Cancer-associated Adipocytes Research Articles

Cancer-associated adipocyte-derived G-CSF promotes breast cancer malignancy via Stat3 signaling

Adipocyte is the most predominant cell type in the tumor microenvironment of breast cancer and plays a pivotal role in cancer progression, yet the underlying mechanisms and functional mediators remain elusive. We isolated primary preadipocytes from mammary fat pads of human breast cancer patients and generated mature adipocytes and cancer-associated adipocytes (CAAs) in vitro. The CAAs exhibited significantly different gene expression profiles as assessed by transcriptome sequencing. One of the highly expressed genes in CAAs is granulocyte colony-stimulating factor (G-CSF). Treatment with recombinant human G-CSF protein or stable expression of human G-CSF in triple-negative breast cancer (TNBC) cell lines enhanced epithelial–mesenchymal transition, migration, and invasion of cancer cells, by activating Stat3. Accordantly, targeting G-CSF/Stat3 signaling with G-CSF-neutralizing antibody, a chemical inhibitor, or siRNAs for Stat3 could all abrogate CAA- or G-CSF-induced migration and invasion of breast cancer cells. The pro-invasive genes MMP2 and MMP9 were identified as target genes of G-CSF in TNBC cells. Furthermore, in human breast cancer tissues, elevated G-CSF expression in adipocytes is well correlated with activated Stat3 signal in cancer cells. Together, our results suggest a novel strategy to intervene with invasive breast cancers by targeting CAA-derived G-CSF.

DT-13 inhibits breast cancer cell migration via non-muscle myosin II-A regulation in tumor microenvironment synchronized adaptations.

Tumor metastasis is a terrifying characteristic of cancer. Numerous studies have been conducted to overcome metastasis by targeting tumor microenvironment (TME). However, due to complexity of tumor microenvironment, it remained difficult for accurate targeting. Dwarf-lillytruf tuber monomer-13 (DT-13) possess good potential against TME. As TME is supportive for tumor metastasis, alternatively it is a challenging for therapeutic intervention. In our present study, we explored molecular mechanism through which TME induced cell migration and how DT-13 interferes in this mechanism. We used a novel model of co-culture system which is eventually developed in our lab. Tumor cells were co-cultured with hypoxia induced cancer-associated fibroblasts (CAF) or with chemically induced cancer-associated adipocytes (CAA). The effect of hypoxia in conditioned medium for CAF was assessed through expression of α-SMA and HIF by western blotting while oil red staining was done to assess the successful chemical induction for adipocytes (CAA), the effect of TME through conditioned medium on cell migration was analyzed by trans-well cell migration, and cell motility (wound healing) analyses. The expression changes in cellular proteins were assessed through western blotting and immunofluorescent studies. Our results showed that tumor microenvironment has a direct role in promoting breast cancer cell migration by stromal cells; moreover, we found that DT-13 restricts this TME regulated cell migration via targeting stromal cells in vitro. Additionally we also found that DT-13 targets NMII-A for its effect on breast cancer cell migration for the regulation of stromal cells in TME.

Cachexia Anorexia Syndrome and Associated Metabolic Dysfunction in Peritoneal Metastasis.

Patients with peritoneal metastasis (PM) of gastrointestinal and gynecological origin present with a nutritional deficit characterized by increased resting energy expenditure (REE), loss of muscle mass, and protein catabolism. Progression of peritoneal metastasis, as with other advanced malignancies, is associated with cancer cachexia anorexia syndrome (CAS), involving poor appetite (anorexia), involuntary weight loss, and chronic inflammation. Eventual causes of mortality include dysfunctional metabolism and energy store exhaustion. Etiology of CAS in PM patients is multifactorial including tumor growth, host response, cytokine release, systemic inflammation, proteolysis, lipolysis, malignant small bowel obstruction, ascites, and gastrointestinal side effects of drug therapy (chemotherapy, opioids). Metabolic changes of CAS in PM relate more to a systemic inflammatory response than an adaptation to starvation. Metabolic reprogramming is required for cancer cells shed into the peritoneal cavity to resist anoikis (i.e., programmed cell death). Profound changes in hexokinase metabolism are needed to compensate ineffective oxidative phosphorylation in mitochondria. During the development of PM, hypoxia inducible factor-1α (HIF-1α) plays a key role in activating both aerobic and anaerobic glycolysis, increasing the uptake of glucose, lipid, and glutamine into cancer cells. HIF-1α upregulates hexokinase II, phosphoglycerate kinase 1 (PGK1), pyruvate dehydrogenase kinase (PDK), pyruvate kinase muscle isoenzyme 2 (PKM2), lactate dehydrogenase (LDH) and glucose transporters (GLUT) and promotes cytoplasmic glycolysis. HIF-1α also stimulates the utilization of glutamine and fatty acids as alternative energy substrates. Cancer cells in the peritoneal cavity interact with cancer-associated fibroblasts and adipocytes to meet metabolic demands and incorporate autophagy products for growth. Therapy of CAS in PM is challenging. Optimal nutritional intake alone including total parenteral nutrition is unable to reverse CAS. Pressurized intraperitoneal aerosol chemotherapy (PIPAC) stabilized nutritional status in a significant proportion of PM patients. Agents targeting the mechanisms of CAS are under development.

