Cancer-associated Adipocytes Research Articles

Abstract A45: Obesity-induced inflammation and desmoplasia promote pancreatic cancer progression and resistance to chemotherapy

Abstract Introduction: With the current epidemic of obesity, the majority of pancreatic cancer patients are overweight or obese at diagnosis. Importantly, obesity worsens treatment outcomes in pancreatic cancer patients. Therefore, understanding the mechanisms that underlie the poorer prognosis of obese cancer patients is of paramount importance. Obesity causes inflammation and fibrosis in the normal pancreas due to the accumulation of dysfunctional hypertrophic adipocytes. Importantly, desmoplasia—a fibro-inflammatory microenvironment—is a hallmark of pancreatic ductal adenocarcinoma (PDAC), and we have shown that activation of pancreatic stellate cells (PSCs) via angiotensin-II type 1 receptor (AT1) pathway is a major contribution to tumor desmoplasia. Whether obesity affects inflammation, PSCs and desmoplasia in PDACs, and interferes with delivery and response of chemotherapeutics is currently unknown. Experimental Design: Using mouse models of PDAC—multiple syngeneic models of PDAC: PAN02, AK4.4, KPC, iKRAS in diet-induced and genetic obese mouse models—we determined the effects of obesity on desmoplasia and inflammation, tumor growth and delivery and response to chemotherapy. We further evaluated whether the obesity-induced effects were mediated by AT1 signaling as well as via immune cell recruitment, and dissected the crosstalk between PSCs, cancer-associated adipocytes (CAAs), and tumor-associated neutrophils (TANs). In addition, we determined if an anti-diabetic drug metformin could counter these effects in vivo, and further dissected the mechanism of action in vitro. Results: We found that obesity aggravates desmoplasia in PDACs in multiple mouse models. In addition, tumors in obese mice presented with elevated levels of activated PSCs and fibrosis, as well as inflammatory cytokines and TANs. These alterations in the tumor microenvironment in obesity associated with accelerated tumor growth, reduced tumor blood perfusion and increased hypoxia, and impaired delivery and efficacy of chemotherapeutics. Genetic ablation and pharmacological inhibition (losartan) of AT1 signaling reversed obesity-augmented desmoplasia and tumor growth, and improved the response to chemotherapy to the level observed in lean mice. We further discovered the underlying mechanisms: 1) obesity increases intra-tumor adipocytes and IL-1ß secretion by these cells; 2) increased IL-1ß induces TAN recruitment; 3) recruited TANs activate PSCs; and 4) activated PSCs enhance desmoplasia. Conversely, activated PSCs also secrete IL-1ß that recruits further TANs. Hence, inactivation of PSCs through AT1 blockade resulted in not only decreased fibrosis but also reduced IL-1ß level and TAN recruitment. Furthermore, reduction of either TANs, IL-1ß, or PSC activation reduced tumor growth in obese mice. These findings suggest that crosstalk between adipocytes, immune cells, and PSCs exacerbates desmoplasia and promotes tumor progression during obesity. Of clinical relevance, we found that metformin not only normalizes the abnormal systemic metabolism, but also alleviates the fibro-inflammatory microenvironment in pancreatic cancer in obesity/diabetes. This occurred via direct reprogramming of PSCs and immune cells by metformin. Importantly, the strategies described above were not effective in the normal weight setting. Conclusion: Here we successfully demonstrated that targeting desmoplasia, including immunomodulation with anti-IL-1ß, or treatment with generic drugs such as losartan and metformin are potential strategies to potentiate treatments in PDAC patients with excess weight. With a better understanding of the mechanisms by which obesity promotes tumor progression and therapy resistance, we will be able to improve the current standard of care in pancreatic cancer. Citation Format: Joao Incio, Hao Liu, Priya Suboj, Shan Min, Ivy Chen, Mei Ng, Hadi Nia, Jelena Grahovac, Shannon Kao, Suboj Babykutty, Yuhui Huang, Keehoon Jung, Nuh Rahbari, Xiaoxing Han, Vikash Chauhan, John Martin, Julia Kahn, Peigen Huang, Vikram Deshpande, James Michaelson, Cristina Ferrone, Raquel Soares, Yves Boucher, Dai Fukumura, Rakesh Jain. Obesity-induced inflammation and desmoplasia promote pancreatic cancer progression and resistance to chemotherapy. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr A45.

Obesity, Inflammation, and Cancer.

