hnRNP L regulates the tumorigenic capacity of lung cancer xenografts in mice via caspase-9 pre-mRNA processing
Rachel Wilson Goehe, … , John D. Minna, Charles E. Chalfant
Rachel Wilson Goehe, … , John D. Minna, Charles E. Chalfant
Published October 25, 2010
Citation Information: J Clin Invest. 2010;120(11):3923-3939. https://doi.org/10.1172/JCI43552.
View: Text | PDF
Research Article

hnRNP L regulates the tumorigenic capacity of lung cancer xenografts in mice via caspase-9 pre-mRNA processing

Abstract

Caspase-9 is involved in the intrinsic apoptotic pathway and suggested to play a role as a tumor suppressor. Little is known about the mechanisms governing caspase-9 expression, but post-transcriptional pre-mRNA processing generates 2 splice variants from the caspase-9 gene, pro-apoptotic caspase-9a and anti-apoptotic caspase-9b. Here we demonstrate that the ratio of caspase-9 splice variants is dysregulated in non–small cell lung cancer (NSCLC) tumors. Mechanistic analysis revealed that an exonic splicing silencer (ESS) regulated caspase-9 pre-mRNA processing in NSCLC cells. Heterogeneous nuclear ribonucleoprotein L (hnRNP L) interacted with this ESS, and downregulation of hnRNP L expression induced an increase in the caspase-9a/9b ratio. Although expression of hnRNP L lowered the caspase-9a/9b ratio in NSCLC cells, expression of hnRNP L produced the opposite effect in non-transformed cells, suggesting a post-translational modification specific for NSCLC cells. Indeed, Ser52 was identified as a critical modification regulating the caspase-9a/9b ratio. Importantly, in a mouse xenograft model, downregulation of hnRNP L in NSCLC cells induced a complete loss of tumorigenic capacity that was due to the changes in caspase-9 pre-mRNA processing. This study therefore identifies a cancer-specific mechanism of hnRNP L phosphorylation and subsequent lowering of the caspase-9a/9b ratio, which is required for the tumorigenic capacity of NSCLC cells.

Authors

Rachel Wilson Goehe, Jacqueline C. Shultz, Charuta Murudkar, Sanja Usanovic, Nadia F. Lamour, Davis H. Massey, Lian Zhang, D. Ross Camidge, Jerry W. Shay, John D. Minna, Charles E. Chalfant

×

Figure 9

Ser52 of hnRNP L is hyperphosphorylated in NSCLC cells and regulates the alternative splicing of caspase-9.

Options: View larger image (or click on image) Download as PowerPoint
Ser52 of hnRNP L is hyperphosphorylated in NSCLC cells and regulates the...
(A) A549 and HBEC-3KT cell lines were seeded at the same confluency and in the same culture media 24 hours before IP. IP hnRNP L was resolved by SDS-PAGE and immunoblotted with phospho-specific and hnRNP L antibodies. (B) A549 cells were transfected with WT-hnRNP L (L-WT) (1 μg), Ser52Ala hnRNP L (S52-A) (1 μg), or Ser52Asp hnRNP L (S52-D) (1 μg). Total RNA was analyzed for caspase-9 splice variants. n = 4 from 3 occasions. (C) A549 cells were transfected with WT-hnRNP L, S52-A, or S52-D. Protein lysates were subjected to SDS-PAGE and immunoblotted for myc-tag and β-actin. Empty vector showed no expression of a myc-tagged protein. (D) A549 and HBEC-3KT cell lines seeded at the same confluency and in the same culture media for 24 hours before IP. Cell lines were transfected with WT-hnRNP L (1 μg) or S52-A (1 μg). Ectopically expressed hnRNP L was IP with c-myc tag Ab, resolved by SDS-PAGE, and immunoblotted with anti–phospho-serine and anti–c-myc tag antibodies. (E) Protein lysates were subjected to SDS-PAGE and immunoblot for phospho-Ser52 hnRNP L and hnRNP L. Phospho-Ser52 antibody for hnRNP L was validated by ELISA, hnRNP shRNA samples, and lack of identifying the Ser52Ala mutant of hnRNP L. (F) Colony formation assays in soft agar. n = 6; error bars represent SEM. *P < 0.005, A549 + S52-A hnRNP L + C9b ectopic versus A549 + S52-A hnRNP L.