unique estrogen-dependent gene signatures.

Bypassing estrogen receptor (ER) signaling during development of endocrine resistance remains the most common cause of disease progression and mortality in breast cancer patients. To date, the majority of molecular research on ER action in breast cancer has occurred in cell line models derived from late stage disease. Here we describe patient-derived ER+ luminal breast tumor models for the study of intratumoral hormone and receptor action. Human breast tumor samples obtained from patients post surgery were immediately transplanted into NOD/SCID or NOD/SCID/ILIIrg(-/-) mice under estrogen supplementation. Five transplantable patient-derived ER+ breast cancer xenografts were established, derived from both primary and metastatic cases. These were assessed for estrogen dependency, steroid receptor expression, cancer stem cell content, and endocrine therapy response. Gene expression patterns were determined in select tumors ±estrogen and ±endocrine therapy. Xenografts morphologically resembled the patient tumors of origin, and expressed similar levels of ER (5-99 %), and progesterone and androgen receptors, over multiple passages. Four of the tumor xenografts were estrogen dependent, and tamoxifen or estrogen withdrawal (EWD) treatment abrogated estrogen-dependent growth and/or tumor morphology. Analysis of the ER transcriptome in select tumors revealed notable differences in ER mechanism of action, and downstream activated signaling networks, in addition to identifying a small set of common estrogen-regulated genes. Treatment of a naïve tumor with tamoxifen or EWD showed similar phenotypic responses, but relatively few similarities in estrogen-dependent transcription, and affected signaling pathways. Several core estrogen centric genes were shared with traditional cell line models. However, novel tumor-specific estrogen-regulated potential target genes, such as cancer/testis antigen 45, were uncovered. These results evoke the importance of mapping both conserved and tumor-unique ER programs in breast cancers. Furthermore, they underscore the importance of primary xenografts for improved understanding of ER+ breast cancer heterogeneity and development of personalized therapies.

Exogenous Thyroid Hormone Is Associated with Shortened Survival and Upregulation of High-Risk Gene Expression Profiles in Steroid Receptor-Positive Breast Cancers.

PURPOSE: Thyroid disease is a frequent comorbidity in women with breast cancer, and many require thyroid hormone replacement therapy (THRT). We postulated that THRT has a deleterious clinical effect mechanistically through hormonal interactions, nuclear receptor cross-talk, and upregulation of high-risk breast cancer genes. EXPERIMENTAL DESIGN: Observational studies of patients with lymph node-negative (LN-) breast cancer (n = 820 and n = 160) were performed to test interactions between THRT and clinical, histologic, outcome, and treatment variables. Differences between the two cohorts include but are not limited to patient numbers, decades of treatment, duration of follow-up/treatment, tumor sizes, incidence, and type and dose/regimen of antihormonal and/or chemotherapeutic agents. In vivo and vitro models, in silico databases, and molecular methods were used to study interactions and define mechanisms underlying THRT effects. RESULTS: THRT significantly and independently reduced disease-free and breast cancer-specific overall survival of only the steroid receptor (SR)-positive (as compared with SR-negative) node-negative patients in both long-term observational studies. Patients with SR+ LN- breast cancer who received THRT and tamoxifen experienced the shortest survival of all treatment groups. A less potent interaction between THRT and aromatase inhibitors was noted in the second patient cohort. Using in vivo and in vitro models, TH administration enhanced estrogen and TH-associated gene expression and proliferation, nuclear colocalization of estrogen receptor and thyroid hormone receptor, and activation of genes used clinically to predict tumor aggression in SR+ breast cancer, including the IGF-IR, WNT, and TGFß pathways. CONCLUSIONS: We show clinically significant adverse interactions between THRT, estrogenic, and oncogenic signaling in patients with SR+ LN- breast cancer.

Genistein induces enhanced growth promotion in ER-positive/erbB-2-overexpressing breast cancers by ER-erbB-2 cross talk and p27/kip1 downregulation.