Obesity, a worldwide epidemic, confers increased risk for multiple serious conditions, including cancer, and is increasingly recognized as a growing cause of preventable cancer risk. Chronic inflammation, a well-known mediator of cancer, is a central characteristic of obesity, leading to many of its complications, and obesity-induced inflammation confers additional cancer risk beyond obesity itself. Multiple mechanisms facilitate this strong association between cancer and obesity. Adipose tissue is an important endocrine organ, secreting several hormones, including leptin and adiponectin, and chemokines that can regulate tumor behavior, inflammation, and the tumor microenvironment. Excessive adipose expansion during obesity causes adipose dysfunction and inflammation to increase systemic levels of proinflammatory factors. Cells from adipose tissue, such as cancer-associated adipocytes and adipose-derived stem cells, enter the cancer microenvironment to enhance protumoral effects. Dysregulated metabolism that stems from obesity, including insulin resistance, hyperglycemia, and dyslipidemia, can further impact tumor growth and development. This review describes how adipose tissue becomes inflamed in obesity, summarizes ways these mechanisms impact cancer development, and discusses their role in four adipose-associated cancers that demonstrate elevated incidence or mortality in obesity.

Confocal fluorescence microscopy to evaluate changes in adipocytes in the tumor microenvironment associated with invasive ductal carcinoma and ductal carcinoma in situ

Adipose tissue is a dynamic organ that provides endocrine, inflammatory and angiogenic factors, which can assist breast carcinoma cells with invasion and metastasis. Previous studies have shown that adipocytes adjacent to carcinoma, known as cancer-associated adipocytes, undergo extensive changes that correspond to an "activated phenotype," such as reduced size relative to adipocytes in non-neoplastic breast tissue. Optical imaging provides a tool that can be used to characterize adipocyte morphology and other features of the tumor microenvironment. In this study, we used confocal fluorescence microscopy to acquire images of freshly excised breast tissue stained topically with proflavine. We developed a computerized algorithm to identify and quantitatively measure phenotypic properties of adipocytes located adjacent to and far from normal collagen, ductal carcinoma in situ and invasive ductal carcinoma. Adipocytes were measured in confocal fluorescence images of fresh breast tissue collected from 22 patients. Results show that adipocytes adjacent to neoplastic tissue margins have significantly smaller area compared to adipocytes far from the margins of neoplastic lesions and compared to adipocytes adjacent to non-neoplastic collagenous stroma. These findings suggest that confocal microscopic images can be utilized to evaluate phenotypic properties of adipocytes in breast stroma which may be useful in defining alterations in microenvironment that may aid in the development and progression of neoplastic lesions.

Exosomal transfer of stroma-derived miR21 confers paclitaxel resistance in ovarian cancer cells through targeting APAF1.

Advanced ovarian cancer usually spreads to the visceral adipose tissue of the omentum. However, the omental stromal cell-derived molecular determinants that modulate ovarian cancer growth have not been characterized. Here, using next-generation sequencing technology, we identify significantly higher levels of microRNA-21 (miR21) isomiRNAs in exosomes and tissue lysates isolated from cancer-associated adipocytes (CAAs) and fibroblasts (CAFs) than in those from ovarian cancer cells. Functional studies reveal that miR21 is transferred from CAAs or CAFs to the cancer cells, where it suppresses ovarian cancer apoptosis and confers chemoresistance by binding to its direct novel target, APAF1. These data suggest that the malignant phenotype of metastatic ovarian cancer cells can be altered by miR21 delivered by exosomes derived from neighbouring stromal cells in the omental tumour microenvironment, and that inhibiting the transfer of stromal-derived miR21 is an alternative modality in the treatment of metastatic and recurrent ovarian cancer.

Abstract P1-03-06: Leptin as a mediator of tumor-stromal interactions promotes breast cancer stem cell activity

Abstract Breast cancer stem cells (BCSCs) play crucial roles in tumor initiation, metastasis and resistance to anticancer therapies. These cells rely for their properties on complex interactions with the tumor microenvironment through networks of cytokines and growth factors. In this study, we investigated how leptin, as a mediator of tumor-stromal interactions, may affect BCSC activity using breast cancer cell lines and patient-derived samples. We found that conditioned media (CM) from cancer associated fibroblasts and breast adipocytes significantly increase mammosphere formation in breast cancer cells. Depletion of leptin from stromal cell-CM as well as inhibition of leptin signaling by using a full leptin receptor antagonist peptide LDFI completely abrogated this effect. Accordingly, mammosphere cultures exhibited increased leptin receptor expression and leptin exposure enhanced mammosphere formation. Microarray analyses revealed a similar expression profile of genes involved in stem cell biology in mammosphere cells treated with stromal cell-CM and leptin. Interestingly, leptin is able to increase the mammosphere formation in metastatic breast cancer cells isolated from patients (n = 10) and this can be blocked by using peptide LDFI. In addition, leptin receptor (OBR) mRNA expression, analyzed in cells from metastatic fluids, directly correlated with mammosphere formation activity ex vivo (r=0.68, p= 0.05; n = 8). Finally, Kaplan–Meier survival curves indicated that OBR expression correlated with reduced overall survival in breast carcinomas (HR=1.9, p=0.022). Together, our results suggest that leptin/leptin receptor may represent a potential therapeutic target that can block the stromal-tumor interactions that drive BCSC-mediated disease progression. Citation Format: Giordano C, Panza S, Chemi F, Barone I, Bonofiglio D, Cordella A, Hashim A, Györffy B, Simões BM, Clarke RB, Weisz A, Catalano S, Andò S. Leptin as a mediator of tumor-stromal interactions promotes breast cancer stem cell activity. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P1-03-06.

