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Background/Objective: Clear cell renal cell carcinoma (ccRCC) is characterized by a high degree of functional intratumoral heterogeneity (ITH). This is highlighted by the finding that tumor cell proliferation and intracellular signaling occur preferentially in the tumor periphery. The driving forces for such a spatial organization are largely unknown. Herein, we investigate the role of the tumor microenvironment in the control of tumor cell proliferation and functional ITH. Methods: Conditioned media (CM) derived from nonmalignant peritumoral kidney tissue were used to stimulate RCC cells in vitro. A neutralization assay was used to characterize the role of FGF-2 in the CM. The molecular mechanisms underlying the action of CM on RCC cells were investigated us-ing immunoblotting, flow cytometry and immunofluorescence microscopy. Lastly, a series of ccRCCs were stained for Ki-67 and p27 Kip1 , and expression was analyzed in both tumor periphery and center. Results: We show that CM derived from nonmalignant kidney cells adjacent to an RCC can downregulate the expression of the CDK inhibitor p27 Kip1 through enhanced protein degradation in an FGF-2-dependent fashion. FGF-2 functions mainly through the PI3K/AKT pathway downstream of its receptors, and RCC cells with constitutively high AKT activity show not only an enhanced degradation of p27 Kip1 through the Emi1-Skp2 axis, but also a subcellular mislocalization of p27 Kip1 to the cytoplasmic compartment. Such a mislocalization was also detected in the tumor periphery in vivo suggesting that p27 Kip1 plays an important role in shaping this spatial niche. Conclusions: Our results suggest that the tumor microenvironment is involved in shaping the tumor peripheral niche by stimulating the enhanced proliferation that is characteristic for this zone.

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