Payal Tiwari

MicroRNAs Reprogram Normal Fibroblasts into Cancer-Associated Fibroblasts in Ovarian Cancer

UNLABELLED Cancer-associated fibroblasts (CAF) are a major constituent of the tumor stroma, but little is known about how cancer cells transform normal fibroblasts into CAFs. microRNAs (miRNA) are small noncoding RNA molecules that negatively regulate gene expression at a posttranscriptional level. Although it is clearly established that miRNAs are deregulated in human cancers, it is not known whether miRNA expression in resident fibroblasts is affected by their interaction with cancer cells. We found that in ovarian CAFs, miR-31 and miR-214 were downregulated, whereas miR-155 was upregulated when compared with normal or tumor-adjacent fibroblasts. Mimicking this deregulation by transfecting miRNAs and miRNA inhibitors induced a functional conversion of normal fibroblasts into CAFs, and the reverse experiment resulted in the reversion of CAFs into normal fibroblasts. The miRNA-reprogrammed normal fibroblasts and patient-derived CAFs shared a large number of upregulated genes highly enriched in chemokines, which are known to be important for CAF function. The most highly upregulated chemokine, CCL5, (C-C motif ligand 5) was found to be a direct target of miR-214. These results indicate that ovarian cancer cells reprogram fibroblasts to become CAFs through the action of miRNAs. Targeting these miRNAs in stromal cells could have therapeutic benefit. SIGNIFICANCE The mechanism by which quiescent fibroblasts are converted into CAFs is unclear. The present study identifies a set of 3 miRNAs that reprogram normal fibroblasts to CAFs. These miRNAs may represent novel therapeutic targets in the tumor microenvironment.

Ligand independent activation of c-Met by fibronectin and α5β1-integrin regulates ovarian cancer invasion and metastasis

The role of the fibronectin receptor, α5β1-integrin, as an adhesion receptor and in angiogenesis is well established. However, its role in cancer cell invasion and metastasis is less clear. We describe a novel mechanism by which fibronectin regulates ovarian cancer cell signaling and promotes metastasis. Fibronectin binding to α5β1-integrin led to a direct association of α5-integrin with the receptor tyrosine kinase, c-Met, activating it in a hepatocyte growth factor/scatter factor (HGF/SF) independent manner. Subsequently, c-Met associated with Src, and activated Src and focal adhesion kinase (FAK). Inhibition of α5β1-integrin decreased the phosphorylation of c-Met, FAK and Src, both in vitro and in vivo. Independent activation of c-Met by its native ligand, HGF/SF, or overexpression of a constitutively active FAK in HeyA8 cells could overcome the effect of α5β1-integrin inhibition on tumor cell invasion, indicating that α5β1-integrin is upstream of c-Met, Src and FAK. Inhibition of α5β1-integrin on cancer cells in two xenograft models of ovarian cancer metastasis resulted in a significant decrease of tumor burden, which was independent of the effect of α5β1-integrin on angiogenesis. These data suggest that fibronectin promotes ovarian cancer invasion and metastasis through an α5β1-integrin/c-Met/FAK/Src-dependent signaling pathway, transducing signals through c-Met in an HGF/SF-independent manner.

Microenvironment-induced downregulation of miR-193b drives ovarian cancer metastasis.

The cross-talk between ovarian cancer (OvCa) cells and the metastatic microenvironment is an essential determinant of successful colonization. MicroRNAs (miRNAs) have several critical roles during metastasis; however, the role of microenvironmental cues in the regulation of miRNAs in metastasizing cancer cells has not been studied. Using a three-dimensional culture model that mimics the human omentum, one of the principal sites of OvCa metastasis, we identified and characterized the microenvironment-induced downregulation of a tumor suppressor miRNA, miR-193b, in metastasizing OvCa cells. The direct interaction of the OvCa cells with mesothelial cells, which cover the surface of the omentum, caused a DNA methyltransferase 1-mediated decrease in the expression of miR-193b in the cancer cells. The reduction in miR-193b enabled the metastasizing cancer cells to invade and proliferate into human omental pieces ex vivo and into the omentum of a mouse xenograft model of OvCa metastasis. The functional effects of miR-193b were mediated, in large part, by the concomitant increased expression of its target, urokinase-type plasminogen activator, a known tumor-associated protease. These findings link paracrine signals from the microenvironment to the regulation of a key miRNA in cancer cells. Targeting miR-193b, which is essential for metastatic colonization of cancer cells could prove effective in the treatment of OvCa metastasis.

