RNA binding motif protein 10 suppresses lung cancer progression by controlling alternative splicing of eukaryotic translation initiation factor 4H

Abstract

Background: RNA splicing defects are emerging molecular hallmarks of cancer. The gene encoding splicing factor RNA binding motif protein 10 (RBM10) has been found frequently mutated in various types of cancer, particularly lung adenocarcinoma (LUAD), but how RBM10 affects cancer pathogenesis remains to be determined. Moreover, the functional roles and clinical significance of RBM10 mutation-associated splicing events in LUAD are largely unknown.

Methods: RBM10 mutations and their functional impacts were examined in LUAD patients from a Chinese patient cohort and The Cancer Genome Atlas (TCGA). Alternative splicing (AS) changes induced by RBM10 mutations in LUAD were identified by RNA sequencing and correlated with patient survival. Functions of RBM10 and the splice variants of eukaryotic translation initiation factor 4H containing or lacking exon 5 (EIF4H-L and EIF4H-S respectively) in LUAD development and progression were examined by cellular phenotypic assays and xenograft tumour formation.

Findings: RBM10 mutations in LUAD generally lead to loss-of-function and cause extensive alterations in splicing events that can serve as prognostic predictors. RBM10 suppresses LUADprogression largely by regulating alternative splicing of EIF4H exon 5. Loss of RBM10 in LUAD enhances the expression of EIF4H-L in LUAD. EIF4H-L, but not EIF4H-S, is critical for LUAD cell proliferation, survival and tumourigenesis.

Interpretation: Our study demonstrates a new molecular mechanism underlying RBM10 suppressive functions in lung cancer and the therapeutic value of RBM10-regulated AS events, providing important mechanistic and translational insights into splicing defects in cancer.

Keywords: Alternative splicing; EIF4H; Lung cancer; RBM10.

Fig. 1

Characterization of RBM10 mutations in lung adenocarcinoma (LUAD). (a) Categories of RBM10 mutations identified in a cohort of Chinese LUAD patients. (b) Locations of mutations within RBM10 protein. RRM: RNA recognition motif; ZF: zinc finger; OCRE, Octamer Repeat; NLS: nuclear localization signal; G-patch: Glycine-patch. (c) RT-qPCR analysis of relative RBM10 RNA levels in LUAD tissue samples harbouring RBM10 nonsense, frameshift or missense mutations or lacking RBM10 mutations (wild type) compared to their adjacent non-tumour tissues. * P < 0.05, ** P < 0.01, ns: not significant, one-way ANOVA followed by Dunnett's tests for comparisons with wild type. (d) Representative images of RBM10 immunohistochemistry analysis of LUAD tissue samples with mutated or wild type RBM10 (RBM10 MUT and RBM10 WT). Scale bar: 50 µm for 20 × magnification, and 20 µm for 40 × magnification. Relative RBM10 staining intensities from low to high were scored 0, 1, 2, and 3. (e) Summary and quantification of immunohistochemistry results in (d). *** P < 0.0001, Fisher's exact tests. (f) Co-mutation plot for RBM10 and known oncogenic mutations identified in the Chinese LUAD patient cohort, with their smoking status, age and gender shown at the bottom. Known oncogenic mutations had previously been identified in these patients [29,50,51].

Fig. 2

RBM10 mutations lead to extensive alternative splicing (AS) changes in LUAD tissues. (a) Experimental design for identifying RBM10 mutation-associated AS events in LUAD patient tissues. LUAD tissues with RBM10 nonsense or frameshift mutations (RBM10 MUT), with wild type RBM10 expression (RBM10 WT) and their matched adjacent non-tumour tissues from Chinese patients were used for RNA-Seq experiments. (b) Splicing changes between RBM10 MUT and RBM10 WT LUAD samples (P < 0.05, |splicing change| > 0.1). CE: cassette exon; A3SS: alternative 3′ splice site; A5SS: alternative 5′ splice site; MEX: mutually exclusive exon; RI: retained intron. (c) The numbers of AS events associated with RBM10 mutations in each AS category. (d) Hierarchical clustering of LUAD tissue samples with or without RBM10 mutation and their adjacent non-tumour tissue samples based on AS events affected by RBM10 mutations. Each row represents an AS event, and each column represents a tissue sample. Two representative AS events were enlarged and shown at the bottom. (e) RT-PCR validation of AS changes in RBM10 mutated, RBM10 wild type LUAD and their adjacent non-tumour tissues from Chinese patients. Left panel: agarose gel image; right panel: quantification result. PSI: percent-spliced-in; T: LUAD tissues, N: non-tumour tissues. Shown are two representative AS events. (f, g) Kaplan-Meier survival curves of TCGA LUAD patients stratified by exon inclusion levels of individual (f) and combination (g) of RBM10 mutation-associated AS events. HR: hazard ratio. P values were calculated by Log-rank tests.

