Hu Junhui, Smith Desmond J, Wu Lily
Molecular and Medical Pharmacology, University of California, Los Angeles, CA 90095, USA.
Institute of Urologic Oncology, University of California Los Angeles, Los Angeles, CA 90095, USA.
Int J Mol Sci. 2023 Sep 14;24(18):14075. doi: 10.3390/ijms241814075.
In the current era of tumor genome sequencing, single amino acid missense variants in the von Hippel-Lindau () tumor suppressor gene are frequently identified in clear cell renal carcinoma (ccRCC). Due to the incomplete knowledge of the structural architecture of protein, the functional significance of many missense mutations cannot be assigned. L169P is one such missense mutation identified in the case of aggressive, metastatic ccRCC. Here, we characterized the biochemical activity, transcriptomic hypoxia signature and biological functions of the L169P variant. Lentiviral vector expressing either wildtype (WT) or L169P were used to transduce two -deficient human ccRCC cell lines, 786-O and RCC4. The stability of the protein and the expression level of , and were analyzed. The impact of restoring L169P or WT on the hypoxia gene expression program in 786-O cells was assessed by mRNA sequencing (RNAseq) and computed hypoxic scores. The impact of restoring expression on the growth of ccRCC models was assessed in cell cultures and in chorioallantoic membrane (CAM) xenografts. In the 786-O cells, the protein stability of L169P was comparable to WT . No obvious difference in the capability of degrading and was observed between WT and L169P in the 786-O or RCC4 cells. The hypoxic scores were not significantly different in the 786-O cells expressing either wildtype or L169P . From the cellular function perspective, both WT and L169P slowed cell proliferation in vitro and in vivo. The L169P variant is comparable to WT in terms of protein stability, ability to degrade factors and ability to regulate hypoxia gene expression, as well as in the suppression of ccRCC tumor cell growth. Taken together, our data indicate that the L169P variant alone is unlikely to drive the oncogenesis of sporadic ccRCC.
在当前肿瘤基因组测序时代，在透明细胞肾细胞癌（ccRCC）中经常发现冯·希佩尔-林道（VHL）肿瘤抑制基因中的单氨基酸错义变体。由于对VHL蛋白结构架构的了解不完整，许多错义突变的功能意义无法确定。L169P就是在侵袭性转移性ccRCC病例中发现的这样一种错义突变。在此，我们对L169P变体的生化活性、转录组缺氧特征和生物学功能进行了表征。使用表达野生型（WT）或L169P VHL的慢病毒载体转导两种VHL缺陷的人ccRCC细胞系，786-O和RCC4。分析了VHL蛋白的稳定性以及HIF-1α、HIF-2α和VEGF的表达水平。通过mRNA测序（RNAseq）和计算缺氧评分评估恢复L169P或WT VHL对786-O细胞中缺氧基因表达程序的影响。在细胞培养物和绒毛尿囊膜（CAM）异种移植中评估恢复VHL表达对ccRCC模型生长的影响。在786-O细胞中，L169P VHL的蛋白稳定性与WT VHL相当。在786-O或RCC4细胞中，WT和L169P VHL在降解HIF-1α和HIF-2α的能力上没有明显差异。在表达野生型或L169P VHL的786-O细胞中，缺氧评分没有显著差异。从细胞功能角度来看，WT和L169P VHL在体外和体内均减缓了细胞增殖。L169P VHL变体在蛋白稳定性、降解HIF因子的能力、调节缺氧基因表达的能力以及抑制ccRCC肿瘤细胞生长方面与WT VHL相当。综上所述，我们的数据表明，单独的L169P VHL变体不太可能驱动散发性ccRCC的肿瘤发生。