Abstract 3372: Molecular characteristics of cancer-associated adipocytes in breast cancer

Abstract Adipose tissue in the tumor microenvironment is now regarded to be an active player in cancer development and progression. Recent studies have suggested critical roles of the cancer-associated adipocytes in breast cancer proliferation and invasion. However, unlike the well-known cancer-associated fibroblasts, the molecular profiles of cancer-associated adipocytes are largely unknown. We studied the histo-morphologic changes in adipose tissue adjacent to the breast cancer cells in human tissues. Adipocytes had smaller fat droplets and often associated with immune cell infiltration. We investigated the changes of mRNA expression profiles of differentiated adipocytes when they were cultured with breast cancer cells (MCF-7 and MDA-MB-231) by using the NIH-3T3-L1 cell line. Indirect coculture with cancer cells resulted in significantly altered expression of genes involved in immune response, extracellular matrix remodeling, angiogenesis, metabolism, and apoptosis. IL6 and its downstream genes, transcription factors such as RUNX1, as well as PTX3 and TIMP1 were most unregulated genes. Increased secretion of IL6 was further validated in the culture media by ELISA. Also, the gene expression data showed that adipocytes cocultured with cancer cells showed decreased adipocyte-differentiation genes suggesting the cells underwent de-differentiation process. Microscopic examination of co-cultured adipocytes showed smaller fat droplets similar to human specimens. The co-cultured adipocytes showed different effect on cancer cells according to the molecular subtypes of the breast cancer cells. The conditioned media from co-cultured adipocytes resulted in increased proliferation of ER+ breast cancer cells, suggesting a subtype-dependent effect of co-cultured adipocytes in breast cancer. Our results suggest that cancer-associated adipocytes show increased expression of genes involved in immune response, extracellular matrix remodeling, angiogenesis, metabolism, and apoptosis. Futhermore, these cancer-associated adipocytes show features toward the preadipocyte state suggesting the possibility of de-differentiation of adipocyte under the influence of adjacent cancer cells. The functional impact of the cancer-associated adipocyte seem to vary according to the molecular subtype of epithelial cancer cells. Citation Format: Hyeong-Gon Moon, Jiwoo Lee, Han-Byoel Lee, Han Suk Ryu, Wonshik Han, Dong-Young Noh. Molecular characteristics of cancer-associated adipocytes in breast cancer. [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 3372. doi:10.1158/1538-7445.AM2015-3372

Cancer-mediated adipose reversion promotes cancer cell migration via IL-6 and MCP-1.

The objective of this study is to investigate interactions between adipocytes and breast cancer cells, and identify the responsible factors for the observed effects. In 27 breast cancer patients undergoing mastectomy, mammary adipose tissue was obtained from the breast quadrant bearing the tumor and corresponding non-tumoral quadrant. Isolated normal breast adipocytes (NBAs) and cancer-associated adipocytes (CAAs) were cultured in collagen gels to mimic the in vivo environment. Immunohistochemistry, qRT-PCR, and cell proliferation assays were performed to analyze adipocyte phenotypes. MCF7 and MDA-MB-231 breast cancer cell lines were co-cultured with adipocytes to detect phenotypic changes. Migration of MCF7 and MDA-MB-231 cells was assessed in NBA- and CAA-conditioned media. Cytokine levels in conditioned media were measured by cytokine array. Migration assays were repeated using conditioned media containing neutralizing antibodies. NBAs and CAAs lost their morphological phenotype in culture, acquiring a spindle-like shape, and CAAs showed higher cell proliferation, suggesting reversion to an immature phenotype. In co-cultures with MCF7 or MDA-MB-231 cells, NBAs exhibited increased cell proliferation, indicating acquisition of the immature phenotype of CAAs. MCF7 and MDA-MB-231 showed higher migration in a CAA-conditioned medium than in an NBA-conditioned medium. Cytokine array analysis of conditioned media revealed higher levels of interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) in the CAA-conditioned medium. Neutralization experiments using antibodies against IL-6 or MCP-1 showed abrogation of migration-enhancing effects of the CAA-conditioned medium. Adipocytes revert to an immature and proliferative phenotype in the presence of breast cancer cells, and promote cancer cell migration via adipokines including IL-6 and MCP-1.

JNK implication in adipocyte-like cell death induced by chemotherapeutic drug cisplatin.

