Fransiscus E. Utama

Cyclin D1 Splice Variants: Polymorphism, Risk, and Isoform-Specific Regulation in Prostate Cancer

Purpose: Alternative CCND1 splicing results in cyclin D1b, which has specialized, protumorigenic functions in prostate not shared by the cyclin D1a (full length) isoform. Here, the frequency, tumor relevance, and mechanisms controlling cyclin D1b were challenged. Experimental Design: First, relative expression of both cyclin D1 isoforms was determined in prostate adenocarcinomas. Second, relevance of the androgen axis was determined. Third, minigenes were created to interrogate the role of the G/A870 polymorphism (within the splice site), and findings were validated in primary tissue. Fourth, the effect of G/A870 on cancer risk was assessed in two large case-control studies. Results: Cyclin D1b is induced in tumors, and a significant subset expressed this isoform in the absence of detectable cyclin D1a. Accordingly, the isoforms showed noncorrelated expression patterns, and hormone status did not alter splicing. Whereas G/A870 was not independently predictive of cancer risk, A870 predisposed for transcript-b production in cells and in normal prostate. The influence of A870 on overall transcript-b levels was relieved in tumors, indicating that aberrations in tumorigenesis likely alter the influence of the polymorphism. Conclusions: These studies reveal that cyclin D1b is specifically elevated in prostate tumorigenesis. Cyclin D1b expression patterns are distinct from that observed with cyclin D1a. The A870 allele predisposes for transcript-b production in a context-specific manner. Although A870 does not independently predict cancer risk, tumor cells can bypass the influence of the polymorphism. These findings have major implications for the analyses of D-cyclin function in the prostate and provide the foundation for future studies directed at identifying potential modifiers of the G/A870 polymorphism. (Clin Cancer Res 2009;15(17):5338–49)

Prolactin Inhibits BCL6 Expression in Breast Cancer through a Stat5a-Dependent Mechanism

BCL6 is a transcriptional repressor that recognizes DNA target sequences similar to those recognized by signal transducer and activator of transcriptions 5 (Stat5). BCL6 disrupts differentiation of breast epithelia, is downregulated during lactation, and is upregulated in poorly differentiated breast cancer. In contrast, Stat5a mediates prolactin-induced differentiation of mammary epithelia, and loss of Stat5 signaling in human breast cancer is associated with undifferentiated histology and poor prognosis. Here, we identify the mammary cell growth factor prolactin as a potent suppressor of BCL6 protein expression in human breast cancer through a mechanism that requires Stat5a, but not prolactin-activated Stat5b, MEK-ERK, or PI3K-AKT pathways. Prolactin rapidly suppressed BCL6 mRNA in T47D, MCF7, ZR75.1, and SKBr3 breast cancer cell lines, followed by prolonged reduction of BCL6 protein levels within 3 hours. Prolactin suppression of BCL6 was enhanced by overexpression of Stat5a but not Stat5b, was mimicked by constitutively active Stat5a, but did not require the transactivation domain of Stat5a. Stat5 chromatin immunoprecipitation demonstrated physical interaction with a BCL6 gene regulatory region, and BCL6 transcript repression required histone deacetylase activity based on sensitivity to trichostatin A. Functionally, BCL6 overexpression disrupted prolactin induction of Stat5 reporter genes. Prolactin suppression of BCL6 was extended to xenotransplant tumors in nude mice in vivo and to freshly isolated human breast cancer explants ex vivo. Quantitative immunohistochemistry revealed elevated BCL6 in high-grade and metastatic breast cancer compared with ductal carcinoma in situ and nonmalignant breast, and cellular BCL6 protein levels correlated negatively with nuclear Stat5a (r = -0.52; P < 0.001) but not with Stat5b. Loss of prolactin-Stat5a signaling and concomitant upregulation of BCL6 may represent a regulatory switch facilitating undifferentiated histology and poor prognosis of breast cancer.

Ultrahigh density microarrays of solid samples

We present a sectioning and bonding technology to make ultrahigh density microarrays of solid samples, cutting edge matrix assembly (CEMA). Maximized array density is achieved by a scaffold-free, self-supporting construction with rectangular array features that are incrementally scalable. This platform technology facilitates arrays of >10,000 tissue features on a standard glass slide, inclusion of unique sample identifiers for improved manual or automated tracking, and oriented arraying of stratified or polarized samples.