Genistein is a major isoflavone with known hormonal and tyrosine kinase-modulating activities. Genistein has been shown to promote the growth of estrogen receptor positive (ER+) MCF-7 cells. In ER-negative (ER-)/erbB-2-overexpressing (erbB-2+) cells, genistein has been shown to inhibit cell growth through its tyrosine kinase inhibitor activity. The effects of genistein on cell growth and tamoxifen response in ER+/erbB-2-altered breast cancers (known as luminal type B and noted in approximately 10 to 20% of breast cancers) have not been well explored. Using erbB-2-transfected ER+ MCF-7 cells, we found that genistein induced enhanced cellular proliferation and tamoxifen resistance when compared with control MCF-7 cells. These responses were accompanied by increased phosphorylation of ERalpha and ER signaling, without increase in ER protein levels. Genistein-treated MCF-7/erbB-2 cells also showed enhanced activation/phosphorylation of erbB-2, Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase. Blockade of the phosphatidylinositol 3-kinase and/or MAPK pathways abrogated genistein-induced growth promotion, suggesting that genistein effects involve both critical signaling pathways. We also found that p27/kip1 was markedly downregulated in genistein-treated MCF-7/erbB-2 cells. Overexpression of p27/kip1 attenuated genistein-mediated growth promotion. In aggregate, our data suggest that the concomitant coexpression of ER and erbB-2 makes breast cancers particularly susceptible to the growth-promoting effects of genistein across a wide range of doses. The underlying mechanisms involve enhanced ER-erbB-2 cross talk and p27/kip1 downregulation.

Downregulation of miR-342 is associated with tamoxifen resistant breast tumors.

BACKGROUND: Tumor resistance to the selective estrogen receptor modulator tamoxifen remains a serious clinical problem especially in patients with tumors that also overexpress HER2. We have recently demonstrated that the clinically important isoform of HER2, HERΔ16, promotes therapeutically refractory breast cancer including resistance to endocrine therapy. Likewise additional breast tumor cell models of tamoxifen resistance have been developed that do not involve HER2 overexpression. However, a unifying molecular mechanism of tamoxifen resistance has remained elusive. RESULTS: Here we analyzed multiple cell models of tamoxifen resistance derived from MCF-7 cells to examine the influence of microRNAs (miRNAs) on tamoxifen resistance. We compared miRNA expression profiles of tamoxifen sensitive MCF-7 cells and tamoxifen resistant MCF-7/HER2Δ16 cells. We observed significant and dramatic downregulation of miR-342 in the MCF-7/HER2Δ16 cell line as well as the HER2 negative but tamoxifen resistant MCF-7 variants TAMR1 and LCC2. Restoring miR-342 expression in the MCF-7/HER2Δ16 and TAMR1 cell lines sensitized these cells to tamoxifen-induced apoptosis with a dramatic reduction in cell growth. Expression of miR-342 was also reduced in a panel of tamoxifen refractory human breast tumors, underscoring the potential clinical importance of miR-342 downregulation. Towards the goal of identifying direct and indirect targets of miR-342 we restored miR-342 expression in MCF-7/HER2Δ16 cells and analyzed changes in global gene expression by microarray. The impact of miR-342 on gene expression in MCF-7/HER2Δ16 cells was not limited to miR-342 in silica predicted targets. Ingenuity Pathways Analysis of the dataset revealed a significant influence of miR-342 on multiple tumor cell cycle regulators. CONCLUSIONS: Our findings suggest that miR-342 regulates tamoxifen response in breast tumor cell lines and our clinical data indicates a trend towards reduced miR-342 expression and tamoxifen resistance. In addition, our results suggest that miR-342 regulates expression of genes involved in tamoxifen mediated tumor cell apoptosis and cell cycle progression. Restoring miR-342 expression may represent a novel therapeutic approach to sensitizing and suppressing the growth of tamoxifen refractory breast tumors.

Subcellular localization of the HER4 intracellular domain, 4ICD, identifies distinct prognostic outcomes for breast cancer patients.