Recent evidence shows that tumor microenvironment containing heterogeneous cells may be involved in cancer initiation, growth and tumor cell response to anticancer therapy. Chemotherapy was designed to make toxic impact on malicious cells in organisms, however, the means to protect healthy cells against chemical toxicity are still unsuccessful. As known, the majority of tumor surrounding cells are cancer-associated adipocytes which influence cancer development, progression and treatment. Targeting the components of tumor microenvironment in combination with conventional cancer treatment may become an effective cancer therapy strategy. However, little is known about adipocyte death mechanisms during combined chemo- and targeted therapy. The importance of c-Jun-NH<inf>2</inf>-terminal kinase (JNK) signaling in tumor development and treatment has been demonstrated using various in vitro and in vivo cancer models. The aim of this study was to ascertain adipocyte viability during simultaneous stress kinase JNK inhibition and exposure to one of the most commonly used anticancer drugs cis-diamminedichloroplatinum II (cisplatin). Our model involved adipocyte-like cells (ADC) which were obtained during in vitro differentiation of adult rabbit muscle-derived stem cells. Cisplatin induced apoptotic cell death. During 24-hr cisplatin treatment gradual, strong and prolonged increase of both JNK and its target protein c-Jun phosphorylation was found in ADC. Pre-treatment of cells with SP600125 decreased cisplatin-induced activation of c-Jun and promoted apoptosis. Upregulation of proapoptotic Bax and downregulation of antiapoptotic Bcl-2 proteins were found to be regulated in JNK-dependent manner. Thus, the results prove the antiapoptotic role of activated JNK in adipocyte-like cells treated with cisplatin.

Abstract 3613: Opposite effects of adipocytes and adenosine on hematological cancer cell survival and proliferation in vitro

Abstract Background: Tumor microenvironment is composed of stroma that contains adipocytes. It is well established that obesity is a cancer risk factor and is associated with a poor prognosis in cancer patients. Cancer-associated adipocytes are thought to interact with cancer cells to promote tumor progression. Moreover, adipocytes express CD73 and can produce adenosine which also has been shown to promote cancer development through its interaction with adenosine receptors on solid cancer cells. As hematological malignancies can proliferate in bone marrow which is rich in adipocytes, we hypothesized that these latter could influence cancer cell survival and proliferation through the action of adenosine. Methods: CD73 enzymatic activity was determined on human pre-adipocyte cells (hMAD, ASC) by the Malachite Green Assay. The expression of adenosine receptor was assessed on human T-acute lymphoblastic leukemia cells (Jurkat) and follicular lymphoma cells (RL) by FACS analysis. Cancer cell proliferation and percentage of cell death were evaluated by FACS using CFSE staining and DAPI respectively. Survival and proliferation was evaluated in the absence or presence of human pre-adipocytes (hMAD, ASC) or adenosine with or without an adenosine deaminase inhibitor (deoxycoformycin 10 µM). Results: CD73 enzymatic activity was observed both in hMAD and ASC cells, while Jurkat and RL cells express the four adenosine receptors A1, A2A, A2B and A3. Hematological cancer cells tend to proliferate more when co-cultivated with pre-adipocytes (Jurkat 63,8% alone vs 69,2% with hMAD; RL 67,7% alone vs 73,9% with hMAD; Jurkat 62,4% alone vs 69,6% with ASC; RL 66,1% alone vs 74,8% with ASC). These cells also tend to have a lower rate of spontaneous cell death in same conditions (Jurkat 31,4% alone vs 23,6% with hMAD; RL 37,1% alone vs 30,1% with hMAD; Jurkat 32,7% alone vs 30,9% with ASC; RL 53,5% alone vs 36,0% with ASC). In contrast, we found that hematological cancer cells proliferate less in the presence of adenosine in a dose-dependent manner starting from 200 µM in media containing 1% serum (Jurkat 71,0% alone vs 68,6% with 200 µM adenosine and 47,6% with 200 µM adenosine + deoxycoformycin; RL 79,0% alone vs 73,3% with 200 µM adenosine and 59,8% with 200 µM adenosine + deoxycoformycin). Finally, adenosine also induced more cell death in these cells (Jurkat 28,6% alone vs 41,2% with 200 µM adenosine and 57,4% with 200 µM adenosine + deoxycoformycin; RL: 28,2% alone vs 47,9% with 200 µM adenosine + deoxycoformycin). Conclusion: Adipose cells have pro-proliferative and protective effects on hematological cancer cells in vitro while acute exposure to adenosine at supraphysiological concentrations has a cytotoxic effect. These results do not suggest that adipocytes favor proliferation through adenosine signaling. Citation Format: Cécile Bossard, Charles Dumontet, Lars P. Jordheim. Opposite effects of adipocytes and adenosine on hematological cancer cell survival and proliferation in vitro. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3613. doi:10.1158/1538-7445.AM2014-3613

