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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 TGFβ pathways., Conclusions: We show clinically significant adverse interactions between THRT, estrogenic, and oncogenic signaling in patients with SR + LN - breast cancer., (©2020 American Association for Cancer Research.)

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 cm 3 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 ., (©2017 American Association for Cancer Research.)

Increasing evidence suggests molecular interactions between erbB2 and other receptor tyrosine kinases, and estrogenic compounds and their cognate receptors. We have recently reported that downregulation of erbB3 abrogates erbB2-mediated tamoxifen resistance in breast cancer cells. On the basis of these data, we hypothesized that erbB3 may play a major role connecting these two sentinel pathways. Interactions were studied using mammary/breast cancer cell lines from wild-type rat c-neu gene transgenic mice and humans. Estradiol promoted cell proliferation and activated erbB2/neu tyrosine kinase, Akt, and mitogen-activated protein kinase signaling exclusively in mammary and breast epithelial cell lines with coexpression of both erbB2 and erbB3. Estradiol action was independent of the transgene promoter (MMTV-LTR) activity, both in vitro and in vivo, as well as c-neu transgene or endogenous erbB2 gene expression. Estrogen induction of cell growth promotion, erbB2/neu activation, and downstream signaling was abrogated by blockade of estrogen receptor (ER) with the pure ER antagonist ICI 182,780 or knockdown of erbB3 expression via specific siRNA. These data suggest that activation of both ER and erbB2/erbB3 signaling is requisite for estrogen-induced mitogenesis and erbB2/neu tyrosine kinase activation.

Greater than 40% of breast cancer patients treated with tamoxifen exhibit de novo or acquired tumor resistance. Recent clinical evidence indicates that loss of expression of HER4 is an independent marker for tamoxifen resistance. In direct corroboration with clinical observations, suppression of HER4 expression in the tamoxifen-sensitive MCF-7 and T47D breast tumor cell lines resulted in resistance to tamoxifen-induced apoptosis. Furthermore, HER4 expression was lost in three independent MCF-7 models of acquired tamoxifen resistance. The HER4 intracellular domain (4ICD) is an independently signaling nuclear protein that functions as a potent ERalpha coactivator. In addition, mitochondrial 4ICD functions as a proapoptotic BH3-only protein. Tamoxifen disrupts an estrogen-driven interaction between ERalpha and 4ICD while promoting mitochondrial accumulation of the 4ICD BH3-only protein. BCL-2 inhibition of tamoxifen-induced apoptosis and tamoxifen activation of BAK, independent of BAX, further supports a role for 4ICD during tamoxifen-induced apoptosis. Finally, reintroduction of HER4, but not HER4 with a mutated BH3 domain, restores tamoxifen sensitivity to tamoxifen-resistant TamR cells in a xenograft model. Clinically, breast cancer patients with tumor expression of nuclear 4ICD responded to tamoxifen therapy with no clinical failures reported after 14 years of follow-up, whereas 20% of patients lacking nuclear 4ICD expression succumbed to their disease within 10 years of diagnosis. Our identification of the HER4/4ICD BH3-only protein as a critical mediator of tamoxifen action provides a clinically important role for 4ICD in human cancer and reveals a potential tumor marker to predict patient response to tamoxifen therapy.

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.

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.

Purpose: erbB-2 and epidermal growth factor receptor (EGFR) may mediate motility via signaling that enables changes in the actin cytoskeleton. A physical basis for this motility may depend on the coexpression of gelsolin, a M(r) 80,000 actin-binding protein., Experimental Design: The expression of erbB-2, EGFR, and gelsolin was analyzed in 790 archival invasive breast cancers. These data were compared with histological, clinical, and outcome data (median follow-up, 16.3 years)., Results: Protein overexpression was observed in overlapping subsets of breast cancers (38% of cases were erbB-2+; 15% of cases were EGFR+; and 56% of cases were gelsolin+). Tumor gelsolin was associated with overexpression of erbB-2 and EGFR, as well as with an aggressive tumor phenotype. By univariate and multivariate analyses, tumor gelsolin alone was not a prognostic factor. Overexpression of all three factors significantly predicted poor clinical outcome by univariate and multivariate analyses. For example, in node-positive patients, coexpression of all three markers was associated with a 3-year disease-specific survival (as compared with erbB-2+, EGFR+, gelsolin- patients, who had a median survival of 6 years)., Conclusions: These data suggest that gelsolin coexpression may be an important additional prognostic factor in erbB-2+, EGFR+ breast cancer patients. We hypothesize that this is due to the role of gelsolin in mediating motility and invasion.

Our objective was to investigate the prognostic significance of cell turnover (apoptosis and proliferation) in breast cancer patients. Apoptosis was microscopically quantitated on histological sections from 791 breast cancer patients with long-term follow-up (median, 16.3 years). Apoptotic counts were also compared with proliferation data (mitotic counts and MIB-1 labeling); apoptosis data derived from terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay; and pathobiological variables, including p53, erbB-2, and estrogen receptor (ER). High apoptotic counts were associated with increased cellular proliferation, ER negativity, immunopositivity of erbB-2 and p53 (P < 0.0001), and shortened disease-specific survival (DSS; P = 0.0009) and disease-free survival (DFS; P = 0.0006). Other factors associated with shortened DFS and DSS by univariate analysis were high tumor grade, nodal metastases, and large tumor size (P < 0.0001 for each). Multivariate analysis of data for all of the patients demonstrated that tumor size, nodal status, ER, histological grade, and erbB-2 showed independent prognostic value. In node-negative patients, tumor size and mitotic rate per 1000 cells independently predicted DFS (P = 0.0055). Tumor grade was the only independent predictor of DSS. For node-positive patients, tumor size, nodal status, ER, and erbB-2 were independent prognostic factors. The number of mitoses per 1000 was independently associated with DFS (P = 0.043) but not with DSS. Apoptosis data did not provide independent prognostic value in any, node-positive or node-negative, breast cancer patients.