Adipocytes and microRNAs Crosstalk: A Key Tile in the Mosaic of Breast Cancer Microenvironment.

Breast cancer (BC) is a disease characterized by a high grade of heterogeneity. Consequently, despite the great achievements obtained in the last decades, most of the current therapeutic regimens still fail. The identification of new molecular mechanisms that will increase the knowledge of all steps of tumor initiation and growth is mandatory in finding new clinical strategies. The BC microenvironment, consisting of endothelial cells, fibroblasts, immune cells and adipocytes, plays an essential role in regulating BC development, and recently it has gained great attention in the scientific community. In particular, adipose tissue is emerging as an important target to investigate among mammary gland components. The mechanisms underlying BC progression driven by adipocytes are predominantly unexplored, especially that involving the switch from normal adipocytes to the so-called cancer-associated adipocytes (CAAs). MicroRNAs (miRNAs), a class of gene expression modulators, have emerged as the regulators of key oncogenes and tumor suppressor genes that affect multiple pathways of the tumor microenvironment and adipose tissue. This review concerns a presentation of the role of adipocytes in breast tissue, and describes the most recent discoveries about the interplay between adipocytes and miRNAs, which collaborate in the arrangement of a pro-inflammatory and cancerous microenvironment, laying the foundations for new concepts in the prevention and treatment of BC.

Adipocyte and lipid metabolism in cancer drug resistance.

Development of novel and effective therapeutics for treating various cancers is probably the most congested and challenging enterprise of pharmaceutical companies. Diverse drugs targeting malignant and nonmalignant cells receive clinical approval each year from the FDA. Targeting cancer cells and nonmalignant cells unavoidably changes the tumor microenvironment, and cellular and molecular components relentlessly alter in response to drugs. Cancer cells often reprogram their metabolic pathways to adapt to environmental challenges and facilitate survival, proliferation, and metastasis. While cancer cells' dependence on glycolysis for energy production is well studied, the roles of adipocytes and lipid metabolic reprogramming in supporting cancer growth, metastasis, and drug responses are less understood. This Review focuses on emerging mechanisms involving adipocytes and lipid metabolism in altering the response to cancer treatment. In particular, we discuss mechanisms underlying cancer-associated adipocytes and lipid metabolic reprogramming in cancer drug resistance.

Abstract 4241: Molecular profile of tumor-associated adipose in obese women with breast cancer

Abstract Breast tumors have a direct physical and vascular interface with white adipose tissue, making them ideal model systems to study the adipose-tumor microenvironment, especially within the context of obesity. As a first initiative to understanding how adipose tissue contributes to breast tumor etiology, we explored cancer-associated adipose tissue in patients with invasive breast cancer. We hypothesized that obesity leads to greater alterations in cancer-associated adipose tissue by expressing genes involved in cellular growth, angiogenesis, and invasion, thus providing support for breast cancer progression. We also proposed that adipocytes adjacent to tumors express a genotype unique to obese breast cancers. Adipose tissue samples (n=10) were obtained from obese and lean women with breast cancer. Within individuals, tissue was sampled proximal and distal (2 cm) to the tumor. Using next-generation whole transcriptome sequencing, we examined genes from cancer-associated adipose tissue in both groups. A heat map of normalized expression values was generated with unsupervised clustering and the fifty most informative genes. Samples from obese individuals with breast cancer tended to cluster together. There were significant gene differences between adipose collected proximal versus distal from the tumor in obese but not in lean women, such as genes associated with myosin heavy chains MYH1, 4, 8, and 11 (adj. p < 0.05). In examining obese women, there were 16 Hallmark pathways that were statistically significant (adj. p < 0.10); whereas a subset of these (11 pathways) were significant in lean women. Of interest, pathways that were significant in both were pathways that regulate EMT, adipogenesis and TGF beta signaling. The molecular signature of cancer-associated adipocytes is different between lean and obese patients. (Supported by NIH P20GM103434 and NIGMS U54GM104942) Citation Format: Niel Infante, James Coad, Donald Primerano, James Denvir, Linda Vona-Davis. Molecular profile of tumor-associated adipose in obese women with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4241.