Adipose tissue and cancer: a high risk tandem

Adipose tissue is found in close proximity whith many invasive cancers. In breast cancer, early local tumour invasion results in close interactions of cancer cells with fully differentiated adipocytes. Aside from their energy-storing function, mature adipocytes are also active endocrine cells prone to influence tumour behaviour through heterotypic signaling processes. After a short description of anatomical depots specificities of adipose tissue, we describe the phenotypic changes induced by tumor secretion in tumour-surrounding adipocytes. These cells (that we named CAA for cancer-associated adipocytes) by their ability to secrete pro-inflammatory cytokines, extra-cellular matrix proteins and proteases involved in its remodeling, as well as to release free fatty acid, stimulate tumor proliferation, invasiveness and drug resistance. These results support the concept that adipocytes participate in a deleterious crosstalk with cancer cells to support tumour progression, that might be amplified in obesity conditions and explain the poor prognosis of cancers observed in this subset of patients.

PSS173 - Modulation of a Targeted Metabolome by Oxidative Stressors: Insights into the Underlying Mechanisms of Atherosclersosis

Adipose tissue influences tumor development in two major ways. First, obese individuals have a higher risk of developing certain cancers (endometrial, esophageal, and renal cell cancer). However, the risk of developing other cancers (melanoma, rectal, and ovarian) is not altered by body mass. In obesity, hypertrophied adipose tissue depots are characterized by a state of low grade inflammation. In this activated state, adipocytes and inflammatory cells secrete adipokines and cytokines which are known to promote tumor development. In addition, the adipocyte mediated conversion of androgens to estrogen specifically contributes to the development of endometrial cancer, which shows the greatest relative risk (6.3-fold) increase between lean and obese individuals. Second, many tumor types (gastric, breast, colon, renal, and ovarian) grow in the anatomical vicinity of adipose tissue. During their interaction with cancer cells, adipocytes dedifferentiate into pre-adipocytes or are reprogrammed into cancer-associated adipocytes (CAA). CAA secrete adipokines which stimulate the adhesion, migration, and invasion of tumor cells. Cancer cells and CAA also engage in a dynamic exchange of metabolites. Specifically, CAA release fatty acids through lipolysis which are then transferred to cancer cells and used for energy production through β-oxidation. The abundant availability of lipids from adipocytes in the tumor microenvironment, supports tumor progression and uncontrolled growth. Given that adipocytes are a major source of adipokines and energy for the cancer cell, understanding the mechanisms of metabolic symbiosis between cancer cells and adipocytes, should reveal new therapeutic possibilities. This article is part of a Special Issue entitled Lipid Metabolism in Cancer.

Abstract 512: p16INK4A down-regulation activates breast stromal adipocytes.

Abstract Obesity, a disease linked to increased number of adipocytes in various tissues, is now recognized as a risk factor for the development of breast cancer. Importantly, obesity has been reported to be associated with the occurrence of larger, more advanced tumors and aggressive cancer pathological features, including lymph node metastasis, advanced tumor stage, and high grade. Cancer-associated adipocytes (CAAs), the most abundant cellular component of breast cancer-associated stroma, promote carcinogenesis through paracrine effects. However, the molecular basis of adipocytes implication in breast carcinogenesis remains elusive. To shed light on this important phenomenon, we established and characterized normal adipocytes from post-plastic surgery of breast tissues. We have shown that these cells express various adipocyte biomarkers, including Leptin, TNF-α and FABP4. Furthermore, we investigated the role of the tumor suppressor p16 INK4A protein in adipocytes activation. We have found that specific down-regulation of p16INK4A by shRNA increased the expression/secretion levels of various adipokines including leptin, activated breast adipocyte and enhanced their migration/invasion abilities. Consequently, media conditioned with these cells stimulated the proliferation and the migration/invasion of breast cancer cells and enhanced endothelial cell differentiation into capillary-like structures. In addition, we have also shown that media conditioned with breast cancer cells decrease the expression of p16INK4A and increase the expression of leptin in adipocytes. These results provide the first indication that p16INK4A down-regulation in breast stromal adipocytes is an important step toward their activation. Citation Format: Huda H. Alkhalaf, Abdelilah Aboussekhra. p16INK4A down-regulation activates breast stromal adipocytes. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 512. doi:10.1158/1538-7445.AM2013-512

Tumour-surrounding adipocytes are active players in breast cancer progression

Accumulating recent evidence highlights the tumour-surrounding adipose tissue as a key component of breast cancer progression. We have recently demonstrated that a bidirectional crosstalk is established with breast cancer cells and tumour-surrounding adipocytes. Tumour cell secretions are able to modify tumour-surrounding adipocytes to an activated state that we have named Cancer-Associated Adipocytes (CAAs). The role of CAAs in breast cancer progression, as well as the potential amplification of this negative effect in obesity conditions will be discussed.

