Laura R. Bohrer

Cyclin-dependent kinases regulate epigenetic gene silencing through phosphorylation of EZH2

The Polycomb group (PcG) protein, enhancer of zeste homologue 2 (EZH2), has an essential role in promoting histone H3 lysine 27 trimethylation (H3K27me3) and epigenetic gene silencing. This function of EZH2 is important for cell proliferation and inhibition of cell differentiation, and is implicated in cancer progression. Here, we demonstrate that under physiological conditions, cyclin-dependent kinase 1 (CDK1) and cyclin-dependent kinase 2 (CDK2) phosphorylate EZH2 at Thr 350 in an evolutionarily conserved motif. Phosphorylation of Thr 350 is important for recruitment of EZH2 and maintenance of H3K27me3 levels at EZH2-target loci. Blockage of Thr 350 phosphorylation not only diminishes the global effect of EZH2 on gene silencing, it also mitigates EZH2-mediated cell proliferation and migration. These results demonstrate that CDK-mediated phosphorylation is a key mechanism governing EZH2 function and that there is a link between the cell-cycle machinery and epigenetic gene silencing.

Inhibition of the Androgen Receptor as a Novel Mechanism of Taxol Chemotherapy in Prostate Cancer

Taxol chemotherapy is one of the few therapeutic options for men with castration-resistant prostate cancer (CRPC). However, the working mechanisms for Taxol are not fully understood. Here, we showed that treatment of 22Rv1, a PTEN-positive CRPC cell line, with paclitaxel and its semisynthetic analogue docetaxel decreases expression of the androgen receptor (AR)-activated genes prostate-specific antigen (PSA) and Nkx3.1 but increases expression of the AR repression gene maspin, suggesting that Taxol treatment inhibits AR activity. This was further supported by the observation that the activity of AR luciferase reporter genes was inhibited by paclitaxel. In contrast, paclitaxel treatment failed to inhibit AR activity in the PTEN-null CRPC cell line C4-2. However, pretreatment of C4-2 cells with the phosphoinositide 3-kinase inhibitor LY294002 restored paclitaxel inhibition of the AR. Treatment of 22Rv1 xenografts in mice with docetaxel induced mitotic arrest and a decrease in PSA expression in tumor cells adjacent to vascular vessels. We further showed that paclitaxel induces nuclear accumulation of FOXO1, a known AR suppressive nuclear factor, and increases the association of FOXO1 with AR proteins in the nucleus. FOXO1 knockdown with small interfering RNA attenuated the inhibitory effect of paclitaxel on AR transcriptional activity, expression of PSA and Nkx3.1, and cell survival. These data reveal a previously uncharacterized, FOXO1-mediated, AR-inhibitory effect of Taxol in CRPC cells that may play an important role in Taxol-mediated inhibition of CRPC growth.

Androgens Suppress EZH2 Expression Via Retinoblastoma (RB) and p130-Dependent Pathways: A Potential Mechanism of Androgen-Refractory Progression of Prostate Cancer

Androgens and the androgen receptor are important for both normal prostate development and progression of prostate cancer (PCa). However, the underlying mechanisms are not fully understood. The Polycomb protein enhancer of zeste homolog 2 (EZH2) functions as an epigenetic gene silencer and plays a role in oncogenesis by promoting cell proliferation and invasion. EZH2 has been implicated in human PCa progression, because its expression is often elevated in hormone-refractory PCa. Here, we demonstrated that expression of EZH2 is lower in androgen-sensitive LNCaP PCa cells compared with Rf and C4-2 cells, two androgen-refractory sublines that are derived from LNCaP cells. Androgen ablation by castration increased the level of EZH2 proteins in LNCaP xenografts in mice. In contrast, treatment of LNCaP cells in culture with the synthetic androgen methyltrieolone (R1881) at doses of 1 nm or higher suppressed EZH2 expression. Moreover, our data suggest that androgen repression of EZH2 requires a functional androgen receptor and this effect is mediated through the retinoblastoma protein and its related protein p130. We further showed that androgen treatment not only increases expression of EZH2 target genes DAB2IP and E-cadherin but also affects LNCaP cell migration. Our results reveal that androgens function as an epigenetic regulator in prostatic cells by repression of EZH2 expression through the retinoblastoma protein and p130-dependent pathways. Our findings also suggest that blockade of EZH2 derepression during androgen deprivation therapy may represent an effective tactic for the treatment of androgen-refractory PCa.