PTP1B suppresses prolactin activation of Stat5 in breast cancer cells

Basal levels of nuclear localized, tyrosine phosphorylated Stat5 are present in healthy human breast epithelia. In contrast, Stat5 phosphorylation is frequently lost during breast cancer progression, a finding that correlates with loss of histological differentiation and poor patient prognosis. Identifying the mechanisms underlying loss of Stat5 phosphorylation could provide novel targets for breast cancer therapy. Pervanadate, a general tyrosine phosphatase inhibitor, revealed marked phosphatase regulation of Stat5 activity in breast cancer cells. Lentiviral-mediated shRNA allowed specific examination of the regulatory role of five tyrosine phosphatases (PTP1B, TC-PTP, SHP1, SHP2, and VHR), previously implicated in Stat5 regulation in various systems. Enhanced and sustained prolactin-induced Stat5 tyrosine phosphorylation was observed in T47D and MCF7 breast cancer cells selectively in response to PTP1B depletion. Conversely, PTP1B overexpression suppressed prolactin-induced Stat5 tyrosine phosphorylation. Furthermore, PTP1B knockdown increased Stat5 reporter gene activity. Mechanistically, PTP1B suppression of Stat5 phosphorylation was mediated, at least in part, through inhibitory dephosphorylation of the Stat5 tyrosine kinase, Jak2. PTP1B knockdown enhanced sensitivity of T47D cells to prolactin phosphorylation of Stat5 by reducing the EC(50) from 7.2 nmol/L to 2.5 nmol/L. Immunohistochemical analyses of two independent clinical breast cancer materials revealed significant negative correlations between levels of active Stat5 and PTP1B, but not TC-PTP. Collectively, our data implicate PTP1B as an important negative regulator of Stat5 phosphorylation in invasive breast cancer.

Insensitivity of Human Prolactin Receptors to Nonhuman Prolactins: Relevance for Experimental Modeling of Prolactin Receptor-Expressing Human Cells

Prolactin (PRL) receptors are expressed in a broad range of human cell types and in a majority of human breast and prostate cancers. Experimentally, normal and malignant human cells are typically cultured in vitro in media containing bovine PRL (bPRL) from fetal bovine serum or as xenotransplants in vivo in the presence of murine PRL (mPRL). The biological efficacy of bPRL toward hPRL receptors (hPRLR) is controversial, and hPRLR are insensitive to mPRL, but the mechanism is not known. To clarify limitations of current in vitro and in vivo experimental model systems for studies of hPRLR-expressing cells, we tested human and relevant subprimate prolactins in multiple hPRLR bioassays. bPRL and ovine PRL were 10-fold less potent hPRLR agonists than hPRL, although maximal responses at high ligand concentrations (efficacies) equaled that of hPRL. mPRL and rat PRL had greater than 50-fold lower potencies toward hPRLR than hPRL and had 50% reduced efficacies. In fact, mPRL and rat PRL were less effective hPRLR agonists than murine GH. Unexpectedly, mPRL was an effective competitive inhibitor of hPRL binding to hPRLR with an inhibitory constant of 1.3 nm and showed partial antagonist activity, suggesting reduced site-2 binding. Collectively, low bioactivities of bPRL and mPRL toward hPRLR suggest that existing laboratory cancer cell lines grown in 10% bovine serum-supplemented media or in mice are selected for growth under lactogen-depleted conditions. The biology and drug responsiveness of existing human cell lines may therefore not be representative of clinical cancers that are sensitive to circulating PRL.

Insulin receptor substrate-1 regulates the transformed phenotype of BT-20 human mammary cancer cells.

Although originating from a human breast cancer, BT-20 cells do not form colonies in soft agar. BT-20 cells do not express insulin receptor substrate-1 (IRS-1), which is known to promote both normal and abnormal growth and to inhibit differentiation. Stable expression of IRS-1 confers to BT-20 cells the ability to form colonies in soft agar. BT-20 cells form tumors in xenografts in mice, but the size of tumors is twice as large when the cells express IRS-1. The increased transformed phenotype is characterized by occupancy of the rDNA and cyclin D1 promoters by IRS-1 and the activation of the cyclin D1, c-myc, and rDNA promoters. In addition, the retinoblastoma protein, which is detectable in the rDNA promoter of quiescent BT-20/IRS-1 cells, is replaced by IRS-1 after insulin-like growth factor-I stimulation. Our results indicate that in BT-20 human mammary cancer cells, expression of IRS-1 activates promoters involved in cell growth and cell proliferation, resulting in a more transformed phenotype. Targeting of IRS-1 could be effective in inhibiting the proliferation of mammary cancer 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.

