Hitefield Naomi L, Mackay Stephen, Hays Lauren E, Chen Shimin, Oduor Ian O, Troyer Dean A, Nyalwidhe Julius O
Department of Microbiology and Molecular Cell Biology, Eastern Virginia Medical School, Norfolk, VA 23507, USA.
Leroy T. Canoles Jr. Cancer Research Center, Eastern Virginia Medical School, Norfolk, VA 23507, USA.
Biomedicines. 2023 Mar 24;11(4):1010. doi: 10.3390/biomedicines11041010.
Renal-cell carcinoma (RCC) is a heterogeneous disease consisting of several subtypes based on specific genomic profiles and histological and clinical characteristics. The subtype with the highest prevalence is clear-cell RCC (ccRCC), next is papillary RCC (pRCC), and then chromophobe RCC (chRCC). The ccRCC cell lines are further subdivided into prognostic expression-based subtypes ccA or ccB. This heterogeneity necessitates the development, availability, and utilization of cell line models with the correct disease phenotypic characteristics for RCC research. In this study, we focused on characterizing proteomic differences between the Caki-1 and Caki-2 cell lines that are commonly used in ccRCC research. Both cells are primarily defined as human ccRCC cell lines. Caki-1 cell lines are metastatic, harboring wild-type , whereas Caki-2 are considered as the primary ccRCC cell lines expressing wild-type von Hippel-Lindau protein (pVHL). Here, we performed a comprehensive comparative proteomic analysis of Caki-1 and Caki-2 cells using tandem mass-tag reagents together with liquid chromatography mass spectrometry (LC/MS) for the identification and quantitation of proteins in the two cell lines. Differential regulation of a subset of the proteins identified was validated using orthogonal methods including western blot, q-PCR, and immunofluorescence assays. Integrative bioinformatic analysis identifies the activation/inhibition of specific molecular pathways, upstream regulators, and causal networks that are uniquely regulated and associated with the two cell lines and RCC subtypes, and potentially the disease stage. Altogether, we have identified multiple molecular pathways, including NRF2 signaling, which is the most significantly activated pathway in Caki-2 versus Caki-1 cells. Some of the differentially regulated molecules and signaling pathways could serve as potential diagnostic and prognostic biomarkers and therapeutic targets amongst ccRCC subtypes.
肾细胞癌（RCC）是一种异质性疾病，根据特定的基因组图谱、组织学和临床特征可分为几种亚型。患病率最高的亚型是透明细胞肾细胞癌（ccRCC），其次是乳头状肾细胞癌（pRCC），然后是嫌色细胞肾细胞癌（chRCC）。ccRCC细胞系进一步细分为基于预后表达的亚型ccA或ccB。这种异质性使得有必要开发、获取和利用具有正确疾病表型特征的细胞系模型用于RCC研究。在本研究中，我们专注于表征ccRCC研究中常用的Caki-1和Caki-2细胞系之间的蛋白质组差异。这两种细胞主要被定义为人类ccRCC细胞系。Caki-1细胞系具有转移性，携带野生型 ，而Caki-2被认为是表达野生型冯·希佩尔-林道蛋白（pVHL）的原发性ccRCC细胞系。在此，我们使用串联质量标签试剂结合液相色谱质谱（LC/MS）对Caki-1和Caki-2细胞进行了全面的比较蛋白质组分析，以鉴定和定量这两种细胞系中的蛋白质。使用包括蛋白质印迹、q-PCR和免疫荧光测定在内的正交方法验证了所鉴定蛋白质子集的差异调节。综合生物信息学分析确定了特定分子途径、上游调节因子和因果网络的激活/抑制，这些途径、调节因子和网络受到独特调节并与这两种细胞系和RCC亚型以及潜在的疾病阶段相关。总之，我们已经鉴定出多个分子途径，包括NRF2信号通路，它是Caki-2细胞与Caki-1细胞中最显著激活的途径。一些差异调节的分子和信号通路可作为ccRCC亚型中潜在的诊断和预后生物标志物以及治疗靶点。