Abstract 2020: Adipocytes up-regulates CD36 expression, fatty acid uptake and oxidation enhancing breast cancer cell progression

Abstract The role of the tumor microenvironment in tumor progression has recently gained enormous attention, with studies reporting heterogeneous interactions between tumor cells and tumor stroma cells driving tumor progression. The breast cancer microenvironment is unique since the breast tissue within which the tumor originates comprises predominantly of adipocytes. This makes adipocytes a unique component in the breast cancer microenvironment. Adipocytes secrete various growth factors and cytokines that influence tumor progression by induction of epithelial-mesenchymal transition (EMT) which enhances cancer cell migration, invasion and metastasis. This study presents an alternative approach by which adipocytes may enhance cancer progression via the transfer of free-fatty acids from tumor-associated adipocytes to breast cancer cells. The uptake of free fatty acid subsequently serves as a secondary source of energy to drive breast cancer progression. The study aimed to provide an in-depth understanding of the molecular mechanism by which free fatty acid are imported and metabolized in cancer cells in an adipocyte-rich microenvironment. Human adipocytes were co-cultured with cancer cells and the fatty acid (FA) translocase (CD36) activity was inhibited by the chemical inhibitor sulfosuccinimidyl oleate (SSO). Silencing RNA and expression plasmids were used to evaluate CD36 activity in cancer cells. Expression levels of key genes and proteins involved in lipid transfer and metabolism were evaluated by qtPCR and western blot. Inhibition of CD36 reduces free fatty acid uptake and induces intracellular lipogenesis with upregulation of fatty acid synthase (FASN). Breast cancer cells co-cultured with human adipocytes express high levels of the CD36 facilitating free fatty acid uptake from adipocytes. Co-cultured cells had increased levels of fatty acid oxidation (FAO) and oxygen consumption, with increased migration and invasion characteristics. We also observed a significant increase in the expression of CPT1A required for mitochondrial fatty acid import and ATGL required for triglyceride metabolism. Our findings indicate an enhanced dependence on FAO in co-cultured cancer cell. Indicating a potential bioenergetic adaptation of breast cancer cells to the adipocyte-rich microenvironment. Inhibition of CD36 and CPT1 significantly decreased breast cancer cell migration and invasion. These results suggest that cancer-associated adipocytes induce free fatty acid uptake via upregulation of CD36 and reprogram breast cancer cell metabolism. Thus, targeting CD36 may have a potential targeted therapy. Citation Format: Junjeong Choi, Jones Gyamfi. Adipocytes up-regulates CD36 expression, fatty acid uptake and oxidation enhancing breast cancer cell progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2020.

Tumor-secreted PAI-1 promotes breast cancer metastasis via the induction of adipocyte-derived collagen remodeling

