Abstract 5474: Sphingosine kinase 2/sphingosine 1-phosphate signaling regulates p16 mediated accelerated aging in normal somatic tissues and tcf21 mediated tumor suppression in lung cancer
Authors: Selvam Shanmugam Panneer , Cooley Marion , Helke Kristi , Garrett-Mayer Elizabeth , Smith Charles D. , Ogretmen Besim 
Published: July 2017

Abstract
Sphingosine 1-phosphate (S1P), a pro-proliferative sphingolipid generated by Sphingosine Kinases is upregulated in many cancers. Telomerase (hTERT), is a ribonucleoprotein that extends the ends of chromosomes (telomeres), to promote lung cancer growth. We discovered that SK2 generated S1P binds and stabilizes telomerase in the nuclear periphery by allosterically mimicking protein phosphorylation. Mechanistically, S1P binding protected telomerase from MKRN1 mediated degradation thereby preventing telomere dysfunction and senescence. The objective of this study is to delineate the molecular mechanism of SK2-S1P mediated telomere dysfunction in aging and lung cancer. SK2 knockout mice tissues showed increased telomeric DNA damage and senescence associated-beta galactosidase activity. Moreover, SK2 knockout mice displayed aging phenotypes by reduced subcutaneous fat, atrophy in spleens and hypoplasia in mice testes. Also, SK2 knockout fibroblasts showed increased senescence and robust p16 expression and interestingly, p16 ablation rescued SK2 knockout fibroblasts from senescence. Importantly, we found SK2 and telomerase protein levels to be upregulated in lung cancer tumor tissues (n=48). Mechanistically, pharmacological inhibition of SK2 by ABC294640 (in Phase II clinical trials) leads to telomere dysfunction in A549 cells and interestingly wild type and a phosphomimicking S921D-hTERT mutant of human telomerase overexpression prevented telomere dysfunction whereas S1P defective mutant D684A-hTERT did not. In vivo, inhibition of SK2 by ABC294640 or shRNA knockdown displayed reduced tumor volume, decreased telomerase protein levels and increased TUNEL positive cells. Interestingly, qPCR based gene array revealed robust increase in TCF21 tumor suppressor in stable shSK2 tumors. Furthermore, shRNA against TCF21 following SCID mice xenograft studies showed protection against ABC294640 induced tumor suppression. Interestingly, SK2 inhibition showed ATM/ATR kinase mediated p-CHK1 increase and subsequent activation of caspase-3 in both in vivo and in vitro experiments and stable TCF21 knockdown prevents apoptotic cell death. Importantly, p16 overexpression in p16 deficient A549 lung cancer cells prevented caspase-3 activation and leading to senescence induction following inhibition of SK2-S1P by ABC294640 or shRNA against SK2. Overall, our data suggest that SK2-S1P regulates telomere dysfunction through p16 mediated aging in normal somatic tissues and TCF21 mediated tumor suppression in lung cancer and interestingly SK2-S1P and p16 acts as a molecular switch between senescence and apoptosis in normal and cancer cells respectively.
Citation Format: Shanmugam Panneer Selvam, Marion Cooley, Kristi Helke, Elizabeth Garrett-Mayer, Charles D. Smith, Besim Ogretmen. Sphingosine kinase 2/sphingosine 1-phosphate signaling regulates p16 mediated accelerated aging in normal somatic tissues and tcf21 mediated tumor suppression in lung cancer [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 5474. doi:10.1158/1538-7445.AM2017-5474