Conflicting reports of the prognostic value of HER4 in breast cancer may be explained by distinct activities of the HER4 intracellular domain, 4ICD. Here, immunohistochemical 4ICD staining of archival invasive breast cancers (n = 923) was scored separately for nuclear and cytosolic expression, and these data were tested for associations with clinicopathological markers, disease-free survival, and disease-specific survival. By univariate analysis, cytosolic 4ICD expression was independently associated with estrogen receptor and progesterone receptor expression and tumor cell apoptosis. Nuclear 4ICD inversely correlated with tumor grade and tumor mitosis. In multivariate analyses cytosolic, but not nuclear 4ICD, significantly correlated with disease-free survival (P = 0.035) and disease-specific survival (P < 0.004) in lymph node-negative patients. Our results demonstrate for the first time that cytosolic 4ICD has significant positive prognostic value in node-negative breast cancer patients. At present, tumor grade and size are the primary clinicopathological parameters commonly used to guide decision making in these patients. Our results suggest that cytosolic 4ICD has important pathological functions and may be used to identify node-negative breast cancer patients at low risk of relapse and an improved survival, thereby avoiding systemic overtreatment of these patients. Our results also suggest that pan-receptor tyrosine kinase inhibitors, currently in clinical trials, or HER4 antagonists, which disengage 4ICD signaling, may have untoward activity in patients whose tumors express cytosolic 4ICD.

Distinct tumor microenvironments of lytic and blastic bone metastases in prostate cancer patients.

The most common metastatic lesions of prostate cancer are in bone and can be classified into three distinct pathology subtypes: lytic, blastic, and an indeterminate mixture of both. We investigated a cohort of decalcified formalin-fixed and paraffin-embedded (FFPE) patient specimens from the bone that contained metastatic prostate cancer with lytic or blastic features. These tissue sections were utilized for immunohistochemistry (IHC) staining, isolation of RNA for gene expression, and Digital Spatial Profiling (DSP) of changes in both the tumor and microenvironment. A diverse set of unique immune cell populations and signaling pathways to both lytic and blastic types of prostate cancer metastases were present. In blastic lesions immune cells were enriched for pSTAT3 and components of the JAK-STAT pathway. In lytic-type lesions, immune cells were enriched for pAKT activity and components of the PI3K-AKT pathway. Enrichment for immune checkpoints including PD-L1, B7-H4, OX40L, and IDO-1 were identified in blastic prostate cancer, providing new therapeutic targets for patients with bone metastases. Biopsies could guide selection of patients into appropriate therapeutic interventions based on protein levels and RNA expression of desired targets in metastatic disease. Molecular pathology has been an excellent complement to the diagnosis, treatment, and management of primary tumors and could be successfully extended to patients with metastatic lesions.

Ganetespib targets multiple levels of the receptor tyrosine kinase signaling cascade and preferentially inhibits ErbB2-overexpressing breast cancer cells.

Although ErbB2-targeted therapeutics have significantly improved ErbB2+ breast cancer patient outcomes, therapeutic resistance remains a significant challenge. Therefore, the development of novel ErbB2-targeting strategies is necessary. Importantly, ErbB2 is a sensitive client protein of heat shock protein 90 (HSP90), which regulates client protein folding, maturation, and stabilization. HSP90 inhibition provides an alternative therapeutic strategy for ErbB2-targeted degradation. In particular, ganetespib, a novel HSP90 inhibitor, is a promising agent for ErbB2+ cancers. Nevertheless, the anti-cancer efficacy and clinical application of ganetespib for ErbB2+ breast cancer is largely unknown. In our study, we examined the anti-cancer effects of ganetespib on ErbB2+ BT474 and SKBR3 breast cancer cells, and isogenic paired cancer cell lines with lentivirus-mediated ErbB2 overexpression. Ganetespib potently inhibited cell proliferation, cell cycle progression, survival, and activation/phosphorylation of ErbB2 and key downstream effectors in ErbB2+ breast cancer cells. Moreover, ganetespib decreased the total protein levels of HSP90 client proteins and reduced ErbB2 protein half-life. ErbB2-overexpressing cancer cells were also more sensitive to ganetespib-mediated growth inhibition than parental cells. Ganetespib also strikingly potentiated the inhibitory effects of lapatinib in BT474 and SKBR3 cells. Ultimately, our results support the application of ganetespib-mediated HSP90 inhibition as a promising therapeutic strategy for ErbB2+ breast cancer.

