The RNA-binding protein LARP1 is a post-transcriptional regulator of survival and tumorigenesis in ovarian cancer

Abstract

RNA-binding proteins (RBPs) are increasingly identified as post-transcriptional drivers of cancer progression. The RBP LARP1 is an mRNA stability regulator, and elevated expression of the protein in hepatocellular and lung cancers is correlated with adverse prognosis. LARP1 associates with an mRNA interactome that is enriched for oncogenic transcripts. Here we explore the role of LARP1 in epithelial ovarian cancer, a disease characterized by the rapid acquisition of resistance to chemotherapy through the induction of pro-survival signalling. We show, using ovarian cell lines and xenografts, that LARP1 is required for cancer cell survival and chemotherapy resistance. LARP1 promotes tumour formation in vivo and maintains cancer stem cell-like populations. Using transcriptomic analysis following LARP1 knockdown, cross-referenced against the LARP1 interactome, we identify BCL2 and BIK as LARP1 mRNA targets. We demonstrate that, through an interaction with the 3' untranslated regions (3' UTRs) of BCL2 and BIK, LARP1 stabilizes BCL2 but destabilizes BIK with the net effect of resisting apoptosis. Together, our data indicate that by differentially regulating the stability of a selection of mRNAs, LARP1 promotes ovarian cancer progression and chemotherapy resistance.

Figure 1.

LARP1 knockdown causes alteration in the cancer cell transcriptome. (A) Frequency distribution of Log₂-fold change in transcript abundance in OVCAR8 cells following transient LARP1 knockdown (LARP1 knockdown relative to control). (B) Venn diagram showing the overlap between genes with altered mRNA abundance on LARP1 knockdown, with the published LARP1–mRNA interactome, together with the P-value for the hypergeometric probability of this overlap, calculated using the R dhyper function (based on 2086 differentially expressed mRNAs and an mRNA interactome with 6784 members) (15). Of the 758 genes present in both datasets molecular ontology enrichment with Ingenuity Pathway Analysis (IPA) was performed (-log[BH-corrected P-value] shown, bold dashed line indicates P = 0.05). (C) RT-qPCR validation of fold changes in mRNA abundance of key survival-associated genes identified from the IPA enrichment analysis following LARP1 knockdown. ***P < 0.001, **P < 0.01, *P < 0.05. Student t-test. Minimum of three experimental repeats. Error bars indicate SEM.

Figure 2.

LARP1 is a component of BCL2-containing mRNP complexes and promotes transcript stability. (A) Schematic of LARP1 RNA-immunoprecipitation (RIP) with representative western blot of LARP1 protein following LARP1-immunoprecipiation in OVCAR8 and SKOV3 cells. (B) RT-qPCR analysis of BCL2 and BIK mRNA obtained from RIP with anti-LARP1 and IgG control antibodies in OVCAR8 cells. OZA1 and 28S were included as negative controls. (C) Representative cell images following LARP1 knockdown and 8 h exposure to actinomycin D (Scale bar 200 μm). (D) Following transient knockdown of LARP1, OVCAR8 cells were treated with actinomycin D to halt transcription and apoptosis (as determined by cleaved Caspase 3/7) was monitored using the CaspaseGlo assay (data normalized to T = 0, at the time of actinomycin-D administration). **P < 0.01, *P < 0.05. (E) Stability of BIK and BCL2 mRNA following treatment with actinomycin D for 6 h. Relative abundance was determined by RT-qPCR (ΔΔCt). MAPK14 was chosen as a negative control as its mRNA abundance did not alter on LARP1 knockdown in the RNA-seq dataset. Student t-test. Minimum of three experimental repeats. Error bars indicate SEM. (F) Western blotting of BIK and BCL2 protein levels following LARP1 knockdown.

Figure 3.

