Juan L. López-Cánovas, Natalia Hermán-Sánchez, Mercedes del Rio-Moreno, Antonio C. Fuentes-Fayos, Araceli Lara-López, Marina E. Sánchez-Frias, VÃctor Amado, Rubén Ciria, Javier Briceño, Manuel de la Mata, Justo P. Castaño, Manuel Rodriguez-Perálvarez, Raúl M. Luque, Manuel D. Gahete | Experimental & Molecular Medicine | 06 January 2023 | Volume 55 | Pages132–142
Abstract
Hepatocellular carcinoma (HCC) pathogenesis is associated with alterations in splicing machinery components (spliceosome and splicing factors) and aberrant expression of oncogenic splice variants. We aimed to analyze the expression and potential role of the spliceosome component PRPF8 (pre-mRNA processing factor 8) in HCC. PRPF8 expression (mRNA/protein) was analyzed in a retrospective cohort of HCC patients (n = 172 HCC and nontumor tissues) and validated in two in silico cohorts (TCGA and CPTAC). PRPF8 expression was silenced in liver cancer cell lines and in xenograft tumors to understand the functional and mechanistic consequences. In silico RNAseq and CLIPseq data were also analyzed. Our results indicate that PRPF8 is overexpressed in HCC and associated with increased tumor aggressiveness (patient survival, etc.), expression of HCC-related splice variants, and modulation of critical genes implicated in cancer-related pathways. PRPF8 silencing ameliorated aggressiveness in vitro and decreased tumor growth in vivo. Analysis of in silico CLIPseq data in HepG2 cells demonstrated that PRPF8 binds preferentially to exons of protein-coding genes, and RNAseq analysis showed that PRPF8 silencing alters splicing events in multiple genes. Integrated and in vitro analyses revealed that PRPF8 silencing modulates fibronectin (FN1) splicing, promoting the exclusion of exon 40.2, which is paramount for binding to integrins. Consistent with this finding, PRPF8 silencing reduced FAK/AKT phosphorylation and blunted stress fiber formation. Indeed, HepG2 and Hep3B cells exhibited a lower invasive capacity in membranes treated with conditioned medium from PRPF8-silenced cells compared to medium from scramble-treated cells. This study demonstrates that PRPF8 is overexpressed and associated with aggressiveness in HCC and plays important roles in hepatocarcinogenesis by altering FN1 splicing, FAK/AKT activation and stress fiber formation.
Introduction
Hepatocellular carcinoma (HCC) is the most prevalent type of primary liver cancer and the fourth most common cancer worldwide1. The majority of HCC cases are associated with chronic liver diseases due to alcohol consumption, chronic viral hepatitis or metabolic syndrome, among other etiologies1. Since the 1990s, the incidence of HCC has increased dramatically, and HCC-related mortality is increasing faster than that of other cancer types1. The elevated death rates observed for HCC could be associated with its late diagnosis despite routine screening strategies with liver ultrasound every 6 months and because the available therapies have a limited impact on overall survival2. Therefore, a better understanding of hepatocellular carcinogenesis could help to identify new diagnostic, prognostic and therapeutic targets.
A common hallmark of cancer is the alteration of important elements regulating cell physiology, especially the presence or aberrant expression of splice variants, which could be associated with development, progression and drug resistance in different types of cancer3. In fact, splice variants of important genes such as CDCC50, KLF6, and FN14,5 are involved in liver carcinogenesis, thus suggesting that an altered splicing process could play an essential role in the development and progression of HCC6.
The splicing process is controlled by the spliceosome, a macromolecular ribonucleoprotein complex that cooperates with hundreds of splicing factors to catalyze this process7. This splicing machinery is essential for appropriate modulation of gene expression, and its dysregulation is associated with oncogenic progression, including in HCC8, and with the generation of an aberrant landscape of alternative splice variants9. In this context, the splicing factor PRPF8 (pre-mRNA processing factor 8) is a key protein in the catalytic nucleus of the spliceosome, which participates in the second step of the splicing process. In Drosophila, loss of PRPF8 decreases cell proliferation, increases cell death and modulates cell differentiation and polarity, and PRPF8-mediated hyperplastic growth is induced by different oncogenes10. Indeed, PRPF8 seems to be crucial for appropriate constitutive and alternative mRNA splicing11. PRPF8 mutations have been associated with severe forms of retinitis pigmentosa as well as with the initiation of various types of myeloid neoplasms and with decreased survival in patients with leukemia12. In solid tumors, silencing of PRPF8 was found to result in cancer subtype-specific implications in breast cancer cell lines13, while in prostate cancer, PRPF8 is involved in androgen receptor splicing14. In addition, recent evidence suggests that PRPF8 may also be overexpressed in HCC and be associated with the tumorigenic potential; however, these conclusions were based on a single HCC cohort and experiments with a single HCC cell line15. Based on this information, and using several in vitro approaches, animal models, and human samples, we aimed to explore the putative dysregulation, association with clinical parameters and functional role of PRPF8 in an ample number of HCC cohorts and cell lines; the implication of PRPF8 in the control of the splicing process in HCC; and the potential utility of its genetic modulation in hepatocarcinogenesis.
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