Alternative splicing of caspase 9 is modulated by the phosphoinositide 3-kinase/Akt pathway via phosphorylation of SRp30a.
Increasing evidence points to the functional importance of alternative splice variations in cancer pathophysiology. Two splice variants are derived from the CASP9 gene via the inclusion (Casp9a) or exclusion (Casp9b) of a four-exon cassette. Here we show that alternative splicing of Casp9 is dysregulated in non-small cell lung cancers (NSCLC) regardless of their pathologic classification. Based on these findings we hypothesized that survival pathways activated by oncogenic mutation regulated this mechanism. In contrast to K-RasV12 expression, epidermal growth factor receptor (EGFR) overexpression or mutation dramatically lowered the Casp9a/9b splice isoform ratio. Moreover, Casp9b downregulation blocked the ability of EGFR mutations to induce anchorage-independent growth. Furthermore, Casp9b expression blocked inhibition of clonogenic colony formation by erlotinib. Interrogation of oncogenic signaling pathways showed that inhibition of phosphoinositide 3-kinase or Akt dramatically increased the Casp9a/9b ratio in NSCLC cells. Finally, Akt was found to mediate exclusion of the exon 3,4,5,6 cassette of Casp9 via the phosphorylation state of the RNA splicing factor SRp30a via serines 199, 201, 227, and 234. Taken together, our findings show that oncogenic factors activating the phosphoinositide 3-kinase/Akt pathway can regulate alternative splicing of Casp9 via a coordinated mechanism involving the phosphorylation of SRp30a.
Department of Biochemistry, Virginia Commonwealth University, VAMC, and Massey Cancer Center, Richmond, Virginia 23298, USA.
Jacqueline C
JC
Shultz
Rachel W
RW
Goehe
D Shanaka
DS
Wijesinghe
Charuta
C
Murudkar
Amy J
AJ
Hawkins
Jerry W
JW
Shay
John D
JD
Minna
Charles E
CE
Chalfant
D017398:Alternative Splicing; D015153:Blotting, Western; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D053453:Caspase 9 / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000069347:Erlotinib Hydrochloride; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008175:Lung Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism*; D011799:Quinazolines / Q000494:pharmacology; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D000068103:Serine-Arginine Splicing Factors; D015398:Signal Transduction
D007527:Isoenzymes; D009687:Nuclear Proteins; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D000069347:Erlotinib Hydrochloride; D019869:Phosphatidylinositol 3-Kinases; D066246:ErbB Receptors; D051057:Proto-Oncogene Proteins c-akt; D053453:Caspase 9