Melanie A. Girondo

Low levels of Stat5a protein in breast cancer are associated with tumor progression and unfavorable clinical outcomes

IntroductionSignal transducer and activator of transcripton-5a (Stat5a) and its close homologue, Stat5b, mediate key physiological effects of prolactin and growth hormone in mammary glands. In breast cancer, loss of nuclear localized and tyrosine phosphorylated Stat5a/b is associated with poor prognosis and increased risk of antiestrogen therapy failure. Here we quantify for the first time levels of Stat5a and Stat5b over breast cancer progression, and explore their potential association with clinical outcome.MethodsStat5a and Stat5b protein levels were quantified in situ in breast-cancer progression material. Stat5a and Stat5b transcript levels in breast cancer were correlated with clinical outcome in 936 patients. Stat5a protein was further quantified in four archival cohorts totaling 686 patients with clinical outcome data by using multivariate models.ResultsProtein levels of Stat5a but not Stat5b were reduced in primary breast cancer and lymph node metastases compared with normal epithelia. Low tumor levels of Stat5a but not Stat5b mRNA were associated with poor prognosis. Experimentally, only limited overlap between Stat5a- and Stat5b-modulated genes was found. In two cohorts of therapy-naïve, node-negative breast cancer patients, low nuclear Stat5a protein levels were an independent marker of poor prognosis. Multivariate analysis of two cohorts treated with antiestrogen monotherapy revealed that low nuclear Stat5a levels were associated with a more than fourfold risk of unfavorable outcome.ConclusionsLoss of Stat5a represents a new independent marker of poor prognosis in node-negative breast cancer and may be a predictor of response to antiestrogen therapy if validated in randomized clinical trials.

Prolactin suppresses a progestin-induced CK5-positive cell population in luminal breast cancer through inhibition of progestin-driven BCL6 expression

Prolactin controls the development and function of milk-producing breast epithelia but also supports growth and differentiation of breast cancer, especially luminal subtypes. A principal signaling mediator of prolactin, Stat5, promotes cellular differentiation of breast cancer cells in vitro, and loss of active Stat5 in tumors is associated with antiestrogen therapy failure in patients. In luminal breast cancer, progesterone induces a cytokeratin-5 (CK5)-positive basal cell-like population. This population possesses characteristics of tumor stem cells including quiescence, therapy resistance and tumor-initiating capacity. Here we report that prolactin counteracts induction of the CK5-positive population by the synthetic progestin (Pg) R5020 in luminal breast cancer cells both in vitro and in vivo. CK5-positive cells were chemoresistant as determined by fourfold reduced rate of apoptosis following docetaxel exposure. Pg-induction of CK5 was preceded by marked upregulation of BCL6, an oncogene and transcriptional repressor critical for the maintenance of leukemia-initiating cells. Knockdown of BCL6 prevented induction of CK5-positive cell population by Pg. Prolactin suppressed Pg-induced BCL6 through Jak2-Stat5 but not Erk- or Akt-dependent pathways. In premenopausal but not postmenopausal patients with hormone receptor-positive breast cancer, tumor protein levels of CK5 correlated positively with BCL6, and high BCL6 or CK5 protein levels were associated with unfavorable clinical outcome. Suppression of Pg-induction of CK5-positive cells represents a novel prodifferentiation effect of prolactin in breast cancer. The present progress may have direct implications for breast cancer progression and therapy as loss of prolactin receptor-Stat5 signaling occurs frequently and BCL6 inhibitors currently being evaluated for lymphomas may have value for breast cancer.

The Paracrine Hormone for the GUCY2C Tumor Suppressor, Guanylin, Is Universally Lost in Colorectal Cancer

