Brian G. Rowan

Phosphorylation of steroid receptor coactivator-1. Identification of the phosphorylation sites and phosphorylation through the mitogen-activated protein kinase pathway.

Steroid receptor coactivator-1 (SRC-1) is a member of a coactivator family that enhance the activation of the steroid/nuclear receptor superfamily of ligand-stimulated transcription factors. To study the regulation of SRC-1 by signaling pathways in the cell, the major phosphorylation sites of SRC-1 were identified in COS-1 cells using a combination of in vivo labeling with [32P]H3PO4, modified manual Edman degradation, phosphoamino acid analysis, endoproteinase digestion, and mutagenesis of the SRC-1 phosphorylation sites. Seven phosphorylation sites were identified in SRC-1: serine 372, serine 395, serine 517, serine 569, serine 1033, threonine 1179, and serine 1185. All the sites contained consensus sequences for the serine/threonine-proline-directed family of protein kinases, and two sites (serine 395 and threonine 1179) contained a perfect consensus sequence for the mitogen-activated protein kinase family (Erk-1 and Erk-2). Furthermore, Erk-2 phosphorylated threonine 1179 and serine 1185 (and to a lesser extent, serine 395) in vitro, suggesting the importance of this pathway for SRC-1 regulation. Treatment of cells expressing SRC-1 with epidermal growth factor enhanced the ligand-dependent, progesterone receptor-mediated activation of a target reporter gene. These results identify phosphorylation as a regulatory modification of SRC-1 and provide a basis upon which to identify signaling pathways that regulate SRC-1 function and, consequently, modify steroid/nuclear receptor action.

Post-translational modifications of nuclear receptors and human disease.

Nuclear receptors (NR) impact a myriad of physiological processes including homeostasis, reproduction, development, and metabolism. NRs are regulated by post-translational modifications (PTM) that markedly impact receptor function. Recent studies have identified NR PTMs that are involved in the onset and progression of human diseases, including cancer. The majority of evidence linking NR PTMs with disease has been demonstrated for phosphorylation, acetylation and sumoylation of androgen receptor (AR), estrogen receptor α (ERα), glucocorticoid receptor (GR) and peroxisome proliferator activated receptor γ (PPARΓ). Phosphorylation of AR has been associated with hormone refractory prostate cancer and decreased disease-specific survival. AR acetylation and sumoylation increased growth of prostate cancer tumor models. AR phosphorylation reduced the toxicity of the expanded polyglutamine AR in Kennedys Disease as a consequence of reduced ligand binding. A comprehensive evaluation of ERα phosphorylation in breast cancer revealed several sites associated with better clinical outcome to tamoxifen therapy, whereas other phosphorylation sites were associated with poorer clinical outcome. ERα acetylation and sumoylation may also have predictive value for breast cancer. GR phosphorylation and acetylation impact GR responsiveness to glucocorticoids that are used as anti-inflammatory drugs. PPARγ phosphorylation can regulate the balance between growth and differentiation in adipose tissue that is linked to obesity and insulin resistance. Sumoylation of PPARγ is linked to repression of inflammatory genes important in patients with inflammatory diseases. NR PTMs provide an additional measure of NR function that can be used as both biomarkers of disease progression, and predictive markers for patient response to NR-directed treatments.

Human Adipose Tissue-Derived Stromal/Stem Cells Promote Migration and Early Metastasis of Triple Negative Breast Cancer Xenografts

Background Fat grafting is used to restore breast defects after surgical resection of breast tumors. Supplementing fat grafts with adipose tissue-derived stromal/stem cells (ASCs) is proposed to improve the regenerative/restorative ability of the graft and retention. However, long term safety for ASC grafting in proximity of residual breast cancer cells is unknown. The objective of this study was to determine the impact of human ASCs derived from abdominal lipoaspirates of three donors, on a human breast cancer model that exhibits early metastasis. Methodology/Principal Findings Human MDA-MB-231 breast cancer cells represents “triple negative” breast cancer that exhibits early micrometastasis to multiple mouse organs [1]. Human ASCs were derived from abdominal adipose tissue from three healthy female donors. Indirect co-culture of MDA-MB-231 cells with ASCs, as well as direct co-culture demonstrated that ASCs had no effect on MDA-MB-231 growth. Indirect co-culture, and ASC conditioned medium (CM) stimulated migration of MDA-MB-231 cells. ASC/RFP cells from two donors co-injected with MDA-MB-231/GFP cells exhibited a donor effect for stimulation of primary tumor xenografts. Both ASC donors stimulated metastasis. ASC/RFP cells were viable, and integrated with MDA-MB-231/GFP cells in the tumor. Tumors from the co-injection group of one ASC donor exhibited elevated vimentin, matrix metalloproteinase-9 (MMP-9), IL-8, VEGF and microvessel density. The co-injection group exhibited visible metastases to the lung/liver and enlarged spleen not evident in mice injected with MDA-MB-231/GFP alone. Quantitation of the total area of GFP fluorescence and human chromosome 17 DNA in mouse organs, H&E stained paraffin sections and fluorescent microscopy confirmed multi-focal metastases to lung/liver/spleen in the co-injection group without evidence of ASC/RFP cells. Conclusions Human ASCs derived from abdominal lipoaspirates of two donors stimulated metastasis of MDA-MB-231 breast tumor xenografts to multiple mouse organs. MDA-MB-231 tumors co-injected with ASCs from one donor exhibited partial EMT, expression of MMP-9, and increased angiogenesis.

