hnRNP L regulates the tumorigenic capacity of lung cancer xenografts in mice via caspase-9 pre-mRNA processing
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
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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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 2

Caspase-9 regulates the tumorigenic capacity in A549 cells.

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Caspase-9 regulates the tumorigenic capacity in A549 cells. (A) Total RN...
(A) Total RNA was isolated from the listed clonal cell lines and analyzed by competitive/quantitative RT-PCR for caspase-9 splice variants. (B) Cells (2 × 103) were plated into 6-well dishes in soft agar and cultured for 14 days before the colony count. (C) Quantization (mean) of the number of colonies for the indicated clonal cell lines. n = 6. Data are mean ± SEM. *P < 0.005, A549 + shRNA control versus A549 + C9b shRNA; **P < 0.001, A549 + vector control versus A549 + C9b ectopic; Student’s t test. (D) Cell lines in A were injected into SCID mice (5 × 106), and tumor volume was measured at the end of 28 days as represented as average size of tumor (cm3). Data are mean ± SEM. *P < 0.05, A549 + control shRNA versus A549 + C9b shRNA; ***P < 0.005, A549 + vector control versus A549 + C9b ectopic Student’s t test. (E) H&E stain of the tumors presented in D with magnification ranging from ×4 to ×40.