BackgroundBreast cancer cells recruit surrounding stromal cells, such as cancer-associated fibroblasts (CAFs), to remodel collagen and promote tumor metastasis. Adipocytes are the most abundant stromal partners in breast tissue, local invasion of breast cancer leads to the proximity of cancer cells and adipocytes, which respond to generate cancer-associated adipocytes (CAAs). These cells exhibit enhanced secretion of extracellular matrix related proteins, including collagens. However, the role of adipocyte-derived collagen on breast cancer progression still remains unclear.MethodsAdipocytes were cocultured with breast cancer cells for 3D collagen invasion and collagen organization exploration. Breast cancer cells and adipose tissue co- implanted mouse model, clinical breast cancer samples analysis were used to study the crosstalk between adipose and breast cancer cells in vivo. A combination of proteomics, enzyme-linked immunosorbent assay, loss of function assay, qPCR, western blot, database analysis and chromatin immunoprecipitation assays were performed to study the mechanism mediated the activation of PLOD2 in adipocytes.ResultsIt was found that CAAs remodeled collagen alignment during crosstalk with breast cancer cells in vitro and in vivo, which further promoted breast cancer metastasis. Tumor-derived PAI-1 was required to activate the expression of the intracellular enzyme procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) in CAAs. Pharmacologic blockade of PAI-1 or PLOD2 disrupted the collagen reorganization in CAAs. Mechanistically, it was observed that PI3K/AKT pathway was activated in adipocytes upon co-culturing with breast cancer cells or treatment with recombinant PAI-1, which could promote the translocation of transcription factor FOXP1 into the nucleus and further enhanced the promoter activity of PLOD2 in CAAs. In addition, collagen reorganization at the tumor-adipose periphery, as well as the positive relevance between PAI-1 and PLOD2 in invasive breast carcinoma were confirmed in clinical specimens of breast cancer.ConclusionIn summary, our findings revealed a new stromal collagen network that favors tumor invasion and metastasis establish between breast cancer cells and surrounding adipocytes at the tumor invasive front, and identified PLOD2 as a therapeutic target for metastatic breast cancer treatment.

Co-culture With Human Breast Adipocytes Differentially Regulates Protein Abundance in Breast Cancer Cells.

Recent research highlights the role of cancer-associated adipocytes (CAA) in promoting breast cancer cell migration, invasion and resistance to therapy. This study aimed at identifying cellular proteins differentially regulated in breast cancer cells co-cultured with CAA. Adipocytes isolated from human breast adipose tissue were co-cultured with hormone receptor-positive (MCF-7) or -negative (MDA-MB-231) breast cancer cells using a transwell co-culture system. Proteomes of co-cultured and control breast cancer cells were compared quantitatively using iTRAQ labelling and tandem mass spectrometry, and the results were validated by western blotting. A total of 1,126 and 1,218 proteins were identified in MCF-7 and MDA-MB-231 cells, respectively. Among these, 85 (MCF-7) and 63 (MDA-MB-231) had an average fold change >1.5 following co-culture. Pathway analysis revealed that CAA-induced enrichment of proteins involved in metabolism, the ubiquitin proteasome, and purine synthesis. This study provides a proteomic platform for investigating the paracrine role of CAA in promoting breast cancer cell metastasis and resistance to therapy.

Interleukin-8 Activates Breast Cancer-Associated Adipocytes and Promotes Their Angiogenesis- and Tumorigenesis-Promoting Effects.

Increasing evidence supports the critical role of active stromal adipocytes in breast cancer development and spread. However, the mediators and the mechanisms of action are still elusive. We show here that cancer-associated adipocytes (CAAs) isolated from 10 invasive breast carcinomas are proinflammatory and exhibit active phenotypes, including higher proliferative, invasive, and migratory capacities compared to their adjacent tumor-counterpart adipocytes (TCAs). Furthermore, all CAAs secreted higher level of interleukin-8 (IL-8), which is critical in mediating the paracrine procarcinogenic effects of these cells. Importantly, ectopic expression of IL-8 in TCA cells activated them and enhanced their procarcinogenic effects both in vitro, in a STAT3-dependent manner, and in vivo In contrast, inhibition of the IL-8 signaling using specific short hairpin RNA, anti-IL-8 antibody, or reparixin suppressed the active features of CAAs, including their non-cell-autonomous tumor-promoting activities both on breast luminal cells and in orthotopic tumor xenografts in mice. IL-8 played also an important role in enhancing the proangiogenic effects of breast adipocytes. These results provide clear indication that IL-8 plays key roles in the activation of breast CAAs and acts as a major mediator for their paracrine protumorigenic effects. Thus, targeting CAAs by inhibiting the IL-8 pathway could have great therapeutic value.

