Genomics Research Center, Academia Sinica, Taipei 11529, Taiwan.
Mol Cell Proteomics. 2012 Oct;11(10):901-15. doi: 10.1074/mcp.M112.020875. Epub 2012 Jul 3.
Mutational activation of KRAS promotes various malignancies, including lung adenocarcinoma. Knowledge of the molecular targets mediating the downstream effects of activated KRAS is limited. Here, we provide the KRAS target proteins and N-glycoproteins using human bronchial epithelial cells with and without the expression of activated KRAS (KRAS(V12)). Using an OFFGEL peptide fractionation and hydrazide method combined with subsequent LTQ-Orbitrap analysis, we identified 5713 proteins and 608 N-glycosites on 317 proteins in human bronchial epithelial cells. Label-free quantitation of 3058 proteins (≥2 peptides; coefficient of variation (CV) ≤ 20%) and 297 N-glycoproteins (CV ≤ 20%) revealed the differential regulation of 23 proteins and 14 N-glycoproteins caused by activated KRAS, including 84% novel ones. An informatics-assisted IPA-Biomarker® filter analysis prioritized some of the differentially regulated proteins (ALDH3A1, CA2, CTSD, DST, EPHA2, and VIM) and N-glycoproteins (ALCAM, ITGA3, and TIMP-1) as cancer biomarkers. Further, integrated in silico analysis of microarray repository data of lung adenocarcinoma clinical samples and cell lines containing KRAS mutations showed positive mRNA fold changes (p < 0.05) for 61% of the KRAS-regulated proteins, including biomarker proteins, CA2 and CTSD. The most significant discovery of the integrated validation is the down-regulation of FABP5 and PDCD4. A few validated proteins, including tumor suppressor PDCD4, were further confirmed as KRAS targets by shRNA-based knockdown experiments. Finally, the studies on KRAS-regulated N-glycoproteins revealed structural alterations in the core N-glycans of SEMA4B in KRAS-activated human bronchial epithelial cells and functional role of N-glycosylation of TIMP-1 in the regulation of lung adenocarcinoma A549 cell invasion. Together, our study represents the largest proteome and N-glycoproteome data sets for HBECs, which we used to identify several novel potential targets of activated KRAS that may provide insights into KRAS-induced adenocarcinoma and have implications for both lung cancer therapy and diagnosis.
KRAS 突变激活可促进多种恶性肿瘤的发生，包括肺腺癌。目前对于激活的 KRAS 下游作用的分子靶标的了解还很有限。在这里，我们使用表达激活的 KRAS（KRAS(V12)）和不表达 KRAS 的人支气管上皮细胞（HBECs）来鉴定 KRAS 靶蛋白和 N-糖蛋白。通过 OFFGEL 肽分级和酰肼方法与随后的 LTQ-Orbitrap 分析相结合，我们鉴定了人支气管上皮细胞中 317 个蛋白上的 608 个 N-糖基化位点和 5713 个蛋白。使用无标记定量法对 3058 个蛋白（≥2 个肽段；CV≤20%）和 297 个 N-糖蛋白（CV≤20%）进行定量分析，发现激活的 KRAS 导致 23 个蛋白和 14 个 N-糖蛋白发生差异调节，其中 84%为新发现的蛋白。IPA-Biomarker®过滤器分析的信息学辅助分析优先考虑了一些差异调节蛋白（ALDH3A1、CA2、CTSD、DST、EPHA2 和 VIM）和 N-糖蛋白（ALCAM、ITGA3 和 TIMP-1）作为癌症生物标志物。此外，对包含 KRAS 突变的肺腺癌临床样本和细胞系的微阵列存储库数据进行的综合计算分析显示，在受 KRAS 调节的蛋白中，有 61%（包括生物标志物蛋白 CA2 和 CTSD）的 mRNA 折叠变化呈阳性（p<0.05）。综合验证的最显著发现是 FABP5 和 PDCD4 的下调。通过 shRNA 敲低实验进一步证实了少数经验证的蛋白，包括肿瘤抑制因子 PDCD4，是 KRAS 的靶标。最后，对 KRAS 调节的 N-糖蛋白的研究揭示了在 KRAS 激活的人支气管上皮细胞中 SEMA4B 的核心 N-聚糖结构的改变，以及 TIMP-1 的 N-糖基化在调节肺腺癌 A549 细胞侵袭中的功能作用。总之，我们的研究代表了最大的 HBEC 蛋白质组和 N-糖蛋白质组数据集，我们使用该数据集鉴定了几个新的激活的 KRAS 的潜在靶标，这些靶标可能为 KRAS 诱导的腺癌提供了新的见解，并对肺癌的治疗和诊断具有重要意义。