Fig. 3

RBM10 suppresses LUAD development and progression. (a-f) Cellular proliferation rates (a; n = 3; inserts are Western blot analysis of RBM10 protein levels, loading control: β-actin, Dox: doxycycline.), cell cycle (b; n = 5), anchorage-dependant colony formation (c; n = 3 for PC9, n = 4 for A549; scale bar: 1 cm), in vitro migration (d; n = 3; scale bar: 50 µm) and invasion (e; n = 4 for PC9, n = 3 for A549; scale bar: 50 µm) and apoptosis (f; n = 3) under inducible overexpression of RBM10 in LUAD cells. (g) Western blot analysis of indicated marker proteins under RBM10 overexpression. Loading control: β-actin. (h) Growth curve of xenograft tumours in nude mice following subcutaneous injection of PC9 cells with or without RBM10 overexpression. Error bar: ±SD. (i) Xenograft tumours were removed at day 27 and weighed. n = 5 for Dox- group, n = 6 for Dox+ group; scale bar: 1 cm; error bar: ±SD. (j) Western blot analysis of indicated marker proteins in xenograft tumours described in (i). Loading control: β-actin. Error bars represent ±SEM unless indicated. * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant. Two-way ANOVA followed by Bonferroni's tests were used in a and h; Student's t-tests were used in b, c, d, e, f and i.

Fig. 4

RBM10 regulates alternative splicing (AS) of cancer-associated genes. (a) Experimental design for identifying AS changes induced by RBM10 knockdown (RBM10_KD) in lung epithelial BEAS-2B cells and overexpression (RBM10_OE) in LUAD PC9 cells. (b) Splicing changes following RBM10 KD in BEAS-2B cells or RBM10 OE in PC9 cells (P < 0.05, |splicing change| > 0.1). AS types were indicated in Fig. 2b. (c) The numbers of altered splicing events in each AS category. (d) RT-PCR validation of RBM10-regulated AS events under the indicated conditions. Agarose gel images (lower panel) and quantification of exon inclusion levels (upper panel) are shown for 3 representative genes. PSI: percent-spliced-in. Error bars represent +SEM, n = 3–4 biological replicates; * P < 0.05, ** P < 0.01, *** P < 0.001; One-way ANOVA followed by Dunnett's tests were used for comparisons with control (siCtrl) in BEAS-2B cells; Student's t-tests were used for comparisons with control (Ctrl) in PC9 and A549 cells.

Fig. 5

RBM10 suppresses LUAD progression by regulating EIF4H splicing. (a) Exon/Intron structures and encoded proteins of EIF4H splice variants containing or lacking exon 5. RRM: RNA recognition motif. Vhs: virion host shutoff (Vhs) protein binding region. (b) Venn diagram of genes whose splicing was affected by RBM10 mutations in Chinese and TCGA LUAD samples (our data and TCGA public data respectively) and regulated by RBM10 knockdown (RBM10_KD) in BEAS-2B and RBM10 overexpression (RBM10_OE) in PC9 cells. (c) Protein expression of two EIF4H splice variants following RBM10 knockdown in BEAS-2B cells or RBM10 overexpression in A549 and PC9 cells examined by Western blot analysis. Shown are the representative results of three biological replicates. Dox: doxycycline. (d) Protein levels of EIF4H splice variants in xenograft tumours shown in Fig. 3i. Loading control: β-actin. (e) Effects of RBM10 silencing and overexpression on EIF4H exon 5 splicing were examined using minigene splicing reporter assays. Upper panel: scheme of minigene reporter. Lower panel: representative agarose gel images (left) and quantification of results (right). (f, g, h) Cell proliferation rates (f; n = 3) and anchorage independent (PC9) or dependant (A549) colony formation (g; Upper panel: representative images, scale bar: 1 mm for PC9 and 1 cm for A549 cells, respectively; Lower panel: quantification of results; n = 4) and apoptosis (h; n = 3) of LUAD cells under conditions of control (Dox-/Ctrl), overexpression of RBM10 alone (Dox+/Ctrl), overexpression of EIF4H-L alone (Dox-/EIF4H-L), or both proteins together (Dox+/EIF4H-L). (i) Expression of indicated proteins under conditions in (f) were examined by Western blot. Loading control: β-actin. Error bars represent ±SEM or +SEM; * P < 0.05, ** P < 0.01, *** P < 0.001, ns: not significant; Student's t-tests in e; One-way ANOVA followed by Turkey's tests for multiple comparisons in g, h; two-way ANOVA followed by Bonferroni's tests in f.

Fig. 6

EIF4H exon 5 inclusion is crucial for LUAD cell proliferation and a potential therapeutic target for LUAD. (a) Western blot analysis of EIF4H protein expression in indicated LUAD cells transfected with control siRNA (siCtrl), siRNAs targeting total EIF4H (si-All) or selectively targeting the long (si-L) or short (si-S) EIF4H variants. (b, c, g) Cell proliferation rates (b, g) and apoptosis (c) of indicated LUAD cells under conditions described in (a). (d, h) Western blot analysis of cell cycle and apoptotic markers under conditions described in (a). Loading control: β-actin. (e, i) Cell proliferation rates dramatically decreased following antisense oligonucleotide (ASO)-mediated blockade of EIF4H exon 5 inclusion in indicated LUAD cells. Targeting location and sequence of the ASO were shown in the upper panel. (f, j) Western blot analysis of indicated proteins under conditions in (e, i) respectively. Loading control: β-actin. (k) Growth curves of xenograft tumours formed by H1944 cells transfected with siRNAs against EIF4H-L or control. The sizes of xenograft tumours were measured at indicated time points. n = 6 per condition. Error bar: ±SD. (l) Xenograft tumours were removed at day 27 and weighed. Error bar: ±SD. Error bars represent ±SEM unless indicated; n = 3 biological replicates unless indicated; * P < 0.05, ** P < 0.01, *** P < 0.001; two-way ANOVA followed by Bonferroni's tests compared to siCtrl in b, e, g, i and k; One-way ANOVA followed by Dunnett's tests compared to siCtrl in c; and Student's t-test in l.