Abstract IA06: Stromal regulation of metastasis

Abstract Most patients with high-grade serous ovarian cancer (HGSOC) develop metastasis early and, while they are initially chemoresponsive, the existence of disseminated, solid tumors generally leads to the grim prognosis associated with the disease. Many studies of metastasis in HGSOC focus on the epithelial cancer cell and its genetic and epigenetic makeup, underestimating the contribution of the host microenvironment. The metastatic pattern of HGSOC is very different from other epithelial cancers (e.g., breast cancer), since metastasis is mostly limited to the mesothelial cell covered surfaces of the abdominal cavity or retroperitoneal lymph nodes. Even before they metastasize, ovarian cancer cells at the primary site use exosomes to prime the metastatic sites. In parallel, the stromal cells of the primary metastatic sites, generally the omentum and peritoneum, release cytokines that attract the cancer cells. Once the individual HGSOC cells or cell spheroids “land” on the omentum or peritoneum, an intensive bidirectional signaling ensues between the cancer cells and host fibroblasts, mesothelial cells, adipocytes, and immune cells. Mesothelial cells and macrophages initially fight the local invasion by blocking the attachment of cancer cells or recruiting various inflammatory cells to the site of micrometastasis, where the immune cells surround the metastasis. Subsequently, the cancer cells activate the dispersal of the mesothelial cells, attach to the sub-mesothelial basement membrane and reprogram fibroblasts. Within a short period of time, cancer cells transform normal fibroblasts within the ECM into cancer-associated fibroblasts (CAF). Eventually, most stromal cells become cancer associated stromal cells, releasing pro-proliferative and migratory cytokines. In addition, metabolic reprogramming takes place, involving reciprocal metabolic interactions between the cancer and stromal cells. The cancer cells are able to extract fatty acids, to be used as fuel, from cancer-associated adipocytes. Also, a hypoxic environment is produced. This advantages the cancer cells, since they readily adapt to a low-oxygen environment and hypoxia is known to impair the efficacy of chemotherapy. Ultimately, the metastatic site is taken over by the tumor organ. We will present data from our use of organotypic 3D cultures of the metastatic site and systematic metabolic and proteomic analysis of the interactions between stromal and cancer cells, which has informed our attempts to understand the complexities of HGSOC metastasis. Only an improved understanding of the biology of metastasis and the metabolic symbiosis within the tumor will allow the identification of novel targets that can be used to disrupt the metastatic process and impair the ability of HGSOC to recruit the stroma. Citation Format: Ernst Lengyel, Mark Eckert, Iris Romero, Hilary Kenny. Stromal regulation of metastasis. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr IA06.

PO-307 Differential secretome analysis of cancer-associated adipocytes (CAA) and mature adipocytes to identify adipocyte-driven micro-environmental regulators of breast cancer progression

IntroductionIn breast cancer, obesity is linked to invasive tumours that respond poorly to chemotherapy. We have explored relationships between breast cancer cells and adipocytes in the tumour microenvironment using a transwell co-culture system. Human breast adipocytes induced chemo-resistance in both hormone receptor positive (MCF-7) and triple negative (MDA-MB-231) breast cancer cells. In addition, cancer-associated adipocytes (CAA) induced a partial epithelial-mesenchymal transition, creating a population of breast cancer cells that were highly migratory. In this study, we used discovery mass spectrometry and antibody arrays to analyse the proteins secreted by CAA to identify proteins that may contribute to breast cancer progression.Material and methodsBreast adipose tissue samples were collected from patients undergoing breast surgery at Christchurch Hospital. Pre-adipocytes were isolated and differentiated into mature adipocytes, followed by co-culture with MCF-7 and MDA-MB-231 cancer cells for 3 days in a transwell co-culture system. Breast cancer cells were removed and adipocytes were washed and serum free media was added for 24 hours. This conditioned media was collected from pre-adipocytes, mature differentiated adipocytes and CAA co-cultured with either MCF-7 or MDA-MB-231 cells. Secretomes were compared using antibody arrays and iTRAQ labelling with LC-coupled LTQ-Orbitrap tandem mass spectrometry. Validation of iTRAQ results was performed using ELISA.Results and discussionsOf the 327 proteins identified using iTRAQ-MS, 11.3% were intracellular and thus excluded; the remainder were either extracellular or secreted proteins. Of the 183 secreted proteins, 45% of these were secreted at higher levels by adipocytes after co-culture with breast cancer cells compared with adipocytes alone. These included proteins associated with inflammation and matrix remodelling. Chitinase 3-like 1 (Chi3L1) levels were increased according to both iTRAQ-MS and antibody array analysis. Recombinant Chi3L1 treatment (4 ng/ml) increased viability, proliferation, migration and resistance to chemotherapy in both breast cancer cell lines.ConclusionOur study describes for the first time, the secretome profile from CAA and provides a comprehensive platform for further research investigating local interactions between breast cancer cells and CAA. Our data identified CAA derived Chi3L1 as a key player in the interactions between adipocytes and cancer cells within the tumour microenvironment.

