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, Ser 52 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.

hnRNP U Enhances Caspase-9 Splicing and Is Modulated by AKT-dependent Phosphorylation of hnRNP L

“…Therefore, we hypothesized that phosphorylation events might impact the association between hnRNP U and C9/E3. Nevertheless, our findings in this study demonstrate that phosphorylation of hnRNP U is unlikely to play a role, and the regulatory mechanism is mainly via phosphorylation of hnRNP L. Together with the findings that caspase-9a/9b ratio is higher in nontransformed than in transformed cells (4,5) and the enhanced association of hnRNP U in nontransformed cells, these data extend the mechanistic insights into the dysregulation of caspase-9 splicing in transformed cells. In this paradigm, certain survival/oncogenic kinases are activated and phosphorylate hnRNP L in transformed cells.…”

“…Moreover, cascape-9b is not significantly expressed in nontransformed cells (4), and the induction of caspase-9b expression is due to activation of hnRNP L via phosphorylation to compete/ inhibit hnRNP U association with C9/E3. Therefore, targeting the phosphorylation of hnRNP L by specific kinase inhibitors to further augment the formation of apoptotic caspase-9a over anti-apoptotic caspase-9b would be an attractive cancer-specific approach for treatment of NSCLC, the leading cause of cancer-related death in both men and women worldwide (24).…”

“…Mass Spectrometry Analysis-Analysis of RNA-protein complexes was performed at the Emory University Mass Spectrometry Center (Atlanta, GA) as previously described (4). Briefly, RNA-protein complexes retrieved from EMSA were excised from the gel following by in-gel trysin digestion.…”

“…The ratio of caspase-9a/9b isoform is dysregulated in nonsmall cell lung cancer (NSCLC) 2 cells (4,5), and the factors implicated in regulating the alternative splicing of caspase-9 include endogenous ceramides (6), SRSF1 (SRp30a or ASF/SF2) (5,7,8), and heterogeneous nuclear ribonucleoprotein L (hnRNP L) (4). De novo ceramide synthesis and SRSF1 have been documented to down-regulate the level of caspase-9b mRNA in contrast to hnRNP L, which represses the inclusion of four-exon cassette to favor the formation of caspase-9b.…”

“…De novo ceramide synthesis and SRSF1 have been documented to down-regulate the level of caspase-9b mRNA in contrast to hnRNP L, which represses the inclusion of four-exon cassette to favor the formation of caspase-9b. Importantly, phosphorylation of SRSF1 or hnRNP L and subsequent effects on the caspase-9 splicing process have been shown to be important in sensitizing NSCLC cells to chemotherapeutics and promoting the tumorigenic capacity of these cells (4,8).…”