FGFR1 underlies obesity-associated progression of estrogen receptor-positive breast cancer after estrogen deprivation.

Obesity increases breast cancer mortality by promoting resistance to therapy. Here, we identified regulatory pathways in estrogen receptor-positive (ER-positive) tumors that were shared between patients with obesity and those with resistance to neoadjuvant aromatase inhibition. Among these was fibroblast growth factor receptor 1 (FGFR1), a known mediator of endocrine therapy resistance. In a preclinical model with patient-derived ER-positive tumors, diet-induced obesity promoted a similar gene expression signature and sustained the growth of FGFR1-overexpressing tumors after estrogen deprivation. Tumor FGFR1 phosphorylation was elevated with obesity and predicted a shorter disease-free and disease-specific survival for patients treated with tamoxifen. In both human and mouse mammary adipose tissue, FGF1 ligand expression was associated with metabolic dysfunction, weight gain, and adipocyte hypertrophy, implicating the impaired response to a positive energy balance in growth factor production within the tumor niche. In conjunction with these studies, we describe a potentially novel graft-competent model that can be used with patient-derived tissue to elucidate factors specific to extrinsic (host) and intrinsic (tumor) tissue that are critical for obesity-associated tumor promotion. Taken together, we demonstrate that obesity and excess energy establish a tumor environment with features of endocrine therapy resistance and identify a role for ligand-dependent FGFR1 signaling in obesity-associated breast cancer progression.

Perinucleolar compartment prevalence has an independent prognostic value for breast cancer.

The perinucleolar compartment (PNC) is a multicomponent nuclear structure enriched with RNAs transcribed by RNA pol III and RNA binding proteins. Studies in cultured cells showed an association between PNC and transformed phenotype. To evaluate the relationship between structure and malignancy in vivo, we examined PNC prevalence (the percentage of cells containing at least one PNC) in normal and cancerous paraffin-embedded breast tissues using immunohistochemistry against a PNC-associated protein. Five hundred nuclei in the most active area of each sample were scored for PNC prevalence. The results show that PNC prevalence significantly correlates with the progression of breast cancer (by the criteria of staging). PNC prevalence in primary tumors, lymph nodes, and distant metastases shows a stepwise increase from a median of 23% in primary tumors to approximately 100% in distant metastases. In addition, univariate and multivariate (controlling for tumor size and grade) analyses show that early-stage patients with invasive ductal carcinomas containing a higher PNC prevalence have a significantly poorer prognosis. These findings link PNC prevalence with the progression of breast cancer in vivo and suggest that PNC-containing cells have metastatic advantages. These findings also show the potential of PNC prevalence as a prognostic marker for breast cancer.

Activation of cancerous inhibitor of PP2A (CIP2A) contributes to lapatinib resistance through induction of CIP2A-Akt feedback loop in ErbB2-positive breast cancer cells.

Lapatinib, a small molecule ErbB2/EGFR inhibitor, is FDA-approved for the treatment of metastatic ErbB2-overexpressing breast cancer; however, lapatinib resistance is an emerging clinical challenge. Understanding the molecular mechanisms of lapatinib-mediated anti-cancer activities and identifying relevant resistance factors are of pivotal significance. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is a recently identified oncoprotein that is overexpressed in breast cancer. Our study investigated the role of CIP2A in the anti-cancer efficacy of lapatinib in ErbB2-overexpressing breast cancer cells. We found that lapatinib concurrently downregulated CIP2A and receptor tyrosine kinase signaling in ErbB2-overexpressing SKBR3 and 78617 cells; however, these effects were attenuated in lapatinib-resistant (LR) cells. CIP2A overexpression rendered SKBR3 and 78617 cells resistant to lapatinib-induced apoptosis and growth inhibition. Conversely, CIP2A knockdown via lentiviral shRNA enhanced cell sensitivity to lapatinib-induced growth inhibition and apoptosis. Results also suggested that lapatinib downregulated CIP2A through regulation of protein stability. We further demonstrated that lapatinib-induced CIP2A downregulation can be recapitulated by LY294002, suggesting that Akt mediates CIP2A upregulation. Importantly, lapatinib induced differential CIP2A downregulation between parental BT474 and BT474/LR cell lines. Moreover, CIP2A shRNA knockdown significantly sensitized the BT474/LR cells to lapatinib. Collectively, our results demonstrate that CIP2A is a molecular target and resistance factor of lapatinib with a critical role in lapatinib-induced cellular responses, including the inhibition of the CIP2A-Akt feedback loop. Further investigation of lapatinib-mediated CIP2A regulation will advance our understanding of lapatinib-associated anti-tumor activities and drug resistance.