Abstract IA30: Metabolic symbiosis: The contribution of adipocytes to visceral metastasis

Abstract Many tumor types (gastric, breast, colon, renal, and ovarian) grow in the anatomical vicinity of adipose tissue and are known to reprogram adjacent normal adipocytes into cancer-associated adipocytes (CAA). Clinical observations indicate that the most common, as well as the largest, site of ovarian cancer (OvCa) metastasis is the omentum, a large fat pad (20x12x3cm) positioned in front of the bowel. The omentum, which is primarily composed of adipocytes, provides energy storage and functions as an endocrine organ. Because cancer cell behavior is highly dependent on the microenvironment, and adipocytes are a major source of energy-dense fatty acids, we hypothesized that adipocytes promote OvCa omental metastasis and growth via altered lipid metabolism. To investigate this hypothesis, primary human visceral adipocytes were cultured from omental tissue specimens collected from patients who underwent surgical procedures for benign conditions. The rate of β-oxidation was assessed by a radioisotopic assay using 3H-palmitate. Sections of human serous ovarian carcinomas and corresponding omental metastases were utilized for immunohistochemistry, RT-PCR, and a reverse-phase protein array. Homozygous fatty acid binding protein 4 (FAPB4)-null mice or wild-type (WT) animals were inoculated orthotopically or intraperitoneally with mouse OvCa cells. Adipocytes were found to promote the invasion, migration, and proliferation of OvCa cells. The homing of OvCa towards adipocytes in vitro, or to the mouse omentum in vivo, was reduced by using inhibitory antibodies against interleukin (IL)-6,IL-8, or their receptors. Co-culture of OvCa cells with adipocytes resulted in cytoplasmic lipid accumulation in OvCa cells, which was a consequence of direct lipid transfer from the adipocytes. Co-injection of SKOV3ip1 OvCa cells with adipocytes enhanced tumor growth in mice, when compared to the injection of SKOV3ip1 cells alone. Moreover, lipolysis was activated in co-cultured adipocytes as evidenced by the secretion of free fatty acids and glycerol. FABP4 was found to be highly expressed in omental metastatic tissues as compared to the respective primary OvCa tumor tissue. When mouse OvCa cells were injected under the ovarian bursa of FABP4 knock-out mice the number of metastases was significantly lower than in WT mice. Treatment with a FABP4 inhibitor also induced apoptosis and inhibited migration and invasion of OvCa cells. The fatty acid translocase (FAT/CD36) was identified as the fatty acid transporter responsible for lipid accumulation in cancer cells co-cultured with adipocytes. Our results suggest that adipocytes advance the progression of OvCa through two major mechanisms: (1) They promote metastasis to the omentum via the secretion of cytokines, and (2) They support cancer cell proliferation by activating lipolysis, thereby mobilizing energy-dense fatty acids which are utilized by the cancer cells in an energy-yielding β-oxidation pathway through a FABP4 and FAT/CD36 dependent mechanism. By better understanding these mechanisms and, more generally, the biology of adipocytes in the tumor microenvironment, we will be able to identify metabolic targets and, ultimately, introduce new compounds for the treatment of cancer. Citation Format: Kristin Nieman, Andras Ladanyi, Ernst Lengyel. Metabolic symbiosis: The contribution of adipocytes to visceral metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr IA30.

Diet, obesity, and cancer progression: are adipocytes the link?

Obesity has been linked to more aggressive characteristics of several cancers, including breast and prostate cancer. Adipose tissue appears to contribute to paracrine interactions in the tumor microenvironment. In particular, cancer-associated adipocytes interact reciprocally with cancer cells and influence cancer progression. Adipokines secreted from adipocytes likely form a key component of the paracrine signaling in the tumor microenvironment. In vitro coculture models allow for the assessment of specific adipokines in this interaction. Furthermore, micronutrients and macronutrients present in the diet may alter the secretion of adipokines from adipocytes. The effect of dietary fat and specific fatty acids on cancer progression in several in vivo model systems and cancer types is reviewed. The more common approaches of caloric restriction or diet-induced obesity in animal models establish that such dietary changes modulate tumor biology. This review seeks to explore available evidence regarding how diet may modulate tumor characteristics through changes in the role of adipocytes in the tumor microenvironment.