Background: Although colorectal cancer is a disease characterized by sequential accumulation of mutations in epithelial cells, mechanisms leading to genomic vulnerability contributing to tumor initiation remain undefined. GUCY2C has emerged as an intestine-specific tumor suppressor controlling epithelial homeostasis through circuits canonically disrupted in cancer. Surprisingly, the GUCY2C tumor suppressor is universally overexpressed by human colorectal cancer cells. This apparent paradox likely reflects silencing of GUCY2C through loss of its paracrine hormone guanylin. Here, we quantified expression of guanylin mRNA and protein in tumors and normal epithelia from patients with colorectal cancer. Methods: Guanylin mRNA was quantified in tumors and normal adjacent epithelia from 281 patients by the reverse transcriptase-polymerase chain reaction. Separately, the guanylin protein was quantified by immunohistochemistry in 54 colorectal tumors and 30 specimens of normal intestinal epithelium. Results: Guanylin mRNA in colorectum varied more than a 100-fold across the population. Guanylin mRNA was reduced 100- to 1,000-fold in >85% of tumors compared with matched normal adjacent mucosa (P < 0.001). Loss of guanylin mRNA was greatest in tumors from patients <50 years old (P < 0.005) and with the highest expression in normal adjacent mucosa (Spearman correlation coefficient = 0.61; P < 0.001). In a separate validation cohort, guanylin protein was detected in all 30 normal colorectal mucosa specimens, but in none of 54 colorectal tumors. Conclusions: Colorectal cancer may initiate as a disease of paracrine hormone insufficiency through loss of guanylin expression, silencing the GUCY2C tumor suppressor and disrupting homeostatic mechanisms regulating colorectal epithelia cells. Impact: Intestinal tumorigenesis may be prevented by oral GUCY2C hormone replacement therapy. Cancer Epidemiol Biomarkers Prev; 23(11); 2328–37. ©2014 AACR.

Jak2-Stat5a/b Signaling Induces Epithelial-to-Mesenchymal Transition and Stem-Like Cell Properties in Prostate Cancer

Active Stat5a/b predicts early recurrence and disease-specific death in prostate cancer (PC), which both typically are caused by development of metastatic disease. Herein, we demonstrate that Stat5a/b induces epithelial-to-mesenchymal transition (EMT) of PC cells, as shown by Stat5a/b regulation of EMT marker expression (Twist1, E-cadherin, N-cadherin, vimentin, and fibronectin) in PC cell lines, xenograft tumors in vivo, and patient-derived PCs ex vivo using organ explant cultures. Jak2-Stat5a/b signaling induced functional end points of EMT as well, indicated by disruption of epithelial cell monolayers and increased migration and adhesion of PC cells to fibronectin. Knockdown of Twist1 suppressed Jak2-Stat5a/b-induced EMT properties of PC cells, which were rescued by re-introduction of Twist1, indicating that Twist1 mediates Stat5a/b-induced EMT in PC cells. While promoting EMT, Jak2-Stat5a/b signaling induced stem-like properties in PC cells, such as sphere formation and expression of cancer stem cell markers, including BMI1. Mechanistically, both Twist1 and BMI1 were critical for Stat5a/b induction of stem-like features, because genetic knockdown of Twist1 suppressed Stat5a/b-induced BMI1 expression and sphere formation in stem cell culture conditions, which were rescued by re-introduction of BMI1. By using human prolactin knock-in mice, we demonstrate that prolactin-Stat5a/b signaling promoted metastases formation of PC cells in vivo. In conclusion, our data support the concept that Jak2-Stat5a/b signaling promotes metastatic progression of PC by inducing EMT and stem cell properties in PC cells.

Prolactin-Stat5 signaling in breast cancer is potently disrupted by acidosis within the tumor microenvironment

IntroductionEmerging evidence in estrogen receptor-positive breast cancer supports the notion that prolactin-Stat5 signaling promotes survival and maintenance of differentiated luminal cells, and loss of nuclear tyrosine phosphorylated Stat5 (Nuc-pYStat5) in clinical breast cancer is associated with increased risk of antiestrogen therapy failure. However, the molecular mechanisms underlying loss of Nuc-pYStat5 in breast cancer remain poorly defined.MethodsWe investigated whether moderate extracellular acidosis of pH 6.5 to 6.9 frequently observed in breast cancer inhibits prolactin-Stat5 signaling, using in vitro and in vivo experimental approaches combined with quantitative immunofluorescence protein analyses to interrogate archival breast cancer specimens.ResultsModerate acidosis at pH 6.8 potently disrupted signaling by receptors for prolactin but not epidermal growth factor, oncostatin M, IGF1, FGF or growth hormone. In breast cancer specimens there was mutually exclusive expression of Nuc-pYStat5 and GLUT1, a glucose transporter upregulated in glycolysis-dependent carcinoma cells and an indirect marker of lactacidosis. Mutually exclusive expression of GLUT1 and Nuc-pYStat5 occurred globally or regionally within tumors, consistent with global or regional acidosis. All prolactin-induced signals and transcripts were suppressed by acidosis, and the acidosis effect was rapid and immediately reversible, supporting a mechanism of acidosis disruption of prolactin binding to receptor. T47D breast cancer xenotransplants in mice displayed variable acidosis (pH 6.5 to 6.9) and tumor regions with elevated GLUT1 displayed resistance to exogenous prolactin despite unaltered levels of prolactin receptors and Stat5.ConclusionsModerate extracellular acidosis effectively blocks prolactin signaling in breast cancer. We propose that acidosis-induced prolactin resistance represents a previously unrecognized mechanism by which breast cancer cells may escape homeostatic control.