Metformin Accumulation Correlates with Organic Cation Transporter 2 Protein Expression and Predicts Mammary Tumor Regression In Vivo.

Several epidemiologic studies have associated metformin treatment with a reduction in breast cancer incidence in prediabetic and type II diabetic populations. Uncertainty exists regarding which patient populations and/or tumor subtypes will benefit from metformin treatment, and most preclinical in vivo studies have given little attention to the cellular pharmacology of intratumoral metformin uptake. Epidemiologic reports consistently link western-style high fat diets (HFD), which drive overweight and obesity, with increased risk of breast cancer. We used a rat model of HFD-induced overweight and mammary carcinogenesis to define intratumoral factors that confer metformin sensitivity. Mammary tumors were initiated with 1-methyl-1-nitrosourea, and rats were randomized into metformin-treated (2 mg/mL drinking water) or control groups (water only) for 8 weeks. Two-thirds of existing mammary tumors responded to metformin treatment with decreased tumor volumes (P < 0.05), reduced proliferative index (P < 0.01), and activated AMPK (P < 0.05). Highly responsive tumors accumulated 3-fold greater metformin amounts (P < 0.05) that were positively correlated with organic cation transporter-2 (OCT2) protein expression (r = 0.57; P = 0.038). Importantly, intratumoral metformin concentration negatively associated with tumor volume (P = 0.03), and each 10 pmol increase in intratumoral metformin predicted >0.11 cm3 reduction in tumor volume. Metformin treatment also decreased proinflammatory arachidonic acid >1.5-fold in responsive tumors (P = 0.023). Collectively, these preclinical data provide evidence for a direct effect of metformin in vivo and suggest that OCT2 expression may predict metformin uptake and tumor response. Cancer Prev Res; 10(3); 198-207. ©2017 AACR.

The Androgen Receptor Supports Tumor Progression After the Loss of Ovarian Function in a Preclinical Model of Obesity and Breast Cancer.

The androgen receptor (AR) has context-dependent roles in breast cancer growth and progression. Overall, high tumor AR levels predict a favorable patient outcome, but several studies have established a tumor promotional role for AR, particularly in supporting the growth of estrogen receptor positive (ER-positive) breast cancers after endocrine therapy. Our previous studies have demonstrated that obesity promotes mammary tumor progression after ovariectomy (OVX) in a rat model of postmenopausal breast cancer. Here, we investigated a potential role for AR in obesity-associated post-OVX mammary tumor progression following ovarian estrogen loss. In this model, we found that obese but not lean rats had nuclear localized AR in tumors that progressed 3 weeks after OVX, compared to those that regressed. AR nuclear localization is consistent with activation of AR-dependent transcription. Longer-term studies (8 weeks post-OVX) showed that AR nuclear localization and expression were maintained in tumors that had progressed, but AR expression was nearly lost in tumors that were regressing. The anti-androgen enzalutamide effectively blocked tumor progression in obese rats by promoting tumor necrosis and also prevented the formation of new tumors after OVX. Neither circulating nor mammary adipose tissue levels of the AR ligand testosterone were elevated in obese compared to lean rats; however, IL-6, which we previously reported to be higher in plasma from obese versus lean rats, sensitized breast cancer cells to low levels of testosterone. Our study demonstrates that, in the context of obesity, AR plays a role in driving ER-positive mammary tumor progression in an environment of low estrogen availability, and that circulating factors unique to the obese host, including IL-6, may influence how cancer cells respond to steroid hormones.

