Sphingosine Kinase‐2/Sphingosine 1‐Phosphate Signaling Regulates Telomere Dysfunction through GADD45 and TCF21 Mediated Tumor Suppressor Mechanisms in Lung Cancer

Abstract

Sphingosine 1‐phosphate (S1P), a pro‐proliferative sphingolipid generated by sphingosine kinases (SK1 and SK2) has been shown to be up regulated in many cancers. Telomerase, a ribonucleoprotein extends the ends of chromosomes (telomeres), which is involved in cancer pathogenesis and hTERT, the catalytic subunit of telomerase localizes to nucleus to promote lung cancer growth/metastasis. Previously, we have demonstrated that SK2 generated S1Pbinds and stabilizes telomerase in the nuclear periphery by C′3‐OH of S1P and D684 residue of hTERT. Mechanistically, nuclear S1P binding prevented E3ubiquitin ligase MKRN1‐hTERT association, and protected hTERT from degradation, preventing telomere damage, and delaying senescence in non‐cancerous lung fibroblasts (Panneer Selvam et al, Sci Signal, 2015). The objective of the following study is to understand the molecular mechanisms of lung tumor suppression mediated by SK2‐S1P pathway. First, we identified the protein levels of SK2 and hTERT to be upregulated in both non‐small cell (N=48) and small cell lung cancer (N=40) tumor tissues. Mechanistically, pharmacological inhibition of SK2 by ABC294640 drug (in Phase I clinical trials) leads to telomere dysfunction induced foci (TIF) formation in A549 cells as observed by colocalization of γ‐H2AX and TRF‐2 and interestingly wild type hTERT overexpression prevented TIF formation whereas hTERTD684A expression (lacks S1P binding) did not. In vivo, shRNA mediated SK2 knockdown tumors displayed reduced tumor volume, decreased hTERT protein levels and increased TUNEL positive cells. Moreover, pharmacological inhibition of SK2 by ABC294640 (100mg/kg/day body weight for 16days) in an orthotopic mice model for lung cancer showed decreased cancer proliferation as measured by bioluminescence. Interestingly, qPCR based gene array studies revealed ~5 fold increase in the expression levels of epigenetically silenced tumor suppressor TCF21 in stable shSK2 tumors compared to scrambled control tumors. Moreover, we observed increased protein expression of GADD45 (growth arrest and DNA damage response protein), an upstream regulator of TCF21 with concomitant increase in the mRNA levels of TCF21 following SK2 inhibition. Interestingly, Stable shRNA mediated knockdown of TCF21 in A549cells following xenograft studies in SCID mice showed significant protection against ABC294640 mediated tumor suppression. Interestingly, in in vitro experiments we found caspase‐3activation following treatment with ABC294640 and stable shRNA mediated knockdown of GADD45 and TCF21 prevented caspase‐3 activation suggesting caspase‐3activation downstream of GADD45 and TCF21. Also, ABC294640 treated cells showed decreased cell viability and shGADD45 and shTCF21 cells showed significant protection against cell death. Overall, our data suggest that SK2 generated S1Pregulates telomere dysfunction in cancer cells through hTERT thereby leading to GADD45 and TCF21 mediated tumor suppression in lung cancer.Support or Funding InformationNCI 88932‐06