Bax and Bcl-2 are proteins that regulate programmed cell death and apoptosis. The expression of these proteins can be regulated, at least in part, by the tumor suppressor p53, but the effects of p53 are highly tissue specific. In an effort to better understand the relation between p53 and the in vivo control of the expression of Bax and Bcl-2 in adenocarcinomas of the breast, we evaluated by immunohistochemistry the expression of Bcl-2 and Bax in 149 invasive ductal carcinomas, 135 of which were chosen because of their p53 immunopositivity. The percentages of Bcl-2-immunopositive tumor cells were significantly lower in the p53-positive (median 20%) subsets as compared to the p53-negative (median 85%) subsets (P = 0. 004). Comparisons of the percentages of p53-immunopositive tumor cells with the percentages of Bcl-2- and Bax-positive cells (as continuous variables) revealed a significant inverse correlation between Bcl-2 and p53 (r = -0.41, P < 0.001) but not between Bax and p53. In the p53-positive subset, the percentages of Bax- and Bcl-2-immunopositive tumor cells were correlated positively (r = 0. 27, P = 0.002), suggesting that the expression of these genes may be co-regulated to some extent in these breast cancers. Higher percentages of Bcl-2-positive tumor cells were also associated with estrogen receptor positivity (P = 0.03), low histological tumor grade (P = 0.03), and low T stage (P = 0.02), whereas Bax immunostaining was associated only with c-erbB-2 immunopositivity (P = 0.02). Although the number of cases was small and treatment was non-uniform, preliminary correlations with clinical outcome data suggest that the prognostic significance of Bcl-2 may be enhanced by inclusion of Bax data in patients with p53-immunopositive adenocarcinoma of the breast, at least for patients with node-negative disease. Taken together, these data suggest that, despite the ability of p53 to bind directly to the Bax gene promoter, the regulation of Bax in human breast cancers does not necessarily correlate with p53 status, implying that regulation of this pro-apoptotic gene in these tumors is complex and probably influenced by several factors.

Seventy-two patients with advanced breast carcinoma (42% bone, 25% visceral, 5.5% soft tissue, and 27.5% multiple site metastases) were evaluated to determine the relationship between tumor expression of the estrogen-regulated protein pS2, estrogen receptor (ER) or progesterone receptor (PgR) content, and response to hormonal therapy. Twenty-nine % of tumors were pS2 positive, 64% were ER positive, and 29% were PgR positive. Of the ER-positive patients (n = 43), 15 (35%) had greater than 10% of the invasive carcinoma which immunostained for pS2 (these were considered pS2 positive). Only 3 of 24 ER-negative tumors were pS2 positive. A weak association between pS2 expression and ER content (P = 0.08) but not PgR content was observed. Of pS2-positive patients, 52% had a partial or complete response to hormonal therapy. In 24% of pS2-positive patients the disease stabilized with treatment. In contrast, 27% of pS2-negative patients had a partial or complete response. In 10% of these patients the disease stabilized. Similar associations between therapeutic response and ER or PgR were not observed. The odds of having a clinical response to hormonal therapy was greater for pS2-positive than for ER- or PgR-positive tumors. pS2 expression may define a subset of ER-positive tumors that are more likely to respond to hormonal treatment.

Various monoclonal antibodies reactive with protooncogene products or tumor-associated antigens have been utilized to investigate breast carcinoma biology or antigen expression with potential prognostic relevance. Murine monoclonal antibody TA1, generated by immunization of BALB/c mice with whole c-erbB-2 (neu) transformed NIH/3T3 cells, recognizes the extracellular domain of the c-erbB-2 protein and binds a Mr 185,000 protein by immunoprecipitation. Using avidin-biotin-peroxidase techniques and monoclonal antibody TA1, 313 archival primary adenocarcinomas of the breast were evaluated for c-erbB-2 overexpression; 290 of these were used for multiparametric statistical analysis. Historical, clinical (age, laterality), histological (nuclear grade, tumor size, lymph node status, lymphatic or blood invasion), and hormone receptor data as well as clinical outcome (minimal follow-up, 6 years; median follow-up, 8.5 years) were compared to TA1 staining. For these 290 patients Cox regression multivariate analysis showed the strongest correlation between lymph node status or estrogen receptor status and overall survival (P = 0.0001 and 0.049, respectively). TA1 staining did not significantly correlate with survival (P = 0.395). However, univariate analysis of certain patient subpopulations showed a significant correlation if the examined tumors were subdivided into negative or focally reactive and those with greater than or equal to 40% cellular reactivity. For T3, T4 patients, strong TA1 immunoreactivity correlated with a shortened disease-free survival (log rank P = 0.0018; Wilcoxon p = 0.0078) and overall survival (log rank P = 0.0002; Wilcoxon P = 0.0013). For these patients the overall survival at 6 years was markedly different between the strongly reactive tumors (0%) and the negative to weakly reactive tumors (55%). In lymph node-positive patients a trend between high TA1 reactivity and a worse overall survival was also noted (log rank P = 0.128; Wilcoxon P = 0.054), with a 6-year survival of 42% in the strongly reactive tumors (n = 16) and 65% in the negative to weakly reactive carcinomas (n = 105). No correlation between TA1 immunoreactivity and other historical, clinical, and histological features were noted. c-erbB-2 overexpression as measured by immunohistochemical techniques, therefore, may have clinical significance in certain patient subpopulations.