Development of Novel Patient-Derived Xenografts from Breast Cancer Brain Metastases.

Brain metastases are an increasing burden among breast cancer patients, particularly for those with HER2+ and triple negative (TN) subtypes. Mechanistic insight into the pathophysiology of brain metastases and preclinical validation of therapies has relied almost exclusively on intracardiac injection of brain-homing cells derived from highly aggressive TN MDA-MB-231 and HER2+ BT474 breast cancer cell lines. Yet, these well characterized models are far from representing the tumor heterogeneity observed clinically and, due to their fast progression in vivo, their suitability to validate therapies for established brain metastasis remains limited. The goal of this study was to develop and characterize novel human brain metastasis breast cancer patient-derived xenografts (BM-PDXs) to study the biology of brain metastasis and to serve as tools for testing novel therapeutic approaches. We obtained freshly resected brain metastases from consenting donors with breast cancer. Tissue was immediately implanted in the mammary fat pad of female immunocompromised mice and expanded as BM-PDXs. Brain metastases from 3/4 (75%) TN, 1/1 (100%) estrogen receptor positive (ER+), and 5/9 (55.5%) HER2+ clinical subtypes were established as transplantable BM-PDXs. To facilitate tracking of metastatic dissemination using BM-PDXs, we labeled PDX-dissociated cells with EGFP-luciferase followed by reimplantation in mice, and generated a BM-derived cell line (F2-7). Immunohistologic analyses demonstrated that parental and labeled BM-PDXs retained expression of critical clinical markers such as ER, progesterone receptor, epidermal growth factor receptor, HER2, and the basal cell marker cytokeratin 5. Similarly, RNA sequencing analysis showed clustering of parental, labeled BM-PDXs and their corresponding cell line derivative. Intracardiac injection of dissociated cells from BM-E22-1, resulted in magnetic resonance imaging-detectable macrometastases in 4/8 (50%) and micrometastases (8/8) (100%) mice, suggesting that BM-PDXs remain capable of colonizing the brain at high frequencies. Brain metastases developed 8-12 weeks after ic injection, located to the brain parenchyma, grew around blood vessels, and elicited astroglia activation characteristic of breast cancer brain metastasis. These novel BM-PDXs represent heterogeneous and clinically relevant models to study mechanisms of brain metastatic colonization, with the added benefit of a slower progression rate that makes them suitable for preclinical testing of drugs in therapeutic settings.

Relationship of epidermal growth factor receptor expression to ErbB-2 signaling activity and prognosis in breast cancer patients.

PURPOSE: To examine the relationship of epidermal growth factor receptor (EGFR) expression to ErbB-2 signaling activity in breast cancer and the impact that this interaction has on the prognosis of patients with early-stage breast cancer. PATIENTS AND METHODS: Paraffin tumor sections were collected retrospectively from 807 breast cancer patients diagnosed between 1976 and 1983. Immunohistochemical assays for ErbB-2, phosphorylated (activated) ErbB-2, and EGFR were performed, and the results were correlated with clinicopathologic variables and outcome. RESULTS: EGFR expression was detectable in 15% of 807 invasive breast cancers, including 35% of the 306 ErbB-2-positive patients. Conversely, the majority (87%) of EGFR-positive tumors co-overexpressed ErbB-2. Ninety-seven percent of tumors with phosphorylated ErbB-2 co-overexpressed EGFR. Patients whose cancers demonstrated ErbB-2 phosphorylation or co-overexpression of ErbB-2 and EGFR had the shortest survival. In contrast, patients whose tumors were negative for all three markers and those tumors that expressed only EGFR or only nonphosphorylated ErbB-2 had a relatively favorable outcome. CONCLUSION: These data provide the first clinical evidence that EGFR expression is linked to activation of ErbB-2 in human breast cancers. We have further shown that the adverse prognostic value of ErbB-2 overexpression is observed only when ErbB-2 is in the phosphorylated (activated) state or coexpressed with EGFR. These data suggest that ligand-dependent mechanisms of ErbB-2 activation are important in human breast cancer. These results also suggest that agents targeting EGFR may be useful in the treatment of tumors with activated ErbB-2.