Steroid induction of therapy-resistant cytokeratin-5-positive cells in estrogen receptor-positive breast cancer through a BCL6-dependent mechanism.

Therapy resistance remains a major problem in estrogen receptor-α (ERα)-positive breast cancer. A subgroup of ERα-positive breast cancer is characterized by mosaic presence of a minor population of ERα-negative cancer cells expressing the basal cytokeratin-5 (CK5). These CK5-positive cells are therapy resistant and have increased tumor-initiating potential. Although a series of reports document induction of the CK5-positive cells by progestins, it is unknown if other 3-ketosteroids share this ability. We now report that glucocorticoids and mineralocorticoids effectively expand the CK5-positive cell population. CK5-positive cells induced by 3-ketosteroids lacked ERα and progesterone receptors, expressed stem cell marker, CD44, and displayed increased clonogenicity in soft agar and broad drug-resistance in vitro and in vivo. Upregulation of CK5-positive cells by 3-ketosteroids required induction of the transcriptional repressor BCL6 based on suppression of BCL6 by two independent BCL6 small hairpin RNAs or by prolactin. Prolactin also suppressed 3-ketosteroid induction of CK5+ cells in T47D xenografts in vivo. Survival analysis with recursive partitioning in node-negative ERα-positive breast cancer using quantitative CK5 and BCL6 mRNA or protein expression data identified patients at high or low risk for tumor recurrence in two independent patient cohorts. The data provide a mechanism by which common pathophysiological or pharmacologic elevations in glucocorticoids or other 3-ketosteroids may adversely affect patients with mixed ERα+/CK5+ breast cancer. The observations further suggest a cooperative diagnostic utility of CK5 and BCL6 expression levels and justify exploring efficacy of inhibitors of BCL6 and 3-ketosteroid receptors for a subset of ERα-positive breast cancers.

Validation of tumor protein marker quantification by two independent automated immunofluorescence image analysis platforms

Protein marker levels in formalin-fixed, paraffin-embedded tissue sections traditionally have been assayed by chromogenic immunohistochemistry and evaluated visually by pathologists. Pathologist scoring of chromogen staining intensity is subjective and generates low-resolution ordinal or nominal data rather than continuous data. Emerging digital pathology platforms now allow quantification of chromogen or fluorescence signals by computer-assisted image analysis, providing continuous immunohistochemistry values. Fluorescence immunohistochemistry offers greater dynamic signal range than chromogen immunohistochemistry, and combined with image analysis holds the promise of enhanced sensitivity and analytic resolution, and consequently more robust quantification. However, commercial fluorescence scanners and image analysis software differ in features and capabilities, and claims of objective quantitative immunohistochemistry are difficult to validate as pathologist scoring is subjective and there is no accepted gold standard. Here we provide the first side-by-side validation of two technologically distinct commercial fluorescence immunohistochemistry analysis platforms. We document highly consistent results by (1) concordance analysis of fluorescence immunohistochemistry values and (2) agreement in outcome predictions both for objective, data-driven cutpoint dichotomization with Kaplan–Meier analyses or employment of continuous marker values to compute receiver-operating curves. The two platforms examined rely on distinct fluorescence immunohistochemistry imaging hardware, microscopy vs line scanning, and functionally distinct image analysis software. Fluorescence immunohistochemistry values for nuclear-localized and tyrosine-phosphorylated Stat5a/b computed by each platform on a cohort of 323 breast cancer cases revealed high concordance after linear calibration, a finding confirmed on an independent 382 case cohort, with concordance correlation coefficients >0.98. Data-driven optimal cutpoints for outcome prediction by either platform were reciprocally applicable to the data derived by the alternate platform, identifying patients with low Nuc-pYStat5 at ~3.5-fold increased risk of disease progression. Our analyses identified two highly concordant fluorescence immunohistochemistry platforms that may serve as benchmarks for testing of other platforms, and low interoperator variability supports the implementation of objective tumor marker quantification in pathology laboratories.

