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.
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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

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Figure 8

The role of hnRNP L in regulating the alternative splicing of caspase-9 in non-transformed human bronchial epithelial cells.

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The role of hnRNP L in regulating the alternative splicing of caspase-9 ...
(A) Total protein lysates from A549, H838, H2030, and HBEC-3KT cell lines were subjected to SDS-PAGE analysis and immunoblotted for hnRNP L and β-actin. (B) HBEC-3KT cells were transfected with 100 nM control siRNA or 100 nM hnRNP L SMARTpool siRNA for 48 hours. Total RNA was isolated and analyzed by competitive/quantitative RT-PCR for caspase-9 splice variants. Simultaneously, protein lysates were also produced, subjected to SDS-PAGE, and immunoblotted for hnRNP L and β-actin. Data are n = 4 from 2 separate occasions. (C) HBEC-3KT cell lines were transfected with either wild-type hnRNP L (0.25 μg) in conjunction with caspase-9 minigene (0.25 μg) or caspase-9 minigene (0.25 μg) in conjunction with empty vector (0.25 μg) for 24 hours. Total RNA was extracted and analyzed by competitive/quantitative RT-PCR for caspase-9 minigene splice variants. Data are n = 3 on 2 separate occasions. Of note: the ratio of caspase-9a/9b minigene mRNA tended to present with a slightly higher ratio than endogenous caspase-9a/9b mRNA.