Abstract 2036: Keratin 17 mediates p27KIP1-nuclear export, proliferative signaling and tumor growth
Authors: Escobar-Hoyos Luisa F. , Shah Ruchi , Roa-Peña Lucia , Najafian Nilofar , Vanner Elizabeth , Banach Anna , Nielsen Erik , Al-Khalil Ramsey , Akalin Ali , Talmage David , Shroyer Kenneth 
Published: August 2015

Abstract
One of the most fundamental traits of cancer cells is the ability to sustain proliferation by circumventing cell cycle regulatory programs normally controlled by tumor suppressors. Keratin 17 (K17) is not expressed in most somatic tissues but is overexpressed and is a negative prognostic marker in cervical, breast, ovarian, and gastric carcinomas. The mechanisms by which K17 contributes to cancer-related signaling, however, remain unknown. Here, we report that nuclear-localized K17 interacts with tumor suppressor p27KIP1 and mediates its nuclear export, maintaining proliferation.
After validating K17 is a prognostic marker in cervical cancer, independent of tumor stage, we performed in vitro and in vivo experiments to investigate the role of K17 in proliferative signaling and tumor growth using loss- and gain- of function approaches in cervical cancer and other cancer-derived cell lines. We found that K17 expression maintains proliferation, while silencing K17 induces G1 arrest by nuclear accumulation and stabilization of tumor suppressors p27KIP1 and pRb. During G1, K17 localizes in the nucleus, mediated by a classical bipartite nuclear localization signal (NLS) (AA385-400) that was identified by in silico analysis. This NLS is specific among keratins and present only in primates but not in other species. To our knowledge, this is the first report that a keratin has a NLS and is capable of undergoing nuclear translocation.
During G1, p27KIP1is exported from the nucleus in a CRM1-dependent manner and is subsequently degraded, triggering G1/S transition. p27KIP1 lacks the classical leucine-rich nuclear export signal (NES) and requires an adaptor for CRM1-exportin binding. After confirming the binding of K17 and p27KIP1 within the nucleus, we performed in silico analyses and identified a leucine-rich NES required for CRM1-binding in K17 (AA194-199), which was further validated by a 3-fold nuclear retention of K17 and p27KIP1 after leptomycin B treatment. We introduced point mutations within the K17-NES (mNES) and the K17-NLS (mNLS). Nuclear p27KIP1 was lost in cells expressing wild-type K17. In contrast, nuclear p27KIP1 levels were 3-fold higher in cells expressing either mNLS or mNES. Furthermore, nuclear localization of K17 was abolished in mNLS cells.
Xenografts of cervical cancer cells showed that tumors derived from K17 expressing cells were 3-times larger than those derived from K17 knockdown cells, the latter showing increased nuclear p27KIP1 and decreased PCNA and Ki67 expression. Finally, we identified an inverse correlation between K17 and p27KIP1 expression in human cervical cancer specimens, intertumorally and intratumorally.
These data suggest a model in which nuclear-localized K17 acts as an oncoprotein promoting G1/S transition in cancer cells, by mediating exportin binding and nuclear translocation of tumor suppressor p27KIP1, contributing to sustained proliferation, tumor aggressiveness and poor-patient outcome.
Citation Format: Luisa F. Escobar-Hoyos, Ruchi Shah, Lucia Roa-Peña, Nilofar Najafian, Elizabeth Vanner, Anna Banach, Erik Nielsen, Ramsey Al-Khalil, Ali Akalin, David Talmage, Kenneth Shroyer. Keratin 17 mediates p27KIP1-nuclear export, proliferative signaling and tumor growth. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2036. doi:10.1158/1538-7445.AM2015-2036