RBFOX2 as Key Factor in Metastatic Signature of Pancreatic Cancer

Pancreatic Cancer, Genomics: Pancreatic Cancer, GenomicsPancreatic ductal adenocarcinoma (PDA) is notoriously difficult to treat and has a poor prognosis with a 5-year survival rate of only 11 percent. Local aggressive invasion and metastatic spread define its high mortality rate. The examination of pancreatic cancer genomes has identified four key driver genes: KRAS, SMAD4, CDKN2A, and TP53, responsible for the initial development of solid tumors. Although these mutations remain consistent as the disease advances, no specific genetic mutation has been found to trigger the spread of cancer to other parts of the body. Additionally, the role of alternative splicing and splicing factors in PDA is not well understood and has been the subject of few reports. Consequently, researchers are exploring alternative causes of disease progression in PDA, as the lack of a genetic driver for metastases necessitates new approaches to understanding the disease. Recently, a multi-institution collaboration investigated the potential role of RNA splicing in PDA. The study was published in Nature (2023; https://doi.org/10.1038/s41586-023-05820-3). A research group at the Hebrew University Faculty of Medicine, Jerusalem, analyzed RNA splicing data from primary and metastatic PDA tumors where they identified splicing events correlating with PDA development. They used principal component analysis determined with profiling alternative splicing instead of gene expression to RNA sequencing datasets from 395 PDA patient samples (local and metastatic disease). Researchers found two clusters not classified according to the mutation status of the driver genes KRAS, SMAD4, CDKN2A, and TP53. Variations in the processing of RNA molecules in the cell, rather than DNA, were responsible for pancreatic tumor metastasis. RBFOX2, a central protein that controls RNA processing, is degraded and present in much lower levels in metastases. This causes hundreds of genes to produce RNA and proteins differently, leading to cell invasion. The researchers demonstrated that restoring RBFOX2 to metastatic cells inhibited metastasis formation, while knocking it down stimulated pancreatic cancer metastasis formation. The disappearance of RBFOX2 affects a group of genes that control the organization of the cellular skeleton, which is important for the motility and invasive ability of the cells. Genes in the RHO GTPase pathways are enriched in RBFOX2 target genes, as shown through an RNA-sequencing and splicing analysis. This reveals that RBFOX2 guides cytoskeletal organization and focal adhesion formation. Moreover, genetic intervention in RNA processing of RBFOX2-affected genes eliminated the metastatic ability of pancreatic cancer cells taken from patients in a mouse model. The team identified two inhibitors, MBQ-167 and azathioprine, which target these pathways and were tested in a mouse model. The immunosuppressant drug azathioprine, which blocks RAC1, a protein in the RHO GTPase pathway, was able to inhibit lung metastases in the mouse model. Similarly, genetically knocking out the RAC1 gene produced similar results. Based on these findings, the researchers suggest that azathioprine and MBQ-167 are potential candidates for therapeutic intervention in metastatic pancreatic cancer. To learn more about molecular mechanisms that lead to the progression of PDA and additional insights into the study, Oncology Times connected with senior author, Rotem Karni, PhD, Chair of Biochemistry and Molecular Biology at the Institute for Medical Research Israel-Canada at the Hebrew University-Hadassah Medical School in Jerusalem, Israel. Oncology Times: What was the rationale for this study and can you describe the multinational collaboration involved in this study and how it contributed to the findings? Karni: “The molecular mechanisms that lead to progression of PDA to metastasis are not well defined. Previous genomic studies have shown limited mutational heterogeneity between primary and metastatic tumors, suggesting the role of non-genetic mechanisms in metastasis. The rationale for this study was to investigate the molecular mechanism of metastasis in PDA, particularly the role of the process of alternative splicing. Alternative splicing is considered one of the hallmarks of cancer, and its dysregulation can contribute to tumor progression. “This study was led by a doctoral student Amina Jbara. The multinational collaboration also included physicians and scientists from Sheba Medical Center and Bar Ilan University in Israel, Cornell University and Cold Spring Harbor Laboratory in the United States, and Toronto University in Canada. Through the joint effort of these groups, we were able to identify that the RNA transcriptome in primary PDA tumors differed from the RNA in metastasized PDA