PO-234 Transwell co-culture with human breast adipocytes alters the proteome expression profiles of MCF-7 and MDA-MB-231 human breast cancer cells

IntroductionBreast tumours grow within adipose tissue, and stromal adipocytes are likely the first cell type encountered by breast cancer cells as they metastasize. Adipocytes at the invasive edge of human breast tumours exhibit a modified phenotype, and consequently these adipocytes have been named Cancer Associated Adipocytes (CAA). Previous in vitro research has shown that co-culture with CAA promotes breast cancer cell resistance to radiation and some therapeutic agents, as well as breast cancer cell invasiveness both in vitro and in vivo. The aim of the current research was to identify cellular proteins that are differentially regulated in breast cancer cells co-cultured with CAA.Material and methodsThis study collected human breast adipose tissue samples, from which pre-adipocytes were isolated, differentiated into mature adipocytes, and co-cultured with hormone receptor positive (MCF-7) and negative (MDA-MB-231) human breast cancer cells for 3 days in a transwell co-culture system. The proteomes of co-cultured and control breast cancer cells were compared quantitatively using iTRAQ labelling and LC-coupled LTQ-Orbitrap tandem mass spectrometry. Validation of iTRAQ results was performed by Western blotting.Results and discussionsThis study identified and quantified a total of 1126 and 1218 proteins expressed in MCF-7 and MDA-MB-231 cells, respectively. Of these, 85 in MCF-7 and 63 in MDA-MB-231 had a fold change >1.5 between co-culture and control breast cancer cell samples. Overall, MCF-7 cells had more proteins downregulated (n=53) than upregulated (n=32) after co-culture, whereas, more proteins were upregulated (n=51) than downregulated (n=12) in MDA-MB-231 cells. Enrichment analysis revealed an upregulation of proteins involved in metabolic pathways, namely TCA cycle proteins in MCF-7 cells and glycolysis proteins in MDA-MB-231 cells. The metabolic enzyme PGK1 was upregulated in response to co-culture with CAA in both cell lines, and is associated with poorer overall survival and resistance to the chemotherapy agent paclitaxel in breast cancer. Western blotting results validated mass spectrometry ratios for three candidate proteins.ConclusionOverall, this study describes for the first time the local effect CAA have on the expression of proteins in breast cancer cells in vitro, and provides a comprehensive platform for further research investigating local interactions between breast cancer cells and CAA.

Abstract 173: Adipocytes-derived collagen reorganization in microenvironment promotes breast cancer progression

Abstract Purposes Breast cancer cells recruit surrounding stromal cells, such as cancer-associated fibroblasts (CAFs), to reorganize collagen and promote tumor metastasis. Adipocytes are the most abundant stromal partners in breast tissue, whether cancer-associated adipocytes (CAAs) involve in the collagen reorganization and ultimately promoting metastasis of breast cancer is still unknown. In this study, we seek to understand whether adipocytes involve in the extracellular matrix collagen reorganization in breast cancer and its underline mechanism. Methodology In vitro mature adipocytes and breast cancer transwell co-culture model and orthotopic xenograft mouse model of human breast tumor formation in vivo were used to study the crosstalk between adipocytes and breast cancer cells, and then using a combination of proteomics, database analysis and clinical breast cancer samples analysis to study the mechanism mediated adipocytes-derived collagen reorganization. Results Here, we found that CAAs co-cultured with breast cancer cells increased the linearly aligned type I collagen fibers deposition in vitro and in vivo, which accompanied with increasing collagen lysyl hydroxylation 2 (LH2) and lysyl oxidase (LOX) expression. Breast cancer cells migrated orderly along the CAAs-derived collagen, while randomly migrated through the mature adipocyte-derived collagen. Simultaneously, clinical breast cancer samples analysis showed LH2 was high expression in the areas where cancer cells infiltrated into fatty tissue, which accompanied with poor prognosis. It is well-known that interaction between adipocytes and cancer altered the secretion of cytokines. In the present study, proteomics analysis showed that IL-6, MIF-1, TECK, MCP-1, PAI-1, IGFBP-1, TIMP1 and TIMP2 are highly secreted in adipocytes-breast cancer system. And using agonist or antagonist of these cytokines showed that these cytokines might involve in the reorganization of adipocyte-derived collagen. Mechanistic investigations demonstrated PI3K/AKT pathway and JAK/STAT3 signaling pathway involved in the rearrangement of adipocyte-derived collagen, which mediated by promoting forkhead-box(FOX) and phosphorylation STAT3(p-STAT3) translocate to the nucleus, respectively. Moreover, FOX and STAT3-dependent induction of LH2 expression further potentiated the adipocytes-derived collagen reorganization, which thus promoting breast cancer metastasis. Conclusion Collectively, our findings reveal new insights underlying increased breast cancer progression and offer new opportunities for stromal-targeted therapies in breast cancer. Citation Format: Wei Xiaohui, He Jinyong, Li Sijing, Yuan Shengtao, Sun Li. Adipocytes-derived collagen reorganization in microenvironment promotes breast cancer progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 173.