Adult human mesenchymal stem cells enhance breast tumorigenesis and promote hormone independence

Adult human mesenchymal stem cells (hMSCs) have been shown to home to sites of breast cancer and integrate into the tumor stroma. We demonstrate here the effect of hMSCs on primary breast tumor growth and the progression of these tumors to hormone independence. Co-injection of bone marrow-derived hMSCs enhances primary tumor growth of the estrogen receptor-positive, hormone-dependent breast carcinoma cell line MCF-7 in the presence or absence of estrogen in SCID/beige mice. We also show hormone-independent growth of MCF-7 cells when co-injected with hMSCs. These effects were found in conjunction with increased immunohistochemical staining of the progesterone receptor in the MCF-7/hMSC tumors as compared to MCF-7 control tumors. This increase in PgR expression indicates a link between MCF-7 cells and MSCs through ER-mediated signaling. Taken together, our data reveal the relationship between tumor microenvironment and tumor growth and the progression to hormone independence. This tumor stroma-cell interaction may provide a novel target for the treatment of estrogen receptor-positive, hormone-independent, and endocrine-resistant breast carcinoma.

Circadian and Melatonin Disruption by Exposure to Light at Night Drives Intrinsic Resistance to Tamoxifen Therapy in Breast Cancer

Resistance to endocrine therapy is a major impediment to successful treatment of breast cancer. Preclinical and clinical evidence links resistance to antiestrogen drugs in breast cancer cells with the overexpression and/or activation of various pro-oncogenic tyrosine kinases. Disruption of circadian rhythms by night shift work or disturbed sleep-wake cycles may lead to an increased risk of breast cancer and other diseases. Moreover, light exposure at night (LEN) suppresses the nocturnal production of melatonin that inhibits breast cancer growth. In this study, we used a rat model of estrogen receptor (ERα(+)) MCF-7 tumor xenografts to demonstrate how altering light/dark cycles with dim LEN (dLEN) speed the development of breast tumors, increasing their metabolism and growth and conferring an intrinsic resistance to tamoxifen therapy. These characteristics were not observed in animals in which the circadian melatonin rhythm was not disrupted, or in animals subjected to dLEN if they received nocturnal melatonin replacement. Strikingly, our results also showed that melatonin acted both as a tumor metabolic inhibitor and a circadian-regulated kinase inhibitor to reestablish the sensitivity of breast tumors to tamoxifen and tumor regression. Together, our findings show how dLEN-mediated disturbances in nocturnal melatonin production can render tumors insensitive to tamoxifen.

Activation of autophagy in mesenchymal stem cells provides tumor stromal support

Recent studies have implicated multipotential mesenchymal stem cells (MSCs) as an aid to breast cancer cell proliferation and metastasis, partly as a result of the MSCs secretome. As the tumor gets beyond 2 mm in diameter, the stromal cells could undergo starvation due to the lack of sufficient nutrients in solid tumor microenvironment. In this study, we investigated the survival mechanisms used by stressed stromal cells in breast cancers. We used serum-deprived mesenchymal stem cells (SD-MSCs) and MCF-7 breast cancer cells as model system with a hypothesis that stromal cells in the nutrient-deprived core utilize survival mechanisms for supporting surrounding cells. We tested this hypothesis using in vivo tumor xenografts in immunodeficient mice, which indicated that SD-MSCs supported MCF-7 tumor growth by protection from apoptosis. Histochemical assays showed that SD-MSCs-injected tumors exhibited higher cellularity, decreased apoptosis and decreased differentiation. Beclin-1 staining indicated autophagic areas surrounded by actively proliferating cells. Furthermore, in vitro studies demonstrate that SD-MSCs survive using autophagy and secrete paracrine factors that support tumor cells following nutrient/serum deprivation. Western blot and immunocytochemistry analysis of SD-MSCs demonstrated upregulation and perinuclear relocation of autophagy key regulators such as beclin-1, ATG10, ATG12, MAP-LC3 and lysosomes. Electron microscopic analysis detected a time-dependent increase in autophagosome formation and HDAC6 activity assays indicated the upregulation of autophagy. Taken together, these data suggest that under nutrient-deprived conditions that can occur in solid tumors, stromal cells utilize autophagy for survival and also secrete anti-apoptotic factors that can facilitate solid tumor survival and growth.