tumors, due to a central RNA processing protein, RBFOX2. Analysis of PDA patient samples showed significantly lower protein expression of RBFOX2 in metastatic tumors compared to primary tumors. From this study, we conclude that RBFOX2 may be responsible for the splicing changes associated with PDA metastasis. This study highlights the importance of splicing changes in PDA metastasis and the potential role of RBFOX2 in this process. Oncology Times: Can you explain how RBFOX2 modulates the alternative splicing of RNA in pancreatic cancer? Karni: “RBFOX2 is an essential protein involved in alternative splicing, a process that enables a single gene to produce various protein isoforms with diverse functions. Dysregulation of alternative splicing has been linked to the progression and metastasis of several cancer types. In this study, we investigated the role of RBFOX2 in pancreatic cancer metastasis using a combination of bioinformatics, cell culture, and animal models. “Our study revealed a signature of alternative splicing events associated with RBFOX2 expression that could differentiate primary PDA tumors from metastatic tumors. We manipulated RBFOX2 expression in primary and metastatic PDA cells in culture and in vivo, and uncovered that RBFOX2 functions as a metastatic tumor suppressor in PDA progression. Through splicing analysis of these cells, we identified a significant set of RBFOX2 target genes in the Rho GTPases pathways. Overall, our findings demonstrate the critical role of RBFOX2 in modulating alternative splicing and its impact on pancreatic cancer metastasis.” Oncology Times: Which group of genes did the disappearance of RBFOX2 specifically affect and what is their function in pancreatic cancer? Karni: “RNA sequencing and splicing analysis of RBFOX2 target genes revealed enrichment of genes in the RHO GTPase pathways, suggesting a role of RBFOX2 splicing activity in cytoskeletal organization and focal adhesion formation. RBFOX2 depletion enhanced focal adhesion and altered cytoskeleton organization in pancreatic cells. “One of the differentially alternatively spliced events that we examined was splicing of myosin phosphatase RHO-interacting protein (MPRIP). RBFOX2 depletion resulted in production of more of the short variant of MPRIP that promotes tumor cell migration and invasion, while the long isoform showed significantly reduced migration and colony formation abilities. Modulation of this RBFOX2-regulated splicing event is associated with PDA metastases, altered cytoskeletal organization, and the induction of focal adhesion formation. “Analysis of the different MPRIP isoforms revealed unique phosphorylation sites predicted to be phosphorylated by various kinases, with the short isoform receiving a high score rank for PKC kinases, which could explain the observed increase in focal adhesion formation in cells expressing mainly the short isoform. Using mass spectrometry, we identified enrichment of proteins in the MAPK family signaling cascades, MAPK1/MAPK signaling, and RAF/MAP kinase cascade and cell cycle pathways binding to the short MRIP isoform and not the long MPRIP isoform.” Oncology Times: What are the potential clinical implications of this research for the prognosis and treatment of pancreatic cancer? Karni: “This study has important clinical implications for the prognosis and treatment of pancreatic cancer. Based on our findings, we propose two different approaches as potential therapies for pancreatic ductal adenocarcinoma (PDA) patients. The first approach involves inhibiting the Rho GTPase pathways by pharmacological means. We identified approximately 100 RBFOX2-regulated genes associated with the cell skeletal organization and the cell's ability to migrate and form tumors in other organs. Azathioprine, a known inhibitor of the Rho GTPase pathway, could potentially inhibit metastasis in pancreatic cells that have lost RBFOX2 expression. “The second approach involves developing Splice-switching antisense oligonucleotides (SSOs) as a precise strategy to modulate alternative splicing events in the Rho GTPases pathways. SSOs are synthetic nucleic acid molecules designed to modulate RNA splicing by binding to the cis-elements at the pre-mRNA and inducing splicing changes that can correct aberrant splicing events associated with certain diseases. In particular, SSOs can alter the balance of oncogenic versus tumor suppressor isoforms and may have potential as a therapeutic approach for treating PDA. “In conclusion, our study provides valuable insights into potential therapeutic approaches for pancreatic cancer patients by targeting splicing alterations associated with RBFOX2 depletion, highlighting the potential of pharmacological inhibition and SSOs as precision therapies.” Dibash Kumar Das is a contributing writer.

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