Introduction Circulating tumor cell (CTC) clusters are potent mediators of metastasis, exhibiting dramatically enhanced survival and colonization potential compared to single CTCs. However, the molecular and metabolic programs that initiate cluster formation remain poorly defined. CD36 is known to be critical for metastasis and metabolism. However, its regulatory role during the Adherent-to-Suspension Transition (AST) and subsequent CTC clustering remains unclear. This study aimed to elucidate how CD36 orchestrates early clustering of AST-derived cancer cells and to explore its potential as a stage-specific therapeutic vulnerability. Methods We utilized an in vitro AST-Cluster Transition Model with cancer cells, monitored by live-cell imaging. The impact of CD36, glycolysis, and ROS inhibition on cluster dynamics and viability was assessed. Efficacy was confirmed in vivo using a cluster-driven metastasis mouse model and patient data (CTC transcriptomes, METABRIC cohort) were analyzed for metabolic and prognostic relevance. Results We found biphasic CD36 expression (upregulated in AST cells, decreased upon clustering) concurrent with a metabolic shift from OXPHOS to glycolysis under hypoxia. This metabolic reprogramming was sustained by the AKT, AMPK, and HIF1α axes. CD36 inhibition impaired early cluster formation, which was accompanied by decreased expression of junctional proteins, and was sufficient to reduce cluster-driven lung metastases in vivo. Patient data linked similar metabolic remodeling signatures, and high expression of CD36 and JUP was associated with poorer early survival. Conclusions This study identifies CD36 as a dynamic regulator linking AST to clustering and metabolic adaptation. CD36 blockade effectively suppresses cluster-driven colonization, revealing CD36 as a tractable, stage-specific therapeutic target to intercept metastatic dissemination at its earliest, most vulnerable stage.

Introduction Adipose tissue constitutes a significant portion of the breast tumor microenvironment (TME), influencing lipid metabolism and cancer progression. Squalene epoxidase (SQLE), a key enzyme in cholesterol biosynthesis, and PRDM16, involved in adipogenesis and metabolic regulation, have been implicated in tumor biology. However, the impact of stromal composition on SQLE and PRDM16 expression in breast cancer remains unclear. Methods We analyzed the expression of SQLE and PRDM16 in breast cancer samples categorized into adipose-rich and fibrous stromal subtypes. Immunohistochemistry (IHC) was performed on tissue microarrays, and co-culture experiments with 3T3-L1 and NIH-3T3 cells were conducted using breast cancer cell lines representing different molecular subtypes. Protein and mRNA levels of SQLE and PRDM16 were assessed using Western blotting and quantitative real-time PCR. The prognostic significance of SQLE and PRDM16 expression was evaluated through survival analysis. Results SQLE and PRDM16 expression were significantly higher in breast cancers with adipose stroma than those with fibrous stroma (p < 0.001 and p = 0.007, respectively). Co-culture with 3T3-L1 induced SQLE and PRDM16 expression in HER2-overexpressing and triple-negative breast cancer (TNBC) cell lines but had minimal effects on Luminal and HER2-positive subtypes. In contrast, NIH-3T3 co-culture did not significantly alter their expression. Survival analysis indicated that PRDM16 expression was associated with shorter overall survival (p = 0.049), while SQLE expression did not considerably impact prognosis. Conclusion Our findings suggest that adipose stroma promotes SQLE and PRDM16 expression, particularly in aggressive breast cancer subtypes. PRDM16 may be a potential prognostic marker, influencing tumor progression through metabolic and stromal interactions. Further studies are needed to elucidate the mechanistic role of PRDM16 and SQLE in the breast cancer microenvironment.

Adipocytes play a dynamic role in the tumor microenvironment (TME) by acting as facilitators, providing cytokines and metabolites that regulate cancer progression and metastasis. Despite metastasis being a major contributor to cancer-associated mortality, our understanding of how adipocytes influence this process remains limited. This study aims to elucidate the regulatory mechanism of Adherent to Suspension Transition (AST) reprogramming within the adipocyte, driven by anchorage dependency. AST facilitates the conversion of adherent tumor cells into suspension cells, thereby contributing to the generation of circulating tumor cells (CTCs). We have evaluated generating AST cells from primary tumors using a dissemination assay that mimics CTCs in vitro. Additionally, we examined AST cell formation when incubated with human adipocyte-conditioned media (ADCM) using the InCucyte live-cell imaging system. Through this approach, we effectively assessed the impact of the tumor-adipocyte interactions on CTC formation from the perspective of AST. As a metastasis-initiating marker, CD36 is pivotal in fatty acid (FA) acquisition and regulates lipid metabolic remodeling during the AST. The generation of AST cells through AST reprogramming is controlled by fatty acid oxidation (FAO), and pharmacological blockade of CD36 and FAO significantly reduced AST cell generation. This demonstrates that CD36 plays a key role in the early stages of AST-induced dissemination. Additionally, promoting cancer cell aggressiveness through ADCM enhances metastatic potency and upregulates the expression of AST reprogramming factors. Inhibition of lipid metabolism not only suppresses AST cell formation but also decreases survival in suspension. This indicates that exogenous lipid uptake and FAO via CD36 play crucial roles in the metastasis process, facilitating the dissemination of primary tumors into the bloodstream. Adipocytes contribute to cancer progression by supplying various metabolites to cancer cells. While primary tumors predominantly rely on glucose as a major energy source, cellular remodeling during dissemination shifts metabolic dependency toward lipids. In the TME, where adipocytes are abundant, tumor cells acquire FA through CD36-mediated uptake for metabolic adaptation. This shift to lipid metabolism is essential for AST, and thus, targeting lipid metabolism via inhibition of CD36 and FAO could serve as a potential therapeutic strategy for AST.

