Lacey M. Litchfield

Molecular Pathways: Trafficking of Metabolic Resources in the Tumor Microenvironment

A model of tumor metabolism is proposed that describes how the complementary metabolic functions of the local stroma and the tumor cells contribute to cancer progression. Cancer cells alter the metabolism of cancer-associated fibroblasts to obtain lactate and amino acids, which are utilized for energy production, rapid growth, and resistance to chemotherapy drugs. Cancer cells use glutamine supplied by cancer-associated fibroblasts to replenish tricarboxylic acid cycle intermediates and as a nitrogen source for nucleotide synthesis. Moreover, adipocytes in the microenvironment attract cancer cells through the secretion of inflammatory cytokines and proteases. The cancer cells then induce metabolic changes in the adipocytes to acquire free fatty acids that are oxidized by cancer cells to generate energy for proliferation. Increasing knowledge about the metabolic symbiosis within the tumor has led to novel therapeutic strategies designed to restrict metabolic adaptation, including inhibiting lactate transporters and repurposing antidiabetic drugs (thiazolidinediones, metformin). Clin Cancer Res; 21(4); 680–6. ©2015 AACR.

Identification and characterization of nucleolin as a COUP-TFII coactivator of retinoic acid receptor β transcription in breast cancer cells.

Introduction The orphan nuclear receptor COUP-TFII plays an undefined role in breast cancer. Previously we reported lower COUP-TFII expression in tamoxifen/endocrine- resistant versus sensitive breast cancer cell lines. The identification of COUP-TFII-interacting proteins will help to elucidate its mechanism of action as a transcriptional regulator in breast cancer. Results FLAG-affinity purification and multidimensional protein identification technology (MudPIT) identified nucleolin among the proteins interacting with COUP-TFII in MCF-7 tamoxifen-sensitive breast cancer cells. Interaction of COUP-TFII and nucleolin was confirmed by coimmunoprecipitation of endogenous proteins in MCF-7 and T47D breast cancer cells. In vitro studies revealed that COUP-TFII interacts with the C-terminal arginine-glycine repeat (RGG) domain of nucleolin. Functional interaction between COUP-TFII and nucleolin was indicated by studies showing that siRNA knockdown of nucleolin and an oligonucleotide aptamer that targets nucleolin, AS1411, inhibited endogenous COUP-TFII-stimulated RARB2 expression in MCF-7 and T47D cells. Chromatin immunoprecipitation revealed COUP-TFII occupancy of the RARB2 promoter was increased by all-trans retinoic acid (atRA). RARβ2 regulated gene RRIG1 was increased by atRA and COUP-TFII transfection and inhibited by siCOUP-TFII. Immunohistochemical staining of breast tumor microarrays showed nuclear COUP-TFII and nucleolin staining was correlated in invasive ductal carcinomas. COUP-TFII staining correlated with ERα, SRC-1, AIB1, Pea3, MMP2, and phospho-Src and was reduced with increased tumor grade. Conclusions Our data indicate that nucleolin plays a coregulatory role in transcriptional regulation of the tumor suppressor RARB2 by COUP-TFII.

Statin therapy is associated with improved survival in patients with non-serous-papillary epithelial ovarian cancer: a retrospective cohort analysis.

Aim To determine whether statin use is associated with improved epithelial ovarian cancer (OvCa) survival. Methods This is a single-institution retrospective cohort review of patients treated for OvCa between 1992 and 2013. Inclusion criteria were International Federation of Gynecology and Obstetrics (FIGO) stage I–IV OvCa. The primary exposures analyzed were hyperlipidemia and statin use. The primary outcomes were progression-free survival (PFS) and disease-specific survival (DSS). Results 442 patients met inclusion criteria. The cohort was divided into three groups: patients with hyperlipidemia who used statins (n = 68), patients with hyperlipidemia who did not use statins (n = 28), and patients without hyperlipidemia (n = 346). OvCa outcomes were evaluated. When we analyzed the entire cohort, we found no significant differences in PFS or DSS among the groups. The median PFS for hyperlipidemics using statins, hyperlipidemics not using statins, and non-hyperlipidemics was 21.7, 13.6, and 14.7 months, respectively (p = 0.69). Median DSS for hyperlipidemics using statins, hyperlipidemics not using statins, and non-hyperlipidemics was 44.2, 75.7, and 41.5 months, respectively (p = 0.43). These findings did not change after controlling for confounders. However, a secondary analysis revealed that, among patients with non-serous-papillary subtypes of OvCa, statin use was associated with a decrease in hazards of both disease recurrence (adjusted HR = 0.23, p = 0.02) and disease-specific death (adjusted HR = 0.23, p = 0.04). To augment the findings in the retrospective cohort, the histology-specific effects of statins were also evaluated in vitro using proliferation assays. Here, statin treatment of cell lines resulted in a variable level of cytotoxicity. Conclusion Statin use among patients with non-serous-papillary OvCa was associated with improvement in both PFS and DSS.

