Neoadjuvant endocrine therapy expands stromal populations that predict poor prognosis in estrogen receptor-positive breast cancer.

The tumor microenvironment (TME) is an important modulator of response and resistance to endocrine therapy in estrogen receptor alpha (ER) positive breast cancer. Endocrine therapy is highly effective at reducing tumor burden and preventing recurrence in most estrogen receptor alpha (ER) positive breast cancers. Existing drugs work either directly by targeting tumor-cell ER or indirectly by inhibiting estrogen production in stromal cells with aromatase inhibitors (AI). However, many stromal cells also express ER and the direct impact of endocrine therapies on ER + stromal cells remain unclear. In this study, we investigated how neoadjuvant endocrine therapy (NET) directly effects stromal cells by measuring changes in stomal components of the TME that favor tumor progression. We previously defined two major subsets of tumor-associated stromal cells (TASCs): CD146 positive/CDCP1 negative (TASCCD146 ), CD146 negative/CDCP1 positive (TASCCDCP1 ), and generated a differentially expressed genes list associated with each type. Here, we applied the TASC gene list for classification and an algorithm that estimates immune cell abundance (TIMEx) to METABRIC transcriptomic data for ER + breast cancer patients coupled with multiplex imaging and analysis of paired tissue samples pre- and post- NET with the AI exemestane. TASCCDCP1 composition predicted for decreased patient survival in the METABRIC cohort. Exemestane treatment significantly increased expression of TASCCDCP1 and decreased expression of TASCCD146 . The posttreatment shift toward TASCCDCP1 composition correlated with increased macrophage infiltration and increased CD8+ T-cell, B cell, and general stromal components. The effectiveness of NET is currently based solely on the reduction of ER+ breast cancer cells. Here, we show NET displays clear TME effects that promote the expansion of the less favorable TASCCDCP1 population which are correlated with TME remodeling and reshaping immune infiltration supportive of tumor progression. Our findings highlight the need to further understand the role of endocrine therapy on TME remodeling, tumor progression, and patient outcomes.

ETS transcription factor ESE-1/Elf3 is an independent prognostic factor of survival in HR+HER2+ breast cancer patients.

PURPOSE: The ETS transcription factor ESE-1 has been shown to be important in HER2+ breast cancer and ESE-1 mRNA expression has been shown to associate with prognostic outcomes in the HER2+ subtype, as well as in ER+, HER2+ luminal B patients. However, the clinical significance of ESE-1 protein expression remains unknown. The purpose of the current exploratory study is to evaluate the prognostic value of ESE-1 protein expression in molecular breast cancer subtypes with special emphasis on hormone receptor positive HER2+(HR+ HER2+) and the HER2 positive (HER2+-only) breast cancer patients. METHODS: We developed a mouse monoclonal anti-ESE-1 antibody, verified its specificity, epitope, and used immunohistochemical staining to assess ESE-1 expression in an IBC approved archive of 957 breast tumor samples. Using Pearson product correlation, contingency analysis, and long rank P value testing, we analyzed the association of ESE-1 expression with clinicopathological features and survival outcomes in HR+HER2-; HR+HER2+; HR- HER2- (Triple negative) and HR-HER2+ (HER2 subtype) patients. RESULTS: ESE-1, nuclear or cytoplasmic, was not significantly associated with survival outcomes in HR+HER2-, triple-negative, or HER2+-only breast cancer patients. However, high nuclear ESE-1 was associated with poor survival outcomes in hormone receptor positive (ERα+, PR+) HER2+ patients and was an independent prognostic marker for that group. CONCLUSIONS: This study provides evidence for prognostic significance of nuclear ESE-1 in ERalpha positive breast cancers patients also positive for HER2 indicating that crosstalk between ERalpha and ESE-1 in HER2+ tumors could be important for prognostic outcomes. Further studies regarding the nature of interaction between ESE-1 and ERalpha in these tumors are warranted.

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.

Postpartum diagnosis demonstrates a high risk for metastasis and merits an expanded definition of pregnancy-associated breast cancer.