Until now, Hippo pathway-mediated nucleocytoplasmic translocation has been considered the primary mechanism by which yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) transcriptional coactivators regulate cell proliferation and differentiation via transcriptional enhanced associate domain (TEAD)-mediated target gene expression. In this study, however, we found that TAZ, but not YAP, is associated with the Golgi apparatus in macrophages activated via Toll-like receptor ligands during the resolution phase of inflammation. Golgi-associated TAZ enhanced vesicle trafficking and secretion of proinflammatory cytokines in M1 macrophage independent of the Hippo pathway. Depletion of TAZ in tumor-associated macrophages promoted tumor growth by suppressing the recruitment of tumor-infiltrating lymphocytes. Moreover, in a diet-induced metabolic dysfunction-associated steatohepatitis model, macrophage-specific deletion of TAZ ameliorated liver inflammation and hepatic fibrosis. Thus, targeted therapies being developed against YAP/TAZ-TEAD are ineffective in macrophages. Together, our results introduce Golgi-associated TAZ as a potential molecular target for therapeutic intervention to treat tumor progression and chronic inflammatory diseases.

The mitochondrial glutamine transporter SLC1A5_var plays a central role in the metabolic reprogramming of cancer cells by facilitating glutamine import into mitochondria for energy production and redox homeostasis. Despite its critical function, the development of effective and selective inhibitors targeting SLC1A5_var has remained a significant challenge. Here, we introduce iMQT_020, a selective allosteric inhibitor identified through structure-based screening. iMQT_020 disrupts the trimeric assembly of SLC1A5_var, causing metabolic crisis in cancer cells and selectively suppressing their growth. Mechanistically, iMQT_020 reduces glutamine anaplerosis and oxidative phosphorylation, resulting in a broad disruption of cancer metabolism. Additionally, iMQT_020 treatment epigenetically upregulates PD-L1 expression, enhancing the efficacy of combination therapies with anti-PD-L1 immune checkpoint inhibitors. These findings highlight the therapeutic potential of targeting SLC1A5_var as a critical metabolic vulnerability in cancer and demonstrate that targeting allosteric interprotomer interactions is a novel and promising therapeutic strategy for cancer treatment.

Adipocytes influence breast cancer behaviour via fatty acid release into the tumour microenvironment. Co-culturing human adipocytes and breast cancer cells increased CD36 expression, with fatty acid import into breast cancer cells. Genetic ablation of CD36 attenuates adipocyte-induced epithelial-mesenchymal transition (EMT) and stemness. We show a feedforward loop between CD36 and STAT3; where CD36 activates STAT3 signalling and STAT3 binds to the CD36 promoter, regulating its expression. CD36 expression results in metabolic reprogramming, with a shift towards fatty acid oxidation. CD36 inhibition induces de novo lipogenesis in breast cancer cells. Increased CD36 expression occurs with increased FABP4 expression. We showed that CD36 directly interacts with FABP4 to regulate fatty acid import, transport, and metabolism. CD36 and FABP4 inhibition induces apoptosis in tumour cells. These results indicate that CD36 mediates fatty acid import from adipocytes into cancer cells and activates signalling pathways that drive tumour progression. Targeting CD36 may have a potential for therapy, which will target the tumour microenvironment.

Abstract The breast cancer microenvironment is unique since the breast tissue within which the tumour originates comprises predominantly adipocytes. Adipocytes secrete various growth factors and cytokines that are reported to influence tumour progression by induction of epithelial-mesenchymal transition (EMT) which enhances cancer cell migration, invasion, and metastasis. Immunohistochemical analysis reveals that the invading breast cancer invades surrounding adipose tissues resulting in the delipidation of adipose tissues. To determine the molecular mechanism underlying these events, we co-cultured human adipocytes and breast cancer cells. Co-cultured cells had increased CD36 expression, with fatty acid import. Genetic ablation of CD36 attenuates adipocyte-induced EMT and stemness. Molecular screening for pathways potentially involved in adipocyte-induced CD36 expression reveals activation of the STAT3 signaling axis. Our study identifies a feedforward loop between CD36 and STAT3; where STAT3 binds to the CD36 promoter, activating its expression and CD36 upregulates STAT3 signaling. Breast cancer cells co-cultured with adipocytes actively accumulate fatty acids and undergo metabolic reprogramming, with a shift towards fatty acid oxidation. Seahorse metabolic analysis reveals that co-cultured CD36-expressing cells assumed an energetic phenotype, indicating enhanced mitochondrial respiration. Thus, breast cancer cells alter their metabolic programs, with increased mitochondrial respiration, to meet their energy needs, with the availability of fatty acids. Analysis of breast cancer patient data reveals that increased CD36 expression occurs with increased FABP4 expression. Mechanistic experiments reveal that CD36 directly interacts with FABP4 to regulate fatty acid import, transport, and metabolism in co-cultured breast cancer cells. In vivo studies in mouse models reveal that combined chemical inhibition of CD36 and FABP4 resulted in a significant reduction in tumour growth rate. The study presents an alternative approach by which adipocytes may enhance cancer progression via the transfer of free fatty acids from tumour-associated adipocytes to breast cancer cells. The uptake of free fatty acid subsequently serves as a secondary source of energy to drive breast cancer cell progression. Targeting CD36 in combination with FABP4 may have a potential for therapies targeting the breast cancer microenvironment. Citation Format: Jones Gyamfi, Junjeong Choi, Doru Kwon, JaSeung Koo. Interaction between CD36 and FABP4 regulates the import and metabolism of fatty acid in breast cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P263.