Hormonal and dietary modulation of mammary carcinogenesis in mouse mammary tumor virus-c-erbB-2 transgenic mice.

Exogenous and dietary estrogens have been associated with modification of breast cancer risk. Mammary cancer model systems can be used to explore interactions between specific transgenes, and hormonal and dietary factors. Transgenic mice bearing the rat wild-type erbB-2 gene were used to study the effects of short-term hormonal exposure [17beta-estradiol (E2) or tamoxifen] or a soy meal diet on mammary carcinogenesis. In mice fed a casein diet, mammary tumors developed at an earlier age after short-term E2 exposure during the early reproductive period. The median mammary tumor latency was shortest (29 weeks) for the high-dose estrogen as compared with the lowest dose of E2 treated or placebo control mice (33 and 37 weeks, respectively). The timing of short-term E2 exposure was also important, with the most significant changes observed in mice exposed to E2 between 8 and 18 weeks of age. E2 exposure was associated with the subsequent development of more aggressive tumors as determined by histologic grade, multifocal tumor development, stromal invasion, and pulmonary metastasis. In contrast, short-term tamoxifen-exposed mice generally failed to develop mammary tumors by 60 weeks of age. Mice fed a soy meal diet developed mammary tumors at a later age than casein-fed animals treated with E2 or placebo, whereas no differences were observed by diet for the tamoxifen-treated mice. Mammary tumor prevention was >80% in tamoxifen-treated mice on either diet. Novel histologic tumor types were identified, suggesting greater phenotypic diversity than described previously. Benign mammary gland morphogenesis was also significantly altered by short-term hormonal exposure or dietary factors, consistent with the modification of mammary tumor risk in specific treatment groups. Estrogenic modulation of the mammary tumor phenotype in wild-type erbB-2 transgenic mice was observed. Histologic tumor types and clinical aggressivity not reported previously in this transgenic model were noted, suggesting greater biologic heterogeneity than reported previously. In addition, dietary phytoestrogens modified mammary development and tumor latency, suggesting a need for greater stringency in dietary assignment for transgenic mouse models of mammary neoplasia.

The erbB3- and IGF-1 receptor-initiated signaling pathways exhibit distinct effects on lapatinib sensitivity against trastuzumab-resistant breast cancer cells.

Both erbB3 and IGF-1 receptor (IGF-1R) have been shown to play an important role in trastuzumab resistance. However, it remains unclear whether erbB3- and IGF-1R-initiated signaling pathways possess distinct effects on the sensitivity of lapatinib, a dual tyrosine kinase inhibitor against both EGFR and erbB2, in trastuzumab-resistant breast cancer. Here, we show that the trastuzumab-resistant SKBR3-pool2 and BT474-HR20 breast cancer sublines, as compared the parental SKBR3 and BT474 cells, respectively, exhibit refractoriness to lapatinib. Knockdown of erbB3 inhibited Akt in SKBR3-pool2 and BT474-HR20 cells, significantly increased lapatinib efficacy, and dramatically re-sensitized the cells to lapatinib-induced apoptosis. In contrast, specific knockdown of IGF-1R did not alter the cells' responsiveness to lapatinib. While the levels of phosphorylated Src (P-Src) were reduced upon IGF-1R downregulation, the P-Akt levels remained unchanged. Furthermore, a specific inhibitor of Akt, but not Src, significantly enhanced lapatinib-mediated anti-proliferative/anti-survival effects on SKBR3-pool2 and BT474-HR20 cells. These data indicate that erbB3 signaling is critical for both trastuzumab and lapatinib resistances mainly through the PI-3K/Akt pathway, whereas IGF-1R-initiated Src activation results in trastuzumab resistance without affecting lapatinib sensitivity. Our findings may facilitate the development of precision therapeutic regimens for erbB2-positive breast cancer patients who become resistant to erbB2-targeted therapy.