Protocol for Constructing Tissue Arrays by Cutting Edge Matrix Assembly

We present a protocol for construction of high-density tissue microarrays, cutting edge matrix assembly, which is based on repetitive sectioning and bonding of tissues. Maximized array density is achieved by a scaffold-free, self-supporting construction with rectangular array features that are bonded edge-to-edge, resulting in minimal wasted space between samples. Construction of the tissue array blocks from paraffin-embedded tissue involves initial bonding of primary tissue plates into multiple primary tissue stacks. This is achieved by taking a shaving of desired thickness from the face of each specimen block, trimming the shavings into a set of rectangular primary tissue plates, and bonding multiple plates into primary stacks of tissue. Each resulting primary tissue stack is then transversely cut to produce a set of secondary tissue plates that contains elements of each tissue represented in the primary stacks. Secondary plates from multiple primary sample stacks are then restacked and bonded into a secondary stack. The assembled secondary stack represents a laminate of laminates, which becomes the final array block. The final array block is then reembedded in paraffin and can be sectioned transversely using a microtome to yield micrometer thin sections that are transferred to glass slides for array display and analysis. This technology has facilitated the construction of arrays containing more than 10,000 tissue features on a standard glass slide.

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 P1-06-10: Characterization of novel activated human mammary fibroblast lines and their protumorigenic effect on human breast cancer xenotransplants in mice

Human breast cancer is typically characterized by an extensive stromal compartment enriched in fibroblasts. In contrast, xenotransplants of human breast cancer cell lines are typically epithelial-rich and characterized by sparse in-growth of murine fibroblast stroma. Stromal fibroblasts are important for structural integrity of normal tissue and constitute a major element of the stromal microenvironment of invasive cancer. Cancer associated fibroblasts (CAFs) frequently undergo activation as they co-evolve with cancer cells, serving to promote tumor growth and angiogenesis through secretion of multiple paracrine factors. Presence of activated CAFs in solid malignancies is generally associated with higher grade tumors and poor prognosis. Activated CAFs are resistant to apoptosis and display a myofibroblastic phenotype, including expression of a-smooth muscle actin (α-SMA). To more accurately model human tumor-stroma interactions in human breast cancer xenograft lines in mice, we aimed to develop immortalized activated human mammary fibroblasts for admixture xenografting with human breast cancer cells. Human mammary fibroblasts (HMFs) were isolated from multiple surgically excised tissues of reduction mammoplasties or mastectomies. Five isolated primary HMF lines were screened for α-SMA expression. HMF1 expressed the highest levels of α-SMA and was immortalized by stable lentiviral-delivered hTERT. Quantitative real-time PCR, anchorage-independent growth assay, and in vivo studies were used to further characterize selected HMFs. hTERT-HMF1 and HMF2, despite lower level of α-SMA expression in HMF2, displayed significantly elevated levels of mRNA of proteins commonly associated with activated fibroblasts, including SDF-α, SDF-β, HGF, IL-6, VEGF and podoplanin, as well as promoted a 10-fold increase in anchorage-independent growth of MCF7 breast cancer cells in vitro . To evaluate the ability of these HMF lines to promote tumor growth in vivo , MCF7 human breast cancer cells were orthotopically injected into murine mammary fat pads in the presence or absence of hTERT-HMF1 or HMF2. Both HMF lines facilitated in vivo MCF7-xenograft growth and induced histological changes, including higher grade and greater stromal development, when compared to pure MCF7 cell xenografts. IHC for SMA and collagen confirmed the presence of activated fibroblasts in all tumors; however HMF/MCF7 xenografts showed a distinct distribution of fibroblast stroma throughout the tumor in contrast to sparser stroma between larger epithelial aggregates in tumors grafted from MCF7 cells alone. hTERT-HFM1 cells promoted tumor growth in vivo more robustly than HFM2 cells. By 60 days post xenografting, hTERT-HMF1/MCF7 tumors were significantly larger (∼4-fold) than control MCF7 tumors. In addition, admixture with hTERT-HMF1 cells promoted increased tumor angiogenesis and cancer cell proliferation as measured by Ki67 expression. hTERT-HMF1 represents a novel mammary myofibroblast line that may be useful for improved preclinical xenotransplant modeling and tumor drug response testing of human breast cancer in vivo . Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-06-10.