Macrophages Promote Fibroblast Growth Factor Receptor- Driven Tumor Cell Migration and Invasion in a Cxcr2-Dependent Manner

Infiltration of immune cells, specifically macrophages, into the tumor microenvironment has been linked to increased mammary tumor formation and progression. Activation of growth factor receptor signaling pathways within mammary epithelial cells, such as the fibroblast growth factor receptor 1 (FGFR1) pathway, induces recruitment of macrophages to the mammary epithelium. These macrophages promote increased epithelial cell proliferation and angiogenesis. However, the specific mechanisms by which these macrophages are regulated by the preneoplastic epithelial cells and the mechanisms of action of the macrophages within the developing FGFR1-driven tumor microenvironment remain unknown. In this study, we show that activation of inducible FGFR1 in mammary glands leads to decreased activity of the TGFβ/Smad3 pathway in macrophages associated with early stage lesions. Further studies show that macrophages have increased expression of inflammatory chemokines that bind Cxcr2 following exposure to conditioned media from mammary epithelial and tumor cells in which the FGF pathway had been activated. The increase in these ligands is inhibited following activation of the TGFβ pathway, suggesting that decreased TGFβ signaling contributes to the upregulation of these chemokines. Using coculture studies, we further show that macrophages are capable of promoting epithelial and tumor cell migration and invasion through activation of Cxcr2. These results indicate that macrophage-derived Cxcr2 ligands may be important for promoting mammary tumor formation regulated by FGFR signaling. Furthermore, these results suggest that targeting Cxcr2 may represent a novel therapeutic strategy for breast cancers that are associated with high levels of infiltrating macrophages. Mol Cancer Res; 10(10); 1294–305. ©2012 AACR.

P300 acetyltransferase regulates androgen receptor degradation and PTEN-deficient prostate tumorigenesis

Overexpression of the histone acetyltransferase p300 is implicated in the proliferation and progression of prostate cancer, but evidence of a causal role is lacking. In this study, we provide genetic evidence that this generic transcriptional coactivator functions as a positive modifier of prostate tumorigenesis. In a mouse model of PTEN deletion-induced prostate cancer, genetic ablation of p300 attenuated expression of the androgen receptor (AR). This finding was confirmed in human prostate cancer cells in which PTEN expression was abolished by RNA interference-mediated attenuation. These results were consistent with clinical evidence that the expression of p300 and AR correlates positively in human prostate cancer specimens. Mechanistically, PTEN inactivation increased AR phosphorylation at serine 81 (Ser81) to promote p300 binding and acetylation of AR, thereby precluding its polyubiquitination and degradation. In support of these findings, in PTEN-deficient prostate cancer in the mouse, we found that p300 was crucial for AR target gene expression. Taken together, our work identifies p300 as a molecular determinant of AR degradation and highlights p300 as a candidate target to manage prostate cancer, especially in cases marked by PTEN loss.

FOXO1 binds to the TAU5 motif and inhibits constitutively active androgen receptor splice variants.

Aberrant activation of the androgen receptor (AR) is a major factor highly relevant to castration‐resistant progression of prostate cancer (PCa). FOXO1, a key downstream effector of PTEN, inhibits androgen‐independent activation of the AR. However, the underlying mechanism remains elusive.

Activation of the FGFR–STAT3 Pathway in Breast Cancer Cells Induces a Hyaluronan-Rich Microenvironment That Licenses Tumor Formation

Aberrant activation of fibroblast growth factor receptors (FGFR) contributes to breast cancer growth, progression, and therapeutic resistance. Because of the complex nature of the FGF/FGFR axis, and the numerous effects of FGFR activation on tumor cells and the surrounding microenvironment, the specific mechanisms through which aberrant FGFR activity contributes to breast cancer are not completely understood. We show here that FGFR activation induces accumulation of hyaluronan within the extracellular matrix and that blocking hyaluronan synthesis decreases proliferation, migration, and therapeutic resistance. Furthermore, FGFR-mediated hyaluronan accumulation requires activation of the STAT3 pathway, which regulates expression of hyaluronan synthase 2 (HAS2) and subsequent hyaluronan synthesis. Using a novel in vivo model of FGFR-dependent tumor growth, we demonstrate that STAT3 inhibition decreases both FGFR-driven tumor growth and hyaluronan levels within the tumor. Finally, our results suggest that combinatorial therapies inhibiting both FGFR activity and hyaluronan synthesis is more effective than targeting either pathway alone and may be a relevant therapeutic approach for breast cancers associated with high levels of FGFR activity. In conclusion, these studies indicate a novel targetable mechanism through which FGFR activation in breast cancer cells induces a protumorigenic microenvironment.

Epiregulin contributes to breast tumorigenesis through regulating matrix metalloproteinase 1 and promoting cell survival