Adipose-derived stromal/stem cells: a primer.

Until recently, the complexity of adipose tissue and its physiological role was not well appreciated. This changed with the discovery of adipokines such as leptin. The cellular composition of adipose tissue is heterogeneous and changes as a function of diabetes and disease states such as diabetes. Tissue engineers view adipose tissue as a rich source of adult stromal/stem cells isolated by collagenase digestion. In vitro and in vivo studies have documented that adipose stromal/stem cells are multipotent, with the ability to differentiate along the adipocyte, chondrocyte, osteoblast and other lineage pathways. The adipose stromal/stem cells secrete a wide range of cytokines and growth factors with potential paracrine actions. Furthermore, adipose stromal/stem cells exert immunomodulatory functions when added to mixed lymphocyte reactions, suggesting that they can be transplanted allogeneically. This review article focuses on these mechanisms of adipose stromal/stem cell action and their potential utility as cellular therapeutics.

Progesterone receptor coactivators

Progesterone action is mediated by intracellular progesterone receptors that regulate target gene transcription. Recently, numerous proteins termed coactivators have been identified that are recruited by the liganded progesterone receptor and enhance receptor-dependent transactivation. Coactivators are a diverse group of molecules that bring multiple structural and enzymatic functions to the promoter. The existence of coactivators represents yet another level of regulation for progesterone receptor activation.

Interleukin-17 Promotes Formation and Growth of Prostate Adenocarcinoma in Mouse Models

The contributions of interleukin (IL)-17 to cancer remain unclear and somewhat controversial. We took a genetic approach to explore its role in prostate cancers by interbreeding IL-17 receptor C (IL-17RC)-deficient mice with mice that are conditionally mutant for PTEN, one established preclinical model for prostate cancer. Mice that were IL-17RC-deficient (IL-17RC(-)) displayed prostates that were smaller than mice that maintained IL-17RC expression (IL-17RC(+)). In addition, IL-17RC(-) mice developed a reduced number of invasive prostate adenocarcinomas with lower rates of cellular proliferation and higher apoptosis than IL-17RC(+) mice. Moreover, the fibromuscular stroma surrounding prostatic glands was relatively thicker in IL-17RC(-) mice and was associated with decreased matrix metalloproteinase (Mmp)7 expression and increased Timp1, 2, and 4 expression, whereas administration of recombinant mouse IL-17 induced prostatic expression of Mmp7. Taken together, our results suggested that IL-17 promotes the formation and growth of prostate adenocarcinoma, and that an IL-17-MMP7 signaling axis is required for the transition of prostatic intraepithelial neoplasia to frank adenocarcinoma.

Estrogen receptor alpha phosphorylation and its functional impact in human breast cancer.

Estrogen receptor α (ERα) is a member of the nuclear receptor superfamily of transcription factors that regulates cell proliferation, differentiation and homeostasis in various tissues. Sustained exposure to estrogen/estradiol (E2) increases the risk of breast, endometrial and ovarian cancers. ERα function is also regulated by phosphorylation through various kinase signaling pathways that will impact various ERα functions including chromatin interaction, coregulator recruitment and gene expression, as well impact breast tumor growth/morphology and breast cancer patient response to endocrine therapy. However, many of the previously characterized ERα phosphorylation sites do not fully explain the impact of receptor phosphorylation on ERα function. This review discusses work from our laboratory toward understanding a role of ERα site-specific phosphorylation in ERα function and breast cancer. The key findings discussed in this review are: (1) the effect of site specific ERα phosphorylation on temporal recruitment of ERα and unique coactivator complexes to specific genes; (2) the impact of stable disruption of ERα S118 and S167 phosphorylation in breast cancer cells on eliciting unique gene expression profiles that culminate in significant effects on breast cancer growth/morphology/migration/invasion; (3) the Src kinase signaling pathway that impacts ERα phosphorylation to alter ERα function; and (4) circadian disruption by light exposure at night leading to elevated ERK1/2 and Src kinase and phosphorylation of ERα, concomitant with tamoxifen resistance in breast tumor models. Results from these studies demonstrate that even changes to single ERα phosphorylation sites can have a profound impact on ERα function in breast cancer. Future work will extend beyond single site phosphorylation analysis toward identification of specific patterns/profiles of ERα phosphorylation under different physiological/pharmacological conditions to understand how common phosphorylation profiles in breast cancer program specific physiological endpoints such as growth, apoptosis, migration/invasion, and endocrine therapy response.