LARP1 regulates stability at the level of the 3′ UTR and binds the BCL2 3′ UTR via its DM15 domain. (A) Schematics outlining construction of 3′-untranslated region (3′ UTR) reporter constructs for BIK and BCL2. Both were compared relative to a control vector with no additional 3′ UTR sequence. (B) OVCAR8 and SKOV3 cells were co-transfected with Renilla luciferase 3′ UTR constructs and a control Firefly luciferase control vector. Renilla luciferase activity following LARP1 knockdown was determined for each 3′ UTR construct cells. Data was normalized to Firefly luciferase activity. (C) OVCAR8 cells were co-transfected with Renilla luciferase 3′ UTR constructs and a control Firefly luciferase control vector and Renilla luciferase mRNA abundance following LARP1 knockdown was determined using RT-qPCR. (D) Electrophoretic mobility shift assay (EMSA) of LARP1 with a fragment of the BCL2 3′ UTR (construct 3). The affinity of this interaction is indicated. (E) Competition experiment analysed by EMSA of LARP1 pre-bound to radiolabelled RNA representing the 5′ TOP of RPS6 and competed with cold competitors, as indicated. (F) Confocal immunofluorescence microscopy of SKOV3 cells treated with sodium arsenite to trigger aggregation of mRNP bodies. Cells were stained for LARP1 protein (green) and either the P-body marker DCP1a or stress granule marker PABP (both red). Scale bar 10 μm (top) and 25 μm (bottom).

Figure 4.

LARP1 knockdown increases basal apoptosis and chemosensitivity. (A) Cell viability following transient LARP1 knockdown in OVCAR8 and SKOV3 cells determined by MTT assays. ***P < 0.001, **P < 0.01, *P < 0.05. (B) Relative levels of cleaved Caspase 3/7 determined by the CaspaseGlo assay in OVCAR8 and SKOV3 cells 24 h after transient LARP1 knockdown. (C) Western blot analysis of cleaved PARP in OVCAR3 cells transduced with lentiviral shLARP1 constructs. (D) Cell cycle distribution determined by propidium iodide staining following transient LARP1 knockdown. (E) Schematic of cell transfection and cisplatin (CDDP) treatment with representative OVCAR8 cell images in each condition (Scale bar 200 μm). (F) Percentage of Annexin V-positive cells following transient LARP1 knockdown and treatment for 24 h with 25 μM cisplatin in platinum-resistant OVCAR8 cells with representative dual-colour flow cytometry plots. (G) Normalized cell viability determined by MTT-based assay in OVCAR8 cells following LARP1 knockdown and treatment with 25 μM cisplatin. ***P < 0.001, **P < 0.01, *P < 0.05. Student t-test. Minimum of three experimental repeats. Error bars indicate SEM.

Figure 5.

Oncogenic effects of LARP1 in vivo. (A) SKOV3 control (shGFP) and LARP1 knockdown (shLARP1) cells were injected subcutaneously in SCID-beige mice and tumour volume monitored over time (inset, western blot of LARP1 knockdown in implanted cells). (B) Final tumour weights at sacrifice. (C) Ki67 percentage nuclear positivity of fixed and embedded xenograft tumours analysed by immunohistochemistry and representative examples xenograft tumours stained with anti-Ki67 antibody (scale bar 100 μm). (D) Western blot of LARP1 knockdown in SKOV3 stable cell lines using lentiviral transduction, with schematic of cell injection protocol and representative tumours (scale bar 1 cm). (E) Limiting dilution assay results from SKOV3 cells injected subcutaneously into NSG mice. (F) Kaplan-Meier curves of tumour-free survival for mice receiving 1 × 10⁶ cells (n = 5) and 1 × 10⁵ cells (n = 6). Log-rank test. (G) Final tumour volumes for mice receiving 1 × 10⁶ cells and 1 × 10⁵ cells, respectively. ***P < 0.001, **P < 0.01, *P < 0.05. Student t-test. Error bars indicate SEM.

Figure 6.