Metabolically activated macrophages in mammary adipose tissue link obesity to triple-negative breast cancer

Obesity is associated with increased incidence and severity of triple-negative breast cancer (TNBC); however, mechanisms underlying this relationship are incompletely understood. Macrophages, which accumulate in adipose tissue and are activated during obesity, are an attractive mechanistic link. Here, we show that, during obesity, murine and human mammary adipose tissue macrophages adopt a pro-inflammatory, metabolically- activated (MMe) macrophage phenotype that promotes TNBC stem-like markers and functions, including increased tumorsphere growth in vitro and tumor-initiating potential in vivo. We demonstrate that MMe macrophages release cytokines in an NADPH oxidase 2 (NOX2)-dependent manner that signal through glycoprotein 130 (GP130) on TNBC cells to promote their stem-like properties. Accordingly, deleting Nox2 in myeloid cells or depleting GP130 in TNBC cells attenuates the ability of obesity to drive TNBC tumor formation. Our studies implicate MMe macrophage accumulation in mammary adipose tissue during obesity as a mechanism for promoting TNBC stemness and tumorigenesis. HIGHLIGHTS ⁘ Obesity promotes TNBC tumor formation and stemness. ⁘ Mammary adipose tissue macrophages are metabolically activated (MMe) in obese mice and humans. ⁘ MMe macrophages in mammary adipose tissue contribute to obesity-induced stemness. ⁘ MMe macrophages promote TNBC stemness through GP130 signaling.

Abstract AS23: Microenvironment mediated downregulation of miR-193b promotes ovarian cancer metastasis

The cross-talk between ovarian cancer cells with the microenvironment of the site of metastasis is an essential determinant of successful metastatic colonization. microRNAs have been well established to play critical roles in various stages of cancer progression, including metastasis. However, the role of the signals from the microenvironment in regulating key microRNAs in metastasizing cancer cells has not been studied. Using a 3D culture model mimicking the human omentum, one of the principal sites of ovarian cancer metastasis, we have identified the microenvironment-induced downregulation of a tumor suppressor microRNA miR-193b in the metastasizing ovarian cancer cells. The direct interaction of the ovarian cancer cells with the mesothelial cells covering the surface of the omentum caused DNA methyltransferase 1 (DNMT1) mediated methylation and decreased expression of miR-193b. The reduction in miR-193b enabled the metastasizing cancer cells to invade and proliferate in the omentum both in vitro and in a mouse xenograft model of ovarian cancer metastasis. The functional effects of miR-193b were mediated at least partly through the concomitant increased expression of its target urokinase-type plasminogen activator (uPA). Our findings link paracrine signals from the microenvironment with the regulation of a key microRNA in cancer cells that is essential for the initial steps of ovarian cancer metastatic colonization. Targeting miR-193b would be a promising approach to treat ovarian cancer metastasis. Citation Format: AK Mitra, CY Chiang, P Tiwari, ME Peter, E Lengyel. Microenvironment mediated downregulation of miR-193b promotes ovarian cancer metastasis [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 AS23.

