VHL missense mutations in the p53 binding domain show different effects on p53 signaling and HIFα degradation in clear cell renal cell carcinoma

Abstract

Clear cell Renal Cell Carcinoma (ccRCC) formation is connected to functional loss of the von Hippel-Lindau (VHL) gene. Recent data identified its gene product, pVHL, as a multifunctional adaptor protein which interacts with HIFα subunits but also with the tumor suppressor p53. p53 is hardly expressed and rarely mutated in most ccRCC. We showed that low and absent p53 expression correlated with the severity of VHL mutations in 262 analyzed ccRCC tissues. In contrast to nonsense and frameshift mutations which abrogate virtually all pVHL functions, missense mutations may rather influence one or few functions. Therefore, we focused on four VHL missense mutations, which affect the overlapping pVHL binding sites of p53 and Elongin C, by investigating their impact on HIFα degradation, p53 expression and signaling, as well as on cellular behavior using ccRCC cell lines and tissues. TP53 mRNA and its effector targets p21, Bax and Noxa, were altered both in engineered cell lines and in tumor tissues which carried the same missense mutations. Two of these mutations were not able to degrade HIFα whereas the remaining two mutations led to HIFα downregulation, suggesting the latter are p53 binding site-specific. The selected VHL missense mutations further enhanced tumor cell survival, but had no effects on cell proliferation. Whereas Sunitinib was able to efficiently reduce cell proliferation, Camptothecin was additionally able to increase apoptotic activity of the tumor cells. It is concluded that systematic characterization of the VHL mutation status may help optimizing targeted therapy for patients with metastatic ccRCC.

Keywords: HIFα; VHL missense mutations; clear cell renal cell carcinoma; p53; pVHL binding sites.

Figure 1

A. p53 protein expression in RCC tissue; B. p53 protein expression in ccRCC and VHL mutation status; and C. p53 protein expression in ccRCC and mutation impact on pVHL stability. Wt: VHL wild type; Mild impact: Destabilizing or neutral or stabilizing; High impact: highly stabilizing or highly destabilizing; LOF (Loss of Function): Frameshift and nonsense mutations. p-value: *<0.05, **<0.01, ***<0.001

Figure 2

A. and B. Western blots showing pVHL, p53, HIF1/2α and HIF1/2α targets expression (CAIX and Glut1) in the established stable cell lines.

Figure 3. mRNA levels of TP53, p21, Bax, and Noxa in the established RCC4 stable cell lines

For each gene the mRNA level of the VHL30 sample was used as reference and compared to the mRNA levels in the other cell lines. p-value: *<0.05, **<0.01 (data are presented as mean +SEM).

Figure 4. mRNA levels of TP53, p21, Bax, Noxa relative to VHL transcription levels in three normal kidney tissues and one VHL wild-type ccRCC (data are presented as mean +SEM)

Figure 5. mRNA levels of TP53, p21, Bax, Noxa relative to VHL wild type transcription levels in ccRCC tissue samples carrying the VHL mutations selected for cell line experiments

No tissue with VHL mutation Ser65Trp (endogenous mutation of RCC4) and Tyr98His was available in our cohort. For each target the mRNA level of the VHL wild-type tumor is used as reference and compared to the mRNA levels in the other tumors. p-value: *<0.05, **<0.01, ***<0.001 (data are presented as mean +SEM)

Figure 6. mRNA levels of TP53, p21, Bax and Noxa in RCC4 expressing VHL (white), VHL deficient (grey), and corresponding HIF1α knockdown (patterned)

p-value: *<0.05, **<0.01, ***<0.001, ****<0.0001 (data are presented as mean +SEM).

Figure 7

A. p21 protein expression in ccRCC tissue and VHL mutation type; B. p53/p21 combined expression and VHL mutation type. Wt: wild-type, ms: missense, LOF: Loss of function. p-value: *<0.05, **<0.01, ***<0.001

Figure 8. Effects of VHL mutations on apoptosis and cell proliferation

A. Apoptotic behavior of VHL wild type and mutated RCC4 cells; B. apoptotic behavior of RCC4 with missense mutations in the p53 and HIF1/2α binding domains; C. Proliferative behavior of VHL wild type and mutated RCC4 cells; D: Proliferative behavior of RCC4 with missense mutations in the p53 and HIF1/2α binding domains. VHL30 expression was used as reference and compared to the other cell lines. p-value: *<0.05, **<0.01 (data are presented as mean +SEM).

Figure 9

A. Apoptotic and B. proliferative behavior of VHL wt and mutated RCC4 cells after treatment with Camptothecin, Camptothecin and Sunitinib (Combination), and Sunitinib. Signals after treatment of each cell line were normalized with corresponding mock signals (without treatment). VHL30 was used as reference and compared to the other cell lines. p-value: *<0.05, **<0.01 (data are presented as mean +SEM).

Figure 10. Comparison of predicted effects and own data of VHL mutations affecting the p53 and the HIF1/2α binding sites of pVHL and possible treatment strategies