Several studies have demonstrated the roles of FABP7 in promoting aggressiveness of cancers, including melanomas, by supporting tumor growth and metastatic process. We discovered for the first time, FABP7’s role in regulating melanoma phenotypic shift, using a combination of in vitro experiments, an animal model, and metabolomic analysis.
FABP7 was originally found to be highly expressed in the brain, remarkably in astrocytes and other glial cells. Since melanomas originate from the neural crest cell-derived melanocytes, they may retain the high FABP7 expression from their origin. Goto, et al, demonstrated that FABP7 mRNA copies are higher in the primary melanoma, than the metastatic site30. While patient-acquired tissue immunohistochemistry by Slipicevic, et al. revealed highest FABP7 expression in benign nevi lesion, followed by primary and metastatic melanomas at similar levels31. These fluctuations hint that FABP7 may be downregulated during the malignant transformation and metastatic processes, either as metastasis initiator, or as a result from metastatic processes.
We found relatively higher FABP7 expression in high-metastatic B16F10 murine melanoma cells compared to B16F1, the low-metastatic variant35,36. Our finding may appear contradictory to the results from patient tissue studies. However, FABP7 expression in cell lines was observed in vitro, in a stable state, therefore, it cannot represent in vivo fluctuations. Nonetheless, it is plausible that high FABP7 expression supports B16F10 invasiveness. Our in vivo models also showed a relatively higher FABP7 expression in the lungs, than at the primary tumor site, which may be due to a different metastatic process and could not represent the naturally occurred metastasis which involves multiple steps, since our method involved direct injection of tumor cells into the bloodstream. The strong FABP7 expressing metastatic foci in our model could be explained by ROS scavenging capacity. Direct exposure to blood stream can be stressful to melanoma cells and lead to reactive oxygen species-induced cell death19, which could be attenuated by lipid droplets37. Bansaad, et al, had shown that FABP3 and FABP7 protect glioblastoma cells from ROS during hypoxia-reoxygenation by enhancing lipid droplet formation38, it is reasonable that melanoma cells with higher FABP7 expression will similarly survive better in harmful environment then successfully form tumor foci at distant sites.
In our study, Fabp7-KO B16F10 cells had higher capability to invade through ECM-coated membrane without noticeable change in proliferation, contradicting to other studies which showed decreased melanoma proliferation, migration, and invasion in absence of FABP7 29,31. Our results showed slower primary tumor growth in Fabp7-KO group, suggesting its importance in cell proliferation in vivo. Presence of complex tumor microenvironment and available nutrients may account for the different findings from in vitro experiments. Higher tumor foci count in the lungs of Fabp7-KO group corresponds to the increased invasiveness found in trans-well experiments, but the tumor sizes were smaller than the WT group. This, once again, supports its role in tumor proliferation, which might also be mitigated in KO group, leading to slower tumor growth despite successfully forming a metastatic focus. Still, a similar deterioration was not apparent in in vitro results. It is very likely that the TME can dramatically affect the outcome. Since melanoma is a solid tumor of cells forming in a spheroid mass, each tumor cells may be affected by the environment differently. For example, cells at the tumor core may be less exposed to nutrients or oxygen than those at the periphery, especially in rapidly growing tumor without sufficient vascularization.39 With limited nutrients, tumor cells will have to switch to different modes of metabolism, such as using lipids as main source of energy, instead of glucose.40 Therefore, Fabp7-KO cells with impaired fatty acid transportation, would be susceptible to such metabolic stress, halting their proliferation or undergoing apoptosis, leading to an overall smaller tumor size. This locational nutrient availability is likely not present in monolayer cell culture format. Additionally, other TME components, including immune cells, fibroblasts, adipocytes, and various ECM molecules and cytokines, further complicate the tumor progression.41–43
Since FABP7 prefers PUFAs as its ligand23, Fabp7-KO cells presumably have impaired uptake and transportation of these fatty acids, leading to lower overall amount of unsaturated fatty acids and eventually, increasing the saturated to unsaturated lipid ratio. Imbalance between saturated (SFA) and unsaturated fatty acids can affect cell behaviors20,44. Cell membrane which incorporates unsaturated fatty acids has higher fluidity, enhancing cell motility during invasion, while excessive cellular SFAs can lead to ER stress, unfolded protein response, and ceramide formation21. These conditions can activate cellular stress signaling and eventually induce phenotypic shifting toward the invasive phenotype45. The evidence of lipid saturation change was only apparent in incorporated lipids, but not FFAs, suggests the role of FABP7 as FA transporter since FFAs may be regulated by other entities. Considering FABP7’s binding affinity toward specific FAs, the reduction of AA in Fabp7-KO cells is not unexpected. As AA is the precursor of multiple inflammatory cytokines that benefit tumor growth,46 a lower level of this FA could hinder tumor growth at primary site as well as at metastatic foci.
We found evidence of phenotypic shifting in KO cells, characterized by an increase in Axl expression and various EMT markers. While there is no change in Mitf, its original expression is very low in the wild type cells and remains low in the KO cells, resulting in the invasive MitfLow/AxlHigh signature.
Among multiple factors that can influence the phenotypes, TGF-β signaling is a well-studied pathways known to induce invasion and de-differentiation8. KO cells have higher expression of TGF-β receptors, therefore, an increase in TGF-β signaling could be expected. Kagawa, et al27,47, showed that FABP7 regulates caveolin-1 (Cav-1) expression in gliomas and astrocytes. Apart from changes in membrane FA component that determine the lipid raft function, Cav-1 also plays a significant role in membrane lipid raft formation and can inhibit TGF-β/SMAD signaling via the TGF-β type I receptor48. As a result, these receptors may be disinhibited in Fabp7-KO cells, leading to increased TGF-β signaling.
In epithelial cancers, TGF-β signaling is also known to induce EMT by formation of SMAD complex that binds to DNA34. Different markers used in this study were expressed at different stages of the transition. In brief, epithelial tumor cells generally express CDH1 which codes for E-cadherin, the main component of cell-cell adhesion proteins in tight junctions, that maintain the integrity of epithelial tissue49. CDH2, on the other hand, codes for N-cadherin, which is upregulated in mesenchymal stage, associated with cancer invasiveness50.
Cdh2 was upregulated in Fabp7-KO cells, hinting the mesenchymal-like transition. Meanwhile, upregulation of the epithelial gene Cdh1 was also observed, suggesting a partial EMT-like transformation, that may support the transition back to the proliferative phenotype after successful metastatic seeding51. ZEB1 and ZEB2 are transcription factors that repress E-cadherin expression, eventually promoting the mesenchymal transition52. Thereby, Zeb2 mRNA upregulation in Fabp7-KO cells can also account for the enhanced invasiveness.
Essentially, changes in these genes indicate that FABP7 can regulate melanoma aggressiveness by preventing it from shifting into the invasive phenotype. This regulation occurs both through direct alterations of structural FA components and indirectly through TGF-β and stress signaling, which lead to EMT-like transitioning alongside the phenotypic shift.
In conclusion, this study explores the roles of FABP7 concerning melanoma phenotypic plasticity and lipid metabolism. FABP7 plays an important role in regulating the balance of lipid saturation by preserving UFA levels within the cell. In the absence of FABP7, SFA to UFA ratio increases, likely triggering stress responses and eventually the phenotypic shift toward the invasive phenotype. Through this mechanism, melanoma cells acquire higher metastatic capability, but with a reduction in primary tumor growth. These insights could be potential for finding candidates regarding lipid metabolism, and development of better melanoma therapies.