Quantitative Proteomics Reveals Regulation of Karyopherin Subunit Alpha-2 (KPNA2) and Its Potential Novel Cargo Proteins in Nonsmall Cell Lung Cancer

Abstract

The process of nucleocytoplasmic shuttling is mediated by karyopherins. Dysregulated expression of karyopherins may trigger oncogenesis through aberrant distribution of cargo proteins. Karyopherin subunit alpha-2 (KPNA2) was previously identified as a potential biomarker for nonsmall cell lung cancer by integration of the cancer cell secretome and tissue transcriptome data sets. Knockdown of KPNA2 suppressed the proliferation and migration abilities of lung cancer cells. However, the precise molecular mechanisms underlying KPNA2 activity in cancer remain to be established. In the current study, we applied gene knockdown, subcellular fractionation, and stable isotope labeling by amino acids in cell culturebased quantitative proteomic strategies to systematically analyze the KPNA2-regulating protein profiles in an adenocarcinoma cell line. Interaction network analysis revealed that several KPNA2-regulating proteins are involved in the cell cycle, DNA metabolic process, cellular component movements and cell migration. Importantly, E2F1 was identified as a potential novel cargo of KPNA2 in the nuclear proteome. The mRNA levels of potential effectors of E2F1 measured using quantitative PCR indicated that E2F1 is one of the "master molecule" responses to KPNA2 knockdown. Immunofluorescence staining and immunoprecipitation assays disclosed colocalization and association between E2F1 and KPNA2. An in vitro protein binding assay further demonstrated that E2F1 interacts directly with KPNA2. Moreover, knockdown of KPNA2 led to subcellular redistribution of E2F1 in lung cancer cells. Our results collectively demonstrate the utility of quantitative proteomic approaches and provide a fundamental platform to further explore the biological roles of KPNA2 in nonsmall cell lung cancer. Molecular & Cellular

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“…Our study provided additional evidence for the interaction between KPNA2 and TP53 in U87 cells. In another recent study in non-small cell lung cancer, the association between KPNA2 and TP53 is also demonstrated, 49 and TP53 downstream targets are also affected by KPNA2 knockdown. TP53, known as the "guardian of the cellular genome" and the "cellular gatekeeper," plays a fundamental role in the regulation of autophagy according to its cellular location.…”

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High Throughput Sequencing and Proteomics to Identify Immunogenic Proteins of a New Pathogen: The Dirty Genome Approach

“…CD-151 also affects the tumor vasculature. Moreover, the overall survival rate of CD-151 positive patients was 45.8% compared to CD-151 negative patients. Further, they have deciphered its molecular modulator network to establish it as a novel drug target [110].…”

“…Kashyap candidate biomarkers in oral squamous cancer using tissue culture-based system [44]. In a similar type of study, Wang et al established the regulatory network of karyopherin subunit alpha-2 (KPNA2) as a novel cargo protein in nonsmall cell lung carcinoma (NSCLC) to further establish KPNA2 as a candidate biomarker for NSCLC [45]. In a different kind of approach, Cuomo et al used this versatile technique for the identification of histone signatures in breast cancer cell lines.…”