氟伐他汀通过抑制PHF8增强肾透明细胞癌对舒尼替尼敏感性OA

Objective Sunitinib resistance is an important factor affecting the efficacy of targeted therapy for clear cell renal cell carcinoma(ccRCC),and its underlying molecular mechanisms and effective intervention strategies have not been fully elucidated.This study aims to investigate the role of histone demethylase PHF8 in sunitinib resistance and to evaluate the effect of fluvastatin in targeting PHF8 and enhancing the sensitivity to sunitinib in ccRCC.Methods A sunitinib-resistant ccRCC cell line(786-O-R)and a patient-derived xenograft(PDX)resistant model were established.The expression of PHF8 in these resistant models was validated with Western blotting and immunohistochemical staining.Then 786-O and A498 cell lines with stable PHF8 overexpression and their corresponding cell line-derived xenograft(CDX)models were constructed.The regulatory effect of PHF8 on sunitinib responsiveness was evaluated through in vitro cell proliferation assay,in vivo tumor volume monitoring,and immunohistochemical detection of Ki-67 and CD31 expression.Transcriptome sequencing,Western blotting,and immunohistochemical staining were performed to elucidate the molecular mechanisms by which PHF8 mediates sunitinib resistance.Tumor specimens from 46 ccRCC patients who received sunitinib treatment in Department of Urology of Daping Hospital were analyzed to evaluate the correlations of PHF8 expression with pathological grade and progression-free survival(PFS).An FDA-approved small-molecule drug library was used for virtual screening.RT-qPCR,cycloheximide chase assay,MG132/chloroquine rescue experiments,cellular thermal shift assay(CETSA),surface plasmon resonance(SPR),and molecular docking were conducted to verify the regulatory effect of fluvastatin on PHF8 protein stability.CCK-8 assay and in vivo animal experiments were performed to evaluate the synergistic sensitizing effect of fluvastatin combined with sunitinib on sunitinib-resistant ccRCC cells.Results Stable sunitinib-resistant cell lines and PDX models were successfully constructed.Compared with sunitinib-sensitive control counterparts,the protein expression of PHF8 was markedly upregulated in 786-O-R cells and resistant PDX tumors(Su-P8),indicating that PHF8 may be involved in sunitinib resistance in ccRCC.Further functional experiments demonstrated that PHF8 overexpression significantly reduced the sensitivity of 786-O(IC50:5.406 vs 15.920 µmol/L)and A498 cells(5.186 vs 16.300 µmol/L)to sunitinib.Conversely,PHF8 knockout substantially restored the sensitivity of resistant cells to sunitinib(IC50:13.470 vs 6.355 µmol/L).Consistent with in vitro findings,in vivo experiments showed that after sunitinib treatment,the PHF8-overexpression group exhibited significantly larger tumor volume(P<0.000 1),higher tumor weight(P=0.001 3),and elevated Ki-67 and CD31 expression levels(both P<0.000 1)compared with the control group,confirming that PHF8 drives sunitinib resistance in ccRCC.Clinical sample analysis revealed that high PHF8 expression was significantly correlated with advanced pathological grade(P=0.000 3)and shorter PFS after targeted therapy(P=0.04).Virtual screening targeting PHF8 identified fluvastatin as a potential candidate inhibitor.Fluvastatin downregulated PHF8 expression,increased H3K9me1/2 level,and significantly shortened the half-life of PHF8(P=0.000 7),this effect could be significantly reversed by the proteasome inhibitor MG132,indicating that fluvastatin facilitates proteasome-dependent degradation of PHF8.CETSA results verified that fluvastatin enhanced the thermal stability of PHF8,and SPR analysis further confirmed the direct binding between fluvastatin and PHF8(KD=23.8 µmol/L),with molecular docking predicting their potential binding pattern.Combination treatment with fluvastatin and sunitinib reduced the IC50 value of sunitinib in 786-O-R cells(from 17.460 to 4.601 µmol/L)and exerted a significantly stronger inhibitory effect on cell viability than sunitinib monotherapy(P<0.000 1).Notably,no significant difference was observed between combination therapy and sunitinib monotherapy after PHF8 knockout,demonstrating that the sunitinib-sensitizing effect of fluvastatin is PHF8-dependent.Moreover,in vivo PDX model experiments further validated that fluvastatin supplementation effectively restored the sensitivity of resistant PDX models to sunitinib.Conclusion PHF8 is a critical driver of sunitinib resistance in ccRCC,and its high expression is correlated with advanced pathological grade and poorer prognosis in ccRCC patients receiving sunitinib treatment.Fluvastatin enhance the sensitivity of sunitinib-resistant ccRCC cells to sunitinib by directly targeting PHF8 and promoting its proteasomal degradation.