BackgroundThe epidermal growth factor (EGF) family of ligands has been implicated in promoting breast cancer initiation, growth and progression. The contributions of EGF family ligands and their receptors to breast cancer are complex, and the specific mechanisms through which different ligands regulate breast tumor initiation and growth are not well-defined. These studies focus on the EGF family member epiregulin (EREG) as a mediator of early stage breast tumorigenesis.MethodsEREG expression levels were assessed in both cell lines and human samples of ductal carcinoma in situ (DCIS) using quantitative RT-PCR, ELISA and immunohistochemistry. Gene knock-down approaches using shRNA-based strategies were used to determine the requirement of EREG for growth of MCF10DCIS cells in vivo, and for identifying mechanisms through which EREG promotes tumor cell survival. Experiments were performed using a combination of two-dimensional culture, three-dimensional culture and tumor growth in vivo.ResultsIn comparison with other EGF family members, EREG was induced in MCF10DCIS cells compared with MCF10A and MCF10AT cells and its expression was partially regulated by fibroblast growth factor receptor (FGFR) activity. Reduced EREG expression in MCF10DCIS cells led to decreased tumor growth in vivo, which was associated with reduced cell survival. Furthermore, treatment of MCF10A cells with exogenous EREG enhanced cell survival both in three-dimensional culture and in response to chemotherapeutic agents. Examination of EREG-induced signaling pathways demonstrated that EREG promoted survival of MCF10A cells through regulating expression of matrix metalloproteinase-1 (MMP-1). To determine the relevance of these findings in human tumors, samples of DCIS were analyzed for EREG and MMP-1 expression. EREG was induced in DCIS lesions compared to normal breast epithelium, and EREG and MMP-1 were correlated in a subset of DCIS samples.ConclusionsTogether, these studies lead to identification of a novel pathway involving EREG and MMP-1 that contributes to the formation of early stage breast cancer. Understanding these complex pathways could ultimately lead to the development of novel biomarkers of neoplastic progression and/or new therapeutic strategies for patients with early stage cancer.

ADAM17 in tumor associated leukocytes regulates inflammatory mediators and promotes mammary tumor formation

The presence of inflammatory cells within the tumor microenvironment has been tightly linked to mammary tumor formation and progression. Specifically, interactions between tumor cells and infiltrating macrophages can contribute to the generation of a pro-tumorigenic microenvironment. Understanding the complex mechanisms that drive tumor cell-macrophage cross-talk will ultimately lead to the development of approaches to prevent or treat early stage breast cancers. As described here, we demonstrate that the cell surface protease a disintegrin and metalloproteinase 17 (ADAM17) is expressed by macrophages in mammary tumors and contributes to regulating the expression of pro-inflammatory mediators, including inflammatory cytokines and the inflammatory mediator cyclooxygenase-2 (Cox-2). Furthermore, we demonstrate that ADAM17 is expressed on leukocytes, including macrophages, within polyoma middle T (PyMT)-derived mammary tumors. Genetic deletion of ADAM17 in leukocytes resulted in decreased onset of mammary tumor growth, which was associated with reduced expression of the Cox-2 within the tumor. These findings demonstrate that ADAM17 regulates key inflammatory mediators in macrophages and that leukocyte-specific ADAM17 is an important promoter of mammary tumor initiation. Understanding the mechanisms associated with early stage tumorigenesis has implications for the development of preventive and/or treatment strategies for early stage breast cancers.

Abstract B68: Contributions of STAT3 to pro-tumorigenic alterations within the microenvironment during mammary tumorigenesis

Inflammation has long been recognized as a possible trigger that contributes to tumor initiation. Numerous studies have shown that cells of both the innate and adaptive immune systems are able to support tumor formation and growth through secretion of growth factors and cytokines and by actively shielding the tumor from wider immune surveillance. As a result, inflammation is generally linked to worse patient outcome. However, depending on factors such as tumor size, extracellular matrix, and the complex composition of immune cell infiltrates, inflammation can promote both pro- and anti-tumor properties. Our studies focus on identifying novel mechanisms through which tumor-derived cytokines act in a paracrine manner to regulate the functions of infiltrating inflammatory cells in the tumor microenvironment. We have previously shown that activation of the fibroblast growth factor receptor-1 (FGFR1) oncogene results in the production of high levels of interleukin 6 (IL-6) family members by mammary epithelial cells. Histological examination of FGFR1-driven tumors revealed high levels of STAT3 activation in both tumor cells and macrophages. Thus, further studies focused on identifying mechanisms through which STAT3 activation in different cellular compartments contributes to mammary tumor growth. Initial studies focused on the consequences of STAT3 activation in epithelial/tumor cells. We found that epithelial cell STAT3 activation resulted in accumulation of the extracellular matrix component hyaluronan, which contributed to epithelial cell proliferation, survival and migration. In macrophages, STAT3 is the transcription factor required for transduction of the anti-inflammatory response initiated by interleukin-10. Ongoing experiments have revealed that in primary STAT3fl/fl cre+ bone marrow-derived macrophages, absence of STAT3 signaling reveals a profoundly enhanced pro-inflammatory phenotype. In vitro, treatment of macrophages lacking STAT3 with recombinant IL-6 results in prolonged activation of the proinflammatory STAT1, as well as upregulation of inflammatory molecules such as TNFα and CCL2. Ongoing experiments are aimed at evaluating the functional consequences of this altered tumor microenvironment in vivo during tumor initiation and growth. As anti-STAT3 therapies have began entering clinical trials for solid tumors (including breast tumors), understanding the mechanisms through which STAT3 promotes tumor growth and progression is critical for developing optimal therapeutic strategies. Citation Format: Polly Chuntova, Laura R. Bohrer, Kaylee L. Schwertfeger. Contributions of STAT3 to pro-tumorigenic alterations within the microenvironment during mammary tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr B68.