Abstract P5-04-05: Preclinical modeling of luminal breast cancer: Recapitulating progression to lethal and tamoxifen-resistant lung metastases in novel patient-derived xenotransplant models in prolactin-humanized mice

Seventy to eighty percent of newly diagnosed breast cancer cases are estrogen receptor(ER)-positive and are classified as luminal. Despite hormone therapy, 25-30% of luminal breast cancers will recur within 15 years of surgical removal of the primary tumor, and many of these patients will die from currently incurable distant metastases. Lung and bone are the most common organ sites for distant breast cancer metastases, with either site affected in approximately 70% of patients based on autopsy studies. A major hurdle for therapeutic progress with luminal breast cancer is the historical difficulty to establish xenograft models of human luminal breast cancer in mice. In particular, there is a need for experimental ER-positive breast cancer models that recapitulate distant metastasis formation from orthotopic tumor implants in mammary glands. Such experimental models will allow surgical resection of the primary tumor followed by clinically relevant testing of targeted adjuvant agents against distant residual disease. We have engineered prolactin-humanized mice that have been backcrossed for ten generations into the Nod-SCOD-IL2Rgamma (NSG) immunodeficient background. These prolactin-humanized mice display improved take rates of patient-derived luminal breast cancer. Using prolactin-humanized mice we have established patient-derived serially transplantable luminal breast cancer models that metastasize to distant sites when grown as primary tumors in the mammary gland. One of the ER-positive lines, PDX2, effectively metastasizes to lungs in 100% of animals within 55 days of grafting into mammary glands. PDX2 lung metastases retain ER and progesterone receptor (PR) expression as well as expression of the luminal marker, GATA3, and display high degree of Ki67 positivity indicating rapidly proliferative lesions. Importantly, metastatic PDX2 lesions show only limited dormancy. In fact, when primary PDX2 tumors are surgically removed at Day 55, mice will die from lung metastases around Day 150. Estrogen supplementation is required for establishment of PDX2 tumors in mice. After primary PDX2 tumors are established in mammary glands in the presence of estradiol, primary tumors respond to tamoxifen with growth suppression but do not undergo tumor regression, and develop resistance to tamoxifen. Treatment of mice with adjuvant tamoxifen following surgical removal of primary PDX2 tumors led to extensive regression of existing lung metastases to barely detectable levels within 30 days of surgery. However, tamoxifen-refractory PDX2 lung metastases regrew during the next 30 day-period in the continued exposure to tamoxifen. Molecular phenotyping of the PDX2 model and other new luminal breast cancer models are ongoing, with the goal of characterizing tamoxifen-responsive and tamoxifen-refractory primary and metastatic lesions. Long-term, our intent is to use the PDX2 and other preclinical xenograft models in prolactin-humanized mice to systematically explore agents for synergy with anti-estrogens to establish curative combination treatments for metastatic luminal breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-04-05.

Abstract P1-08-20: Increased risk of hormone therapy failure in breast cancers expressing low phospho-Stat5: Validation of quantitative immunofluorescence assay parameters

Previous analyses of three breast cancer cohorts revealed that loss of phospho-Stat5 in breast cancer is associated with significantly elevated risk of hormone therapy failure (1, 2). Nuclear localized tyrosine phosphorylated Stat5 (Nuc-pYStat5) may therefore have clinical value as a predictive marker. Analysis of two of the three previously reported anti-estrogen treated patient cohorts used pathologist scoring of diaminobenzidine (DAB) chromogen-stained Stat5. However the third cohort, analyzed by quantitative immunofluorescence analysis (QIF) on the Genoptix/HistoRx AQUA platform, revealed a greater hazard ratio than the cohorts analyzed by pathologist DAB-scoring. To extend and validate these observations, we applied the Nuc-pYStat5 cutpoint derived in our previous study (2) to an independent cohort of anti-estrogen-treated breast cancer patients using two distinct QIF software platforms, AQUA and Definiens Tissue Studio. Tissue Studio relies on supervised machine learning and multiparametric features of a high-resolution whole slide image to identify cancer cell regions, while AQUA software relies on costaining of a tumor marker to identify cancer cell regions. The two QIF platforms produced highly concordant Nuc-pYStat5 levels (R2 linear = 0.96, P References: 1) Yamashita et al. Stat5 expression predicts response to endocrine therapy and improves survival in estrogen receptor-positive breast cancer. Endocr Relat Cancer. 2006;13:885-93. 2) Peck et al. Loss of nuclear localized and tyrosine phosphorylated Stat5 in breast cancer predicts poor clinical outcome and increased risk of antiestrogen therapy failure. J Clin Oncol. 2011;29:2448-58. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-08-20.