Abstract 2888: Metabolically activated macrophages in obesity associated TNBC

Triple negative breast cancer (TNBC) accounts for about 20% of all breast cancers. TNBC patients have an extremely poor prognosis due to their high metastatic potential and lack of targeted drug therapies. Emerging epidemiological data suggest that obesity is strongly linked to the incidence and severity of TNBC; obese women have a 35% higher risk of developing TNBC and 46% higher risk of developing distant metastases. Thus, understanding the biological processes that link obesity and TNBC has important clinical applications for prognosis and treatment. Mechanisms by which obesity worsens TNBC prognosis are incompletely understood. One clue to its action is that obesity causes chronic inflammation, and macrophage infiltration into adipose tissue is a key mediator of this inflammation. Recent studies demonstrated that macrophages are enriched in breast adipose tissue of obese humans and mice. Moreover, depletion of macrophages in mice diminished the effects of obesity on TNBC growth and metastasis. Although anti-inflammatory ‘M2-like’ TAMs are key effector cells that promote tumorigenesis, it is well accepted that obesity stimulates macrophages to adopt a pro-inflammatory ‘M1-like’ state. However, M1 macrophages exhibit potent anti-tumor functions. From these discordant observations an important paradox emerges: How can obesity promote breast cancer if it elicits an M1, anti-tumor macrophage phenotype? Using a combination of proteomics, immunology, and cell biology, we recently identified a novel metabolically activated (MMe) macrophage phenotype produced by exposure to high levels of insulin, glucose, and palmitate, conditions characteristic of obese and diabetic patients. Here we show that gene expression patterns in MMe (but not M1) macrophages are strongly associated with pathways involved in breast cancer. Moreover, pre-treating BM1 cells with conditioned media derived from MMe macrophages promotes TNBC cell invasion by 2.3 fold, suggesting that MMe macrophages potentiate metastasis. We further show that breast adipose tissue macrophages isolated from obese women express cell surface markers of MMe (CD36, ABCA1), but not M1 (CD38, CD319, CD274), macrophages. Furthermore, treating naive macrophages with media conditioned by human mammary adipose tissue from an obese subject (BMI = 37), but not a lean subject (BMI = 19), induced genes diagnostic of the MMe phenotype. Thus, mammary adipose tissue from obese women supports metabolic activation of macrophages. Together, these observations suggest that obesity-induced changes to adipose tissue reprogram macrophages to an MMe phenotype that potentiates TNBC. A comprehensive understanding of signaling mechanisms involved in metabolic activation would enable development of directed therapies towards this specific pro-tumorigenic macrophage phenotype, thereby leaving the immune system of cancer patients intact. Citation Format: Payal Tiwari, Kelly Schoenfelt, Swati Kulkarni, Marsha Rosner, Lev Becker. Metabolically activated macrophages in obesity associated TNBC. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2888. doi:10.1158/1538-7445.AM2015-2888

Abstract 1481: microRNA mediated reprograming of fibroblasts into cancer associated fibroblasts in ovarian cancer

Cancer associated fibroblasts (CAFs) are a major constituent of the tumor stroma and promote the invasion and growth of cancer cells. However, the mechanism by which cancer cells transform normal fibroblasts into CAFs is unclear. While it is clearly established that microRNAs are deregulated in cancer and are involved in cellular reprograming, it is not known if they play a role in the transformation of normal fibroblasts into CAFs. Therefore, we investigated the novel possibility of microRNA mediated reprogramming of normal fibroblasts into CAFs. Primary CAFs extracted from the metastasis of patients with ovarian cancer and normal omental fibroblasts (NOFs) extracted from non-cancer patients were profiled for microRNA expression. The functional role of microRNAs in CAFs was established by assessing their ability to promote ovarian cancer co-invasion, colony formation and tumor growth in mice. The microRNA targets were identified using gene array analysis, validated by 3′UTR luciferase reporter assay, and functionally verified both in vitro and in mouse xenografts. Comparing CAFs and NOFs we found that, in ovarian cancer CAFs miR-31 and miR-214 were downregulated while miR-155 was upregulated as compared to normal or tumor-adjacent fibroblasts. Mimicking this deregulation by transfecting microRNAs and microRNA inhibitors induced a phenotypic and functional conversion of normal fibroblasts into CAFs, and the reverse experiment resulted in the reprograming of CAFs into normal fibroblasts. The microRNA induced CAFs and patient-derived CAFs shared multiple upregulated genes highly enriched in chemokines important for CAF function. The most highly upregulated chemokine, CCL5/RANTES, was found to be a direct target of miR-214. CCL5 was functionally important for both CAFs and normal fibroblasts reprogrammed into CAFs, in vitro and in mouse xenografts. These results indicate that tumor cells can reprogram fibroblasts to become CAFs through the regulation of their microRNAs, suggesting that targeting microRNA in stromal cells, as well as in tumor cells, could be beneficial for cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1481. doi:1538-7445.AM2012-1481