Dehydroepiandrosterone Activation of G-protein-coupled Estrogen Receptor Rapidly Stimulates MicroRNA-21 Transcription in Human Hepatocellular Carcinoma Cells

Background: MicroRNA-21 (miR-21) is an oncomiR in human hepatocellular carcinoma and is highly expressed in liver, but its regulation is uncharacterized. Results: Dehydroepiandrosterone (DHEA) rapidly increases miR-21 transcription in HepG2 cells by activating G-protein-coupled estrogen receptor (GPER). Conclusion: miR-21 transcription is regulated by DHEA through GPER. Significance: GPER may be among the activators of miR-21 expression in human hepatocellular carcinoma. Little is known about the regulation of the oncomiR miR-21 in liver. Dehydroepiandrosterone (DHEA) regulates gene expression as a ligand for a G-protein-coupled receptor and as a precursor for steroids that activate nuclear receptor signaling. We report that 10 nm DHEA increases primary miR-21 (pri-miR-21) transcription and mature miR-21 expression in HepG2 cells in a biphasic manner with an initial peak at 1 h followed by a second, sustained response from 3–12 h. DHEA also increased miR-21 in primary human hepatocytes and Hep3B cells. siRNA, antibody, and inhibitor studies suggest that the rapid DHEA-mediated increase in miR-21 involves a G-protein-coupled estrogen receptor (GPER/GPR30), estrogen receptor α-36 (ERα36), epidermal growth factor receptor-dependent, pertussis toxin-sensitive pathway requiring activation of c-Src, ERK1/2, and PI3K. GPER antagonist G-15 attenuated DHEA- and BSA-conjugated DHEA-stimulated pri-miR-21 transcription. Like DHEA, GPER agonists G-1 and fulvestrant increased pri-miR-21 in a GPER- and ERα36-dependent manner. DHEA, like G-1, increased GPER and ERα36 mRNA and protein levels. DHEA increased ERK1/2 and c-Src phosphorylation in a GPER-responsive manner. DHEA increased c-Jun, but not c-Fos, protein expression after 2 h. DHEA increased androgen receptor, c-Fos, and c-Jun recruitment to the miR-21 promoter. These results suggest that physiological concentrations of DHEA activate a GPER intracellular signaling cascade that increases pri-miR-21 transcription mediated at least in part by AP-1 and androgen receptor miR-21 promoter interaction.

Abstract POSTER-BIOL-1305: Lovastatin decreases macrophage IL-6 and CXCL1 production in an ovarian tam-like model reducing ovarian cancer cell invasion

Introduction: Accumulating epidemiologic evidence suggests that cancer patients taking statins for elevated cholesterol and have improved cancer survival. In vitro studies have also shown that invasion, proliferation and cell growth are inhibited by statin treatment in several cancer cell lines. Here, we report that lovastatin inhibits of ovarian cancer cell proliferation and invasion towards ovarian tumor associated macrophages (TAM). Methods: Ovarian cancer cells (ID8, HeyA8, SKOV3ip1, and OVCAR5) were grown to confluency and treated with varying dosages of lovastatin (10 - 40 uM). Proliferation was measured using an MTT assay at 24, 48, and 72 hours after treatment in both complete DMEM media. Invasion was measured as the average number of cancer cells that invaded collagen (I)-coated wells. Lovastatin (20 uM for 16 hours) greatly reduced invasion of all ovarian cancer cell types. THP-1 macrophages were exposed to serum-free media from ovarian cancer cells to induce a pro-tumor phenotype. After 24 hours, the ability of the conditioned macrophages to induce both human and murine ovarian cancer cell invasion was assessed. A chemokine array was performed on media from macrophages to determine which chemokines were altered during the transformation from naive to a TAM-like phenotype, and what effect lovastatin had on those chemokines. The results of the array was confirmed with ELISA for candidate markers. Results: Lovastatin produced a dose-dependent decrease in cell proliferation in HeyA8, OVCAR5, SKOV3ip1 and ID8 cancer cell lines. Lovastatin inhibited OVCAR5, HeyA8, and SKOV3ip1 cell invasion with exposure to 20 uM concentrations for 24 hours before and during an overnight invasion assay. When macrophages (THP-1) were incubated in ovarian cancer cell conditioned media, more invasion of HeyA8 cancer cells was observed compared to either non-stimulated (naive) macrophages or HeyA8 conditioned media alone; indicating that it was the presence of macrophages with conditioned media that stimulated invasion. Invasion of cancer cells towards the stimulated macrophages was significantly reduced when the macrophages were treated with low-dose lovastatin (10 uM for 16 hours). To understand the mechanisms by which stimulated macrophages promote cancer cell invasion chemokine arrays were performed. The results showed that IL-6 and CXCL1 were up regulated during TAM transformation and reduced with lovastatin therapy. The results observed with regard to IL-6 were confirmed in an ELISA where lovastatin treatment produced a dose-dependent reduction in IL-6 production. Discussion: Our studies demonstrate two possible mechanisms by which lovastatin might have a protective effect in ovarian cancer: a) by reducing cancer cell proliferation and b) by interrupting cancer cell invasion promoted by tumor associated macrophages. The second finding is particularly provocative as the primary site of OvCa metastasis is the omentum, a site rich in macrophages that may be facilitating tumor seeding and growth. This study raisers the question of whether statins could be repurposed as a cancer therapeutic targeting the tumor-promoting effects of macrophages in the tumor microenvironment. Citation Format: Michael J Bradaric, PhD; Lacey Litchfield, PhD; Alyssa Johnson, BS; Mohammed Habis, MD; Nadia Ismail, BS; Ernst Lengyel, MD, PhD; Iris Romero, MD. Lovastatin decreases macrophage IL-6 and CXCL1 production in an ovarian tam-like model reducing ovarian cancer cell invasion [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1305.