Sphingosine Kinase-2/Sphingosine 1-Phosphate Signaling Regulates Telomerase in Lung Cancer

Sphingosine 1-phosphate (S1P), a pro-proliferative sphingolipid generated by sphingosine kinases (SK1 and SK2) is up regulated in lung cancer. The role of SK1 generated S1P has been well studied whereas nuclear SK2/S1P function remains understudied. Telomerase is a ribonucleoprotein that extends the ends of chromosomes (telomeres). Telomerase is involved in cancer pathogenesis and hTERT, the catalytic subunit of telomerase localizes to nucleus to promote lung cancer growth/proliferation/metastasis. In our experiments tissue microarrays (TMA's) of lung adenocarcinomas showed elevated SK2 protein levels compared to their paired normal tissues, also up regulation of SK2 was significantly associated with hTERT expression. Moreover RNAi against SK2 decreased hTERT protein levels whereas SK1 RNAi had no measurable effect. Ectopic expression of hTERT in SK2-/- MEFs showed decreased half-life when compared to wild type and SK1-/- MEFs upon cycloheximide treatment indicating hTERT protein instability. Interestingly, wild type SK2 expression rescued hTERT from degradation whereas the catalytic mutant SK2G212E did not. SK2 inhibition by molecular approach or by ABC294640 drug showed increased association of hTERT with MKRN1, an E3 ligase that ubiquitinates and targets hTERT for degradation and interestingly exogenous addition of S1P prevented hTERT-MKRN1 association. Accordingly, siRNA mediated knockdown of MKRN1 prevented hTERT degradation in response to SK2 inhibition. Moreover stable SK2 knockdown in tumor xenografts displayed significantly decreased tumor volume with concomitant decrease in hTERT levels in vivo. Additionally, inhibition of SK2 using ABC294640 in an orthotopic mice model for lung cancer showed decreased cancer proliferation as measured by bioluminescence. Interestingly, SK2-S1P regulates telomerase stability and function in a telomere replication independent mechanism (non-canonical), in vivo SCID mice xenograft models using stable expression of wild type and dominant-negative telomerase showed significant protection against ABC294640 compared to vector controls, without altering its telomere length. Furthermore, SK2-hTERT knockdown lead to increased activation of an emerging tumor suppressor TCF21 and in vivo knockdown of TCF21 in tumor xenografts lead to significant protection against ABC294640 treatment. In summary, these data indicate that SK2 stabilizes telomerase in a telomere-independent mechanism in lung cancer and targeting SK2 induces rapid ubiquitination/degradation of hTERT leading to suppression of lung tumor growth.