Metabolic cooperation between cancer and non-cancerous stromal cells is pivotal in cancer progression.

The way cancer cells adapt to microenvironment is crucial for the success of carcinogenesis, and metabolic fitness is essential for a cancer cell to survive and proliferate in a certain organ/tissue. The metabolic remodeling in a tumor niche is endured not only by cancer cells but also by non-cancerous cells that share the same microenvironment. For this reason, tumor cells and stromal cells constitute a complex network of signal and organic compound transfer that supports cellular viability and proliferation. The intensive dual-address cooperation of all components of a tumor sustains disease progression and metastasis. Herein, we will detail the role of cancer-associated fibroblasts, cancer-associated adipocytes, and inflammatory cells, mainly monocytes/macrophages (tumor-associated macrophages), in the remodeling and metabolic adaptation of tumors.

Targeting Metabolic Cross Talk between Cancer Cells and Cancer-Associated Fibroblasts.

Although tumorigenesis has classically been regarded as a genetic disease of uncontrolled cell growth, the importance of the tumor microenvironment (TME) is continuously emphasized by the accumulating evidence that cancer growth is not simply dependent on the cancer cells themselves [1, 2] but also dependent on angiogenesis [3–6], inflammation [7, 8], and the supporting roles of cancer-associated fibroblasts (CAFs) [9, 10]. After the discovery that CAFs are able to remodel the tumor matrix within the TME and provide the nutrients and chemicals to promote cancer cell growth [11], many studies have aimed to uncover the cross talk between cancer and CAFs. Moreover, a new paradigm in cancer metabolism shows how cancer cells act like “metabolic parasites” to uptake the high-energy metabolites, such as lactate, ketone bodies, free fatty acid, and glutamine from supporting cells, including CAFs and cancer-associated adipocytes (CAAs) [12, 13]. This chapter provides an overview of the metabolic coupling between CAFs and cancer to further define the therapeutic options to disrupt the CAF-cancer cell interactions.

Characterisation of a Mouse Model of Breast Cancer with Metabolic Syndrome.

Patients with breast cancer and metabolic syndrome have poorer outcomes. We aimed to develop and characterise an apolipoprotein E-null/aromatase knockout (ApoE-/-/ArKO) mouse model of breast cancer with metabolic syndrome to aid research of the mechanisms behind poor prognosis. Wild-type, ApoE-/- and ApoE-/-/ArKO mice were orthotopically implanted with EO771 murine breast cancer cells. Tumour growth was monitored and tumours investigated for pathological features such as cancer-associated adipocytes, hypoxia and cancer cell proliferation. Tumours from ApoE-/-/ArKO mice were significantly more proliferative than those from wild-type mice (p=0.003), and exhibited reduced expression of insulin-like growth factor binding protein-5 (p=0.002). However, ApoE-/-/ArKO mice also had a reduced rate of metastasis compared to wild-type and ApoE-/- mice. Tumour hypoxia and the number of cancer-associated adipocytes did not differ. The ApoE-/-/ArKO model with EO771 breast cancer provides a novel mouse model to investigate the effects of metabolic syndrome on aspects of breast tumour biology.

Interaction with adipocyte stromal cells induces breast cancer malignancy via S100A7 upregulation in breast cancer microenvironment

