RBM10 promotes transformation-associated processes in small cell lung cancer and is directly regulated by RBM5

Abstract

Lung cancers are the leading cause of cancer-related deaths worldwide, with small cell lung cancer (SCLC) being the most aggressive type. At the time of diagnosis, SCLC has usually already metastasized, and an astonishing 95% of patients eventually succumb to the disease. This highlights the need for more effective SCLC screening and treatment options. Interestingly, the earliest and most frequent genetic alteration associated with lung cancers involves a lesion in the region to which the RNA binding protein RBM5 maps. We have recently shown that a decrease in RBM5 expression may be a key step in SCLC development, as RBM5 regulated many transformation-associated processes in SCLC cells. RBM5 is structurally and functionally similar to another RNA binding protein, RBM10. Both proteins have tumor-suppressor properties in a variety of cancer cell lines, and it has been suggested that RBM5 expression can influence RBM10. Due to their similarities, and the recent evidence that RBM10 is mutated in up to 21% of lung cancers, we hypothesized that RBM10 would share RBM5’s tumor-suppressor properties in SCLC. Using transcriptome analysis and functional assays, we show, however, that RBM10’s function was opposite to what we hypothesized; in the endogenously RBM5-null GLC20 SCLC cell line, RBM10 actually promoted cell proliferation and other transformation-associated processes. Using RNA immunoprecipitation followed by next generation sequencing (RIP-Seq) and Western blotting, we demonstrate that RBM5 post-transcriptionally regulated RBM10 expression via direct interaction with specific RBM10 splice variants. We propose a working model describing the impact of this interaction on cellular processes. Our results provide evidence that RBM10 expression, in RBM5-null tumors, may contribute to tumor growth and metastasis. Measurement of both RBM10 and RBM5 expression in clinical samples may therefore hold prognostic and/or potentially predictive value.

RBM10: Harmful or helpful‐many factors to consider

“…As the GLC20 cells used in this study almost exclusively expressed RBM10v1 at the protein level, it was most likely RBM10v1 that was interacting with the RBM10v2 RNA. Strikingly, although RBM10v1 protein levels were substantially higher than RBM10v2 protein levels, mRNA expression levels of RBM10v1 and RBM10v2 were very similar, suggesting that the interaction of RBM10v1 protein with RBM10v2 RNA is integral to an RBM10 translational self‐regulation mechanism, at least in GLC20 cells . Of note, in the study by Sun et al described above, RBM10v1 overexpression was followed by decreased RBM10v2 expression at both the mRNA and protein levels, suggesting that multiple levels of RBM10 self‐regulation exist in HEK293 cells.…”

“…Interestingly, an earlier manuscript by Bechara et al showed that mutation of this alternatively spliced valine residue to glutamic acid did alter RBM10 function, supporting the notion that the alternative splicing of this codon could have functional implications for RBM10. In addition, our group recently showed that RBM5 can distinguish between these +/− GTG RBM10 splice variants; RBM5 specifically bound only RBM10v2(V277del) . Taken together, these results suggest that the alternative splicing of RBM10 results in changes in protein tertiary structure that either directly (eg, +/− valine in the RRM domain) or indirectly (eg, RBM10v1 versus RBM10v2) influence function.…”

“…This is highlighted in Figure , which demonstrates that very little is known regarding RBM10‐splice variant interacting factors. In light of the fact that different isoforms may have opposing functions, the ratio of the various RBM10 isoforms in a patient could be of predictive significance for disease incidence and/or progression.…”

“…For instance, both positive and negative expression correlations have been noted: (1) in HEK293 human embryonic kidney cells, RBM10 overexpression correlated with increased RBM5 exon 6 exclusion and overall decreased RBM5 mRNA levels, whereas RBM10 knockdown correlated with a slight decrease in RBM5 exon 6 exclusion and overall higher RBM5 mRNA expression levels; (2) in SHSY5Y human neuronal cells, RBM10 knockdown correlated with increased levels of RBM5 protein; (3) in H9c2 rat myoblast cells, RBM5 knockdown correlated with decreased RBM10 mRNA expression; and (4) in GLC20 RBM5 ‐null small cell lung cancer cells, increased RBM5 expression correlated with increased protein expression of specific RBM10 splice variants . Of note, the first and fourth study described not only showed a correlation between RBM5 and RBM10 expression, but also direct interactions between them: (1) in HEK293 cells, RBM10 bound the RBM5 intron 5 5′‐splice site and intron 6 3′‐splice site, influencing RBM5 alternative splicing and ultimately leading to RBM5 AS‐NMD; and (2) in GLC20 cells, RBM5 bound only one specific RBM10 splice variant, and was associated with increased protein expression of RBM10v2 . Considered together, these results demonstrate that relationships between RBM5 and RBM10 occur across cell types and species, highlighting their potential fundamental importance to the cell.…”

“…For instance, many initial functional studies associated RBM10 expression with increased apoptosis, decreased cell proliferation, decreased colony formation, and decreased xenograft tumor growth . Counterintuitively, however, very recent studies have suggested a tumor promoter role for RBM10, and associated RBM10 expression with additional processes and mechanisms of action . This functional dichotomy highlights the importance of not only gaining a deeper understanding of the downstream consequences of RBM10 expression, but identifying the mechanisms responsible for regulating RBM10 itself.…”