Mitochondrial metabolism in cancer metastasis

We have recently proposed a new two-compartment model for understanding the Warburg effect in tumor metabolism. In this model, glycolytic stromal cells produce mitochondrial fuels (L-lactate and ketone bodies) that are then transferred to oxidative epithelial cancer cells, driving OXPHOS and mitochondrial metabolism. Thus, stromal catabolism fuels anabolic tumor growth via energy transfer. We have termed this new cancer paradigm the “reverse Warburg effect,” because stromal cells undergo aerobic glycolysis, rather than tumor cells. To assess whether this mechanism also applies during cancer cell metastasis, we analyzed the bioenergetic status of breast cancer lymph node metastases, by employing a series of metabolic protein markers. For this purpose, we used MCT4 to identify glycolytic cells. Similarly, we used TO MM20 and COX staining as markers of mitochondrial mass and OXPHOS activity, respectively. Consistent with the “reverse Warburg effect,” our results indicate that metastatic breast cancer cells amplify oxidative mitochondrial metabolism (OXPHOS) and that adjacent stromal cells are glycolytic and lack detectable mitochondria. Glycolytic stromal cells included cancer-associated fibroblasts, adipocytes and inflammatory cells. Double labeling experiments with glycolytic (MCT4) and oxidative (TO MM20 or COX) markers directly shows that at least two different metabolic compartments co-exist, side-by-side, within primary tumors and their metastases. Since cancer-associated immune cells appeared glycolytic, this observation may also explain how inflammation literally “fuels” tumor progression and metastatic dissemination, by “feeding” mitochondrial metabolism in cancer cells. Finally, MCT4(+) and TO MM20(-) “glycolytic” cancer cells were rarely observed, indicating that the conventional “Warburg effect” does not frequently occur in cancer-positive lymph node metastases.

Abstract A27: MicroRNA 21 in adipocyte-derived microvesicles confers taxol-resistance of ovarian cancer cells through down-regulation of Apoptotic Protease Activating Factor 1

Abstract Over the past 30 years, there has been a dramatic increase in the incidence of obesity worldwide. As of 2010, approximately one-third of U.S. adults are obese, while another one-third of adults are overweight. Obesity is one of the risk factors for ovarian cancer. Greater than 75% of ovarian cancer patients present with advanced stage disease. Tumors preferentially spread to the omentum, a major site of abdominal fat, where ovarian cancer cells infiltrate omental adipose tissue. Omental adipocytes may interact directly with cancer cells to create a microenvironment niche that supports cancer growth. Recent studies have shown that cells can communicate through the exchange of bioactive molecules via microvesicles (exosomes and ectosomes). Microvesicles fuse with target cell membranes and transfer proteins, lipids, mRNAs and non-coding RNAs from donor to recipient cells. We hypothesize that adipocytes secrete microvesicles containing tumor-supporting factors that promote ovarian cancer progression. Ion Torrent RNA sequencing and subsequent qRT-PCR analyses were used to identify non-coding microvesicle RNAs secreted by primary cultures of cancer associated adipocytes (CAA), cancer associated fibroblasts (CAF), normal adipocytes, normal fibroblasts and ovarian cancer cells. Of the non-coding microvesicle RNAs identified, microRNA 21 (miR21) showed significantly higher expression in microvesicles secreted from CAA when compared to those from CAF and normal omental adipocytes, fibroblasts, and ovarian cancer cells. Using qRT-PCR of RNA isolated from microdissected frozen tissue and In-situ hybridization on FFPE tissue sections, CAA demonstrated significant higher miR21 expression levels compared to the adjacent cancer cells and normal adipocytes. MiR21 was up-regulated in metastatic ovarian cancer cells in the omentum as compared to those in the primary tumor site. Moreover, strong miR21 staining was observed in omental cancer cells particularly at the invasion front, suggesting that miR21 may be transferred from CAA to their neighboring ovarian cancer cells. To test this hypothesis, SKOV3 ovarian cancer cells were co-cultured with adipocytes transfected with miR21-FAM or incubated with fluorescently labeled adipocyte-derived microvesicles. Confocal microscopy confirmed the delivery of miR21 by adipocyte-derived microvesicles to cancer cells. Since miR21 expression in tumor cells has been linked with resistance to a variety of chemotherapeutic agents, we further delineated the direct effect of miR21 on the chemoresistance of ovarian cancer cell. The ovarian cancer cell lines, OVCA432 and SKOV3, transfected with miR21 precursor showed an increase in cell survival and a decrease in apoptosis in the presence of taxol. Furthermore, miR21 was significantly up-regulated in taxol-resistant HeyA8-MDR and SKOV3-TR cells as compared to the parental cell lines, HeyA8 and SKOV3, respectively. These data strongly suggest that miR21 may confer taxol resistance to ovarian cancer cells. Using microarray analysis of RNA isolated from SKOV3 cells transfected with the miR21 precursor, we identified a set of chemoresistance-related genes associated with miR21 expression. One of the most significantly down-regulated genes is Apoptotic Protease Activating Factor 1 (APAF1). By qRT-PCR, we confirmed that miR21 over-expression decreased APAF1 mRNA expression levels in OVCA432 and SKOV3 cells. A strong miR21 binding site is predicted on the 3′-UTR of APAF1, suggesting APAF1 is the direct target of miR21. In summary, up-regulation of miR21 in omental ovarian cancer cells is mediated through transport of miR21 in adipocyte-derived microvesicles from CAA in the omentum. Further, miR21 confers taxol-resistance in ovarian cancer cells by down-regulating APAF1 expression in ovarian cancer cells to decrease taxol-induced apoptosis. Citation Format: Ngai Na Co, Rosemarie Schmandt, Karen Lu, Samuel Mok. MicroRNA 21 in adipocyte-derived microvesicles confers taxol-resistance of ovarian cancer cells through down-regulation of Apoptotic Protease Activating Factor 1 [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A27.