Previous studies report conflicting data on outcomes of pregnancy-associated breast cancer (PABC). Our aim was to examine the effect of a postpartum diagnosis on maternal prognosis in a young women's breast cancer cohort. We conducted a retrospective cohort study of women age ≤45 years, diagnosed with breast cancer (n = 619) during 1981-2011 at the University of Colorado Hospital and The Shaw Cancer Center in Edwards, CO. Breast cancer cases were grouped according to time between giving birth and diagnosis: nulliparous (n = 125), pregnant (n = 24), < 5 years postpartum (n = 136), >5-<10 postpartum (n = 130), and ≥10 years postpartum (n = 147), to examine the clinicopathologic features and the risk of distance recurrence and death. Cases diagnosed after pregnancy, but within five-years postpartum, had an approximate three fold increased risk of distant recurrence (HR 2.80, 95 % CI: 1.12-6.57) and death (HR 2.65, 95 % CI: 1.09-6.42) compared to nulliparous cases. Postpartum cases diagnosed within five years of last childbirth demonstrated a higher five-year distant recurrence probability (31.1 %) and a markedly lower five-year overall survival probability (65.8 %) compared to nulliparous cases (14.8 and 98.0 %, respectively). A diagnosis of breast cancer during the first five-years postpartum confers poorer maternal prognoses after adjustment for biologic subtype, stage, and year of diagnosis. We propose that the definition of PABC should include cases diagnosed up to at least five-years postpartum to better delineate the increased risk imparted by a postpartum diagnosis. Based on emerging preclinical and epidemiologic data, we propose that pregnant and postpartum cases be researched as distinct subsets of PABC to clarify the risk imparted by pregnancy and the events subsequent to pregnancy, such as breast involution, on breast cancer. Further, we highlight the importance of postpartum breast cancer as an area for further research to reduce the increased metastatic potential and mortality of PABC.

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.

HER2 and response to paclitaxel in node-positive breast cancer.

BACKGROUND: The status of human epidermal growth factor receptor type 2 (HER2) in breast-cancer cells predicts clinical outcomes in women who receive adjuvant anthracycline-based chemotherapy. We hypothesized that HER2 positivity predicts a benefit from adjuvant doxorubicin doses above standard levels, from the addition of paclitaxel after adjuvant chemotherapy with doxorubicin plus cyclophosphamide, or from both. METHODS: We randomly selected 1500 women from 3121 women with node-positive breast cancer who had been randomly assigned to receive doxorubicin (60, 75, or 90 mg per square meter of body-surface area) plus cyclophosphamide (600 mg per square meter) for four cycles, followed by four cycles of paclitaxel (175 mg per square meter) or observation. Tissue blocks from 1322 of these 1500 women were available. Immunohistochemical analyses of these tissue specimens for HER2 with the CB11 monoclonal antibody against HER2 or with a polyclonal-antibody assay kit and fluorescence in situ hybridization for HER2 amplification were performed. RESULTS: No interaction was observed between HER2 positivity and doxorubicin doses above 60 mg per square meter. HER2 positivity was, however, associated with a significant benefit from paclitaxel. The interaction between HER2 positivity and the addition of paclitaxel to the treatment was associated with a hazard ratio for recurrence of 0.59 (P=0.01). Patients with a HER2-positive breast cancer benefited from paclitaxel, regardless of estrogen-receptor status, but paclitaxel did not benefit patients with HER2-negative, estrogen-receptor-positive cancers. CONCLUSIONS: The expression or amplification, or both, of HER2 by a breast cancer is associated with a benefit from the addition of paclitaxel after adjuvant treatment with doxorubicin (<60 mg per square meter) plus cyclophosphamide in node-positive breast cancer, regardless of estrogen-receptor status. Patients with HER2-negative, estrogen-receptor-positive, node-positive breast cancer may gain little benefit from the administration of paclitaxel after adjuvant chemotherapy with doxorubicin plus cyclophosphamide.

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.

Low-dose dietary phytoestrogen abrogates tamoxifen-associated mammary tumor prevention.

Wild-type erbB-2/neu transgenic mice were used to study the interactions between tamoxifen and dietary phytoestrogens (or isoflavones) by dose and form in vivo. Mice were randomized to one of four dietary formulas and implanted with an 8-week continuous-release tamoxifen or placebo pellet at 8 weeks of age. In placebo-treated mice, soy meal diet (but not diets supplemented with low-dose or high-dose isoflavones or a casein diet) resulted in prolongation of tumor latency. In tamoxifen-treated mice fed the soy meal, casein, or high-dose isoflavone enriched diets, the majority (>80%) showed no tumor formation by 60 weeks of age. Of the mice that developed tumors, latency was significantly prolonged. In tamoxifen-treated mice fed the low-dose isoflavone enriched diet, a much higher rate of mammary tumor development (>50%; P < 0.002) and a shorter tumor latency were observed. In vitro studies of human and mouse mammary tumor cell lines confirm that low doses of genistein, co-administered with tamoxifen, promote cell proliferation. This is in contrast to tamoxifen alone or tamoxifen with higher doses of genistein that are growth inhibitory. In summary, low-dose dietary isoflavones abrogated tamoxifen-associated mammary tumor prevention in vivo. These interactions are supported by in vitro data from human and mouse mammary tumor cell lines. These dose-associated interactions likely have relevance to the human use of tamoxifen for prevention or treatment of breast cancer.

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.