Spliceosomic dysregulation in pancreatic cancer uncovers splicing factors PRPF8 and RBMX as novel candidate actionable targets

Abstract

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, characterized by late diagnosis and poor treatment response. Surgery is the only curative approach, only available to early-diagnosed patients. Current therapies have limited effects, cause severe toxicities, and minimally improve overall survival. Understanding of splicing machinery alterations in PDAC remains incomplete. Here, we comprehensively examined 59 splicing machinery components, uncovering dysregulation in pre-mRNA processing factor 8 (PRPF8) and RNA-binding motif protein X-linked (RBMX). Their downregulated expression was linked to poor prognosis and malignancy features, including tumor stage, invasion and metastasis, and associated with poorer survival and the mutation of key PDAC genes. Experimental modulation of these splicing factors in pancreatic cancer cell lines reverted their expression to non-tumor levels and resulted in decreased key tumor-related features. These results provide evidence that the splicing machinery is altered in PDAC, wherein PRPF8 and RBMX emerge as candidate actionable therapeutic targets.

Keywords: PRPF8; RBMX; pancreatic cancer; splicing; splicing factor.

Fig. 1

Splicing dysregulation in Pancreatic Ductal Adenocarcinoma. (A) Fold Change of mRNA levels expressions of Spliceosome Components and Splicing Factors of PDAC FFPE samples compared with non‐tumor adjacent tissue. Data are represented by Fold Change mRNA levels normalized by ACTB expression levels ± SEM. Asterisks indicate significantly differences between groups by Mann–Whitney U test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). (B) Principal Components Analysis (PCA) of the splicing machinery components analyzed in PDAC FFPE samples cohort. (C) sPLSDA analysis showing the best classifying factors between tumor and non‐tumor adjacent tissue in our cohort. Higher expression is shown in red and lower expression in green. (D) STRING analysis of relationships among altered components based on the top genes showing the most differences between the expression in tumor and non‐tumor adjacent tissues.

Fig. 2

PRPF8 and RBMX expression in external cohort and association with clinical parameters. (A) PRPF8 (orange) and RBMX (blue) relative mRNA levels in an external validation Pancreatic Ductal Adenocarcinoma (PDAC) cohort (“Jandaghi, 2016”) [20]. Asterisks indicate significant differences between groups by Mann–Whitney U test (**P < 0.01; ***P < 0.001). (B) Distribution of PRPF8 and RBMX Log₁₀ expression levels normalized by ACTB expression levels in the different Histological grades of PDAC in the in‐house cohort. Median and interquartile range are represented. Asterisks indicate significant differences between groups by Dunn's test (*P < 0.05).

Fig. 3

Survival analysis expression levels in PDAC. (A–C) Kaplan–Meier Survival analysis, Overall Survival and Relapse Free Survival associated with PRPF8 (A) and RBMX (B), mRNA expression levels and their combination (C), respectively, in PanCancer cohort [2]. The respective curves at high (pink) and low (blue) levels of each factor are shown, as well as the P‐value calculated by log‐rank test, the cut‐off point to separate the expression groups and the number at risk in each group.

Fig. 4

Relationship of PRPF8 and RBMX mRNA expression levels with splicing event patterns in PDAC. (A) Volcano‐plot where ΔΨ of total events calculated is plotted against the –log₁₀ P‐value of the Fisher's Exact Test to assay differential splicing events between high and low PRPF8 (orange) and RBMX (blue) expression groups of samples, showing their alternative splicing pattern. (B) Alternative Splicing events characterization of RNA‐seq samples. Total splicing events detected (black) and significantly different events between PRPF8 (orange) and RBMX (blue) expression groups are classified depending on their type, showing different frequencies (%) between both conditions. A5/A3, alternative 5′/3′ splice sites; AF/AL, alternative first/last exons; MXE, mutually exclusive exons; RI, retained intron; SE, skipping exon.

Fig. 5

Relationship of PRPF8 and RBMX mRNA expression levels with expression and mutations of key genes in PDAC. (A) Spearman correlations between PRPF8 (orange) and RBMX (blue) mRNA expression levels and Genome alteration and Mutations in PanCancer cohort. (B) Correlations between PRPF8 (orange) and RBMX (blue) Log₁₀ mRNA expression levels and TP53, KRAS, CDKN2A and SMAD4 mutations in PanCancer cohort. Median and interquartile range are represented. Asterisks indicate significant differences between groups by Mann–Whitney U test (*P < 0.05; **P < 0.01; ****P < 0.0001). (C) Spearman correlations between PRPF8 (orange) and RBMX (blue) mRNA expression levels and TP53, KRAS, CDKN2A, and SMAD4, mRNA expression levels in PanCancer cohort.

Fig. 6

Effect of PRPF8 and RBMX modulation in PDAC. (A, B) RNA expression levels of PRPF8 (A) and RBMX (B) measured in Capan‐2 (n = 4; n = 7; respectively) and BxPC‐3 (n = 7; n = 9) cell lines after overexpression with their respective plasmid compared with mock (control; set at 100%). (C, D) Proliferation rates of Capan‐2, and BxPC‐3 cell lines after PRPF8 (n = 7; n = 6) and RBMX (n = 6; n = 6) overexpression respectively at 24, 48 and 72 h compared with mock (control; set at 100%), represented as a dot line. (E, F) Migration rates of Capan‐2, and BxPC‐3 cell lines after PRPF8 (n = 3; n = 4) and RBMX (n = 6; n = 4) overexpression respectively compared with mock (control; set at 100%), for 24 h. Representative images of wound closures. (G, H) Colony formation capacity of Capan‐2, and BxPC‐3 cell lines after PRPF8 (n = 4; n = 5) and RBMX (n = 5; n = 3) overexpression respectively compared with mock (control; set at 100%). Representative images of colony formation. (I, J) Sphere formation capacity of Capan‐2, and BxPC‐3cell lines after overexpression of PRPF8 (n = 4; n = 6) and RBMX (n = 5; n = 7) respectively compared with mock (control; set at 100%). Representative images of spheres. Data represents mean ± SEM. Asterisks indicate significant differences between groups by Mann–Whitney U test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).