Cancer-associated adipocytes promotes breast tumor radioresistance

Mature adipocytes are excellent candidates to influence tumor behavior through heterotypic signaling processes since these cells produce hormones, growth factors, cytokines and other molecules, a heterogeneous group of molecules named adipokines. Using a 2D coculture system, we demonstrate that breast tumor cells previously co-cultivated with mature adipocytes exhibit radioresistance and an earlier and higher increase in the effector kinase Chk1, a phenotype that was associated with decreased cell death as compared to tumor cells grown alone. Interestingly, the adipocytes-induced tumor changes taking place during the coculture time preceding the exposure to IR were sufficient to confer the radioresistant effect. Notorious among the changes brought by adipocytes was the significant increase of IL-6 expression in tumor cells, whose activity may well account for the observed tumor cell protection from IR toxicity. Indeed, our data confirmed the protective role of this cytokine as tumor cells incubated after irradiation with recombinant IL-6 exhibit an increased in Chk1 phosphorylation and a radioresistant phenotype, thus far recapitulating the effects observed in the presence of adipocytes. Our current study sheds light on a new role of tumor-surrounding adipocytes in fostering a radioresistant phenotype in breast tumors, a finding that might have important clinical implications in obese patients that frequently exhibit aggressive diseases.

Cancer-associated adipocytes exhibit an activated phenotype and contribute to breast cancer invasion.

Early local tumor invasion in breast cancer results in a likely encounter between cancer cells and mature adipocytes, but the role of these fat cells in tumor progression remains unclear. We show that murine and human tumor cells cocultivated with mature adipocytes exhibit increased invasive capacities in vitro and in vivo, using an original two-dimensional coculture system. Likewise, adipocytes cultivated with cancer cells also exhibit an altered phenotype in terms of delipidation and decreased adipocyte markers associated with the occurrence of an activated state characterized by overexpression of proteases, including matrix metalloproteinase-11, and proinflammatory cytokines [interleukin (IL)-6, IL-1β]. In the case of IL-6, we show that it plays a key role in the acquired proinvasive effect by tumor cells. Equally important, we confirm the presence of these modified adipocytes in human breast tumors by immunohistochemistry and quantitative PCR. Interestingly, the tumors of larger size and/or with lymph nodes involvement exhibit the higher levels of IL-6 in tumor surrounding adipocytes. Collectively, all our data provide in vitro and in vivo evidence that (i) invasive cancer cells dramatically impact surrounding adipocytes; (ii) peritumoral adipocytes exhibit a modified phenotype and specific biological features sufficient to be named cancer-associated adipocytes (CAA); and (iii) CAAs modify the cancer cell characteristics/phenotype leading to a more aggressive behavior. Our results strongly support the innovative concept that adipocytes participate in a highly complex vicious cycle orchestrated by cancer cells to promote tumor progression that might be amplified in obese patients.

Adipocyte is a non-trivial, dynamic partner of breast cancer cells

While the participation of adipocytes is well known in tissue architecture, energy supply and endocrine processes, their implication during natural cancer history is just beginning to unfold. An extensive review of the literature concerning the impact of resident adipocytes on breast cancer development/progression was performed. This review provides in vitro and in vivo evidence that adipocytes located close to invasive cancer cells, referred to as cancer-associated adipocytes (CAAs), are essential for breast tumor development/progression. Their deleterious function is dependent, at least partly, on their crosstalk with invasive cancer cells. Indeed, this event leads to dramatic phenotypic and/or functional modifications of both cell types. Adipocytes exhibit delipidation and acquire a fibroblast-like shape. In parallel, cancer cell aggressiveness is exacerbated through increased migratory and invasive properties. Moreover, obesity is currently a sign of poor prognosis in human carcinomas. In this context, a high number of "obese" resident adipocytes might be predicted to be detrimental. Accordingly, there are some similarities between the molecular alterations observed in hypertrophied adipocytes and in CAAs. How adipocytes function to favor tumorigenesis at the molecular level remains largely unknown. Nevertheless, progress has been made recently and molecular clues are starting to emerge. Deciphering the cellular and molecular mechanisms behind the adipocyte-cancer cell heterotypic crosstalk is of great interest since it might provide new targets for improving diagnosis/prognosis and for the design of innovative therapeutic strategies. They might also improve our understanding of the relationship between obesity/metabolic disorders and cancer risk and/or poor patient outcome.