LARP1 is required for the maintenance of populations positive for putative cancer stem cell markers. (A) Representative histogram plot of CD133⁺ OVCAR3 cells determined by flow cytometry (a line with <10% CD133-positivity) following LARP1 knockdown. (B) Mean CD133⁺ populations determined by flow cytometry following LARP1 knockdown in OVCAR3, IGROV1 and HeLa (cervical carcinoma-derived) cells. (C) Percentage of CD133⁺ OVCAR3 cells following treatment with the anti-CSC agent salinomycin. (D) Representative flow cytometry plots of ALDEFLUOR^bright OVCAR3 cells, with high aldehyde dehydrogenase activity, following LARP1 knockdown, determined using the Aldefluor assay. (E) Percentage of ALDEFLUOR^bright OVCAR3 cells following LARP1 knockdown determined using the Aldefluor assay. (F) Relative mRNA expression of key stem cell-associated transcription factors following LARP1 knockdown (ΔΔCt). ***P < 0.001, **P < 0.01, *P < 0.05. Student t-test. Minimum of three experimental repeats. Error bars indicate SEM.

Figure 7.

BCL2 can rescue the LARP1 apoptotic phenotype. (A) BCL2 promoter activity following LARP1 knockdown (firefly luciferase mRNA normalized to renilla luciferase mRNA control). (B) Western blot analysis of BCL2 protein levels following LARP1 knockdown. (C) Western blot analysis of BCL2 protein levels following BCL2 knockdown. (D) Percentage of Annexin V-positive OVCAR8 cells, determined by flow cytometry, following transient BCL2 knockdown, with and without co-treatment with cisplatin (25 μM). (E) Percentage of CD133-posititve OVCAR3 cells, determined by flow cytometry, following transient knockdown of BCL2. (F) Percentage of Annexin V-positive OVCAR8 cells, determined by flow cytometry, following LARP1 knockdown or transfection with control siRNA and treatment with cisplatin (25 μM), with co-transfection of a Flag-tagged control (F-empty) or BCL2-overexpression (F-BCL2) construct. Minimum of three experimental repeats. Student t-test. Error bars indicate SEM.

Figure 8.

LARP1 is highly expressed in ovarian cancers and predicts poor prognosis. (A) Representative TMA cores of normal ovarian tissue and EOC samples, stained with anti-LARP1 antibody (scale bar 250 μm). (B) LARP1 immunohistochemical scoring in healthy ovaries and ovarian cancers. (C) Multivariate cox-regression analysis of overall survival in 67 cases of ovarian cancer. Hazard ratios for LARP1 score, as determined by IHC analysis of sectioned tumours, given for a 10-point change in score. (D) A TMA of pre-treatment ovarian tumour tissue from 281 participants in the SCOTROC4 study was stained with anti-LARP1 antibody, and scored independently. Kaplan-Meier analysis of progression-free survival (PFS) and overall survival (OS) is shown, with patients stratified by LARP1 score (Median PFS 16.5 versus 11.0 months and OS 48.5 versus 27.4 months in LARP1 low and high tumours, respectively).

Figure 9.

LARP1 expression in ovarian tumors correlates with BIK and BCL2 expression. (A) Correlation of mRNA expression (Log₂RPKM) in ovarian tumors (n = 412) between LARP1 and BCL2 or BIK (Pearson R). Data from TCGA Ovarian RNAseq cohort (tcga-data.nci.nih.gov). (B) Comparison of upper and lower quartiles of LARP1 expression (Log₂RPKM, n = 103 in each), by BCL2 or BIK expression (Wilcoxon test). Data as before. (C) A summary of the role of LARP1 in the ovarian cancer cell. As a component of mRNP complexes containing transcripts of survival-associated genes, LARP1 acts to simultaneously stabilize anti-apoptotic mRNAs, whilst destabilizing pro-apoptotic transcripts. The net effect is to promote apoptosis evasion, enhancing tumorigenicity, chemoresistance and cancer stem cell (CSC)-like traits.

Figure 10.

LARP1 protein is detectable in patient blood plasma. (A) A schematic of the LARP1 sandwich enzyme-linked immunosorbent assay (ELISA). (B) The LARP1 ELISA standard curve. (C) Plasma LARP1 concentration in healthy female controls and patients with primary ovarian malignancies sampled prior to primary surgery. (D) Plasma LARP1 concentration for 19 patients with underlying ovarian malignancy sampled before and after primary surgery. Students t-test. Error bars indicate SEM.