Abstract LB-272: KPNA7 nuclear import protein - a key regulator of cancer cell growth and nuclear morphology

Abstract

Abstract Background: Bidirectional nucleocytoplasmic transport unique for eukaryotic cells is a tightly regulated process carried out by specific transport machinery. Its defects result in incorrect localization of proteins that might subsequently lead to diversity of diseases including cancer. Karyopherin alpha 7 (KPNA7) is the newest karyopherin alpha nuclear importer family member, mainly expressed in early embryogenesis and oocytes. However, its expression is reactivated in some cancer cells. We previously showed that KPNA7 promotes the malignant properties of pancreatic cancer cells overexpressing the gene. Here, we identified KPNA7 cargo proteins and further explored the functional consequences of KPNA7 expression for cell proliferation, cell cycle distributions and maintenance of proper nuclear morphology in a panel of pancreatic and breast cancer cell lines with varying KPNA7 expression levels. Methods: To isolate KPNA7 cargo proteins, an affinity-based protein pull-down was performed from stable, tagged KPNA7-overexpressing pancreatic cancer cell lines and the KPNA7 binding partners were identified with mass spectrometry. For functional studies, KPNA7 was silenced in pancreatic and breast cancer cells using siRNAs. Cell numbers and cell cycle distributions were determined 72 to 96 h after transfection and compared to those in corresponding controls. To study mitotic spindle assembly and nuclear morphology, immunofluorescent labeling of γ-tubulin and different nuclear envelope proteins was performed 96 h after transfections. Results: The protein pull-down and mass spectrometry yielded multiple proteins co-purifying with KPNA7. Two cargos, zinc finger protein 414 and major vault protein were successfully validated to bind KPNA7 in vitro and shown to be transported into the nucleus by KPNA7. Additionally, these cargos were shown to possess growth regulatory roles in pancreatic cancer cells. The silencing of KPNA7 in six pancreatic and breast cell lines decreased cell proliferation in all cell lines expressing the gene, whereas both cancer and normal cell lines without endogenous KPNA7 expression were not affected. Also, a decrease in the fraction of proliferating S-phase cells was detected. KPNA7 depletion led to aberrant mitotic spindle assembly and abnormal number of centrosomes. Interestingly, a reorganization of nuclear envelope proteins was observed, resulting in distinct change from round to lobular nuclear morphology. Conclusions: The present results indicate KPNA7 as a central regulator of cancer cell growth and cell cycle, and identify KPNA7 cargo proteins participating in this regulation. We also show that KPNA7, probably via its cargos, has roles beyond nuclear transfer. Our results suggest that KPNA7 functions in the regulation of proper mitosis and organization of the mitotic spindle and acts in the maintenance of nuclear envelope structure and nuclear morphology. This study provides additional evidence on the role of altered nuclear transfer in cancer pathogenesis. Citation Format: Elisa M. Vuorinen, Nina Rajala, Teemu Ihalainen, Hanna E. Rauhala, Anssi Nurminen, Vesa P. Hytonen, Anne Kallioniemi. KPNA7 nuclear import protein - a key regulator of cancer cell growth and nuclear morphology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-272. doi:10.1158/1538-7445.AM2017-LB-272