BackgroundBreast adipocytes play important roles in both the development and function of mammary epithelial cells. Therefore, carcinoma–adipose stromal cell (ASC) interactions have been considered pivotal in supporting tumor growth in breast cancer. In addition, it has been demonstrated that the biological features of cancer-associated adipocytes differ from those of normal ASCs. Therefore, we investigated an interaction between ASCs and carcinoma cell lines to identify genes associated with ASC invasion of carcinoma cells.Methods3T3-L1 ASC-derived conditioned medium (CM) was treated to measure the proliferation rate of breast cancer cells. To further examine the effect of ASCs, breast cancer cells were cocultivated with either primary human or 3T3-L1 ASCs for migration assays, DNA microarrays, quantitative real-time polymerase chain reactions, and Western blotting experiments. Furthermore, immunoreactivity of S100A7, the most upregulated gene in MCF7, after coculture with ASCs was evaluated for 150 breast cancer tissues to statistically analyze its association with clinicopathological parameters.ResultsWe first confirmed that ASC-derived CM treatment enhanced the cell proliferation rate of MCF7, T47D, SK-BR-3, and ZR-75-1 cell lines, whereas the migration rate of breast cancer cells was promoted by coculture with ASCs. We identified that a small calcium-binding protein, S100A7, was markedly upregulated (by 5.8-fold) in MCF7 cells after coculture with primary human ASCs. Knockdown of S100A7 significantly suppressed ASC-stimulated cell proliferation and migration rate, indicating a possible involvement of S100A7 in the carcinoma–ASC interaction in breast tumors. Furthermore, strong S100A7 immunoreactivity was detected at the invasive front of adipose stromal tissues compared with that at the intratumoral area. The status of S100A7 was also significantly correlated with adverse pathological parameters, and multivariate analysis revealed that S100A7 could be an independent prognostic marker for a poor relapse-free survival rate. Moreover, induction of oncostatin M was detected in cancer-stimulated ASCs, whereas the downstream S100A7 binding proteins/receptor for advanced glycation endproducts were significantly upregulated in correspondence with S100A7 expression in breast cancer cells after coculture with ASCs.ConclusionsThe results of our study suggest that paracrine production of cytokines from ASCs stimulates breast carcinoma cell growth via upregulation of S100A7 expression in breast cancer cell lines.

The fat and the bad: Mature adipocytes, key actors in tumor progression and resistance.

Growing evidence has raised the important roles of adipocytes as an active player in the tumor microenvironment. In many tumors adipocytes are in close contact with cancer cells. They secrete various factors that can mediate local and systemic effects. The adipocyte-cancer cell crosstalk leads to phenotypical and functional changes of both cell types, which can further enhance tumor progression. Moreover, obesity, which is associated with an increase in adipose mass and an alteration of adipose tissue, has been established as a risk factor for cancer incidence and cancer-related mortality. In this review, we summarize the mechanisms of the adipocyte-cancer cell crosstalk in both obese and lean conditions as well as its impact on cancer cell growth, local invasion, metastatic spread and resistance to treatments. Better characterization of cancer-associated adipocytes and the key molecular events in the adipocyte-cancer cell crosstalk will provide insights into tumor biology and suggest efficient therapeutic opportunities.

Transition into inflammatory cancer-associated adipocytes in breast cancer microenvironment requires microRNA regulatory mechanism.

The role of adipocytes in cancer microenvironment has gained focus during the recent years. However, the characteristics of the cancer-associated adipocytes (CAA) in human breast cancer tissues and the underlying regulatory mechanism are not clearly understood. We reviewed pathology specimens of breast cancer patients to understand the morphologic characteristics of CAA, and profiled the mRNA and miRNA expression of CAA by using indirect co-culture system in vitro. The CAAs in human breast cancers showed heterogeneous topographic relationship with breast cancer cells within the breast microenvironment. The CAAs exhibited the characteristics of de-differentiation determined by their microscopic appearance and the expression levels of adipogenic markers. Additionally, the 3T3-L1 adipocytes indirectly co-cultured with breast cancer cells showed up-regulation of inflammation-related genes including Il6 and Ptx3. The up-regulation of IL6 in CAA was further observed in human breast cancer tissues. miRNA array of indirectly co-cultured 3T3-L1 cells showed increased expression of mmu-miR-5112 which may target Cpeb1. Cpeb1 is a negative regulator of Il6. The suppressive role of mmu-miR-5112 was confirmed by dual luciferase reporter assay, and mmu-miR-5112-treated adipocytes showed up-regulation of Il6. The transition of adipocytes into more inflammatory CAA resulted in proliferation-promoting effect in ER positive breast cancer cells such as MCF7 and ZR-75-1 but not in ER negative cells. In this study, we have determined the de-differentiated and inflammatory natures of CAA in breast cancer microenvironment. Additionally, we propose a miRNA-based regulatory mechanism underlying the process of acquiring inflammatory phenotypes in CAA.