Endocrine Abstracts

Pancreatic neuroendocrine tumors (PanNETs) are heterogeneous neoplasms with a relatively low but increasing incidence, mainly due to the difficulty in diagnosing this disease in its early stages. This notably complicates the treatment of this pathology and leads to a poor prognosis. One of the main reasons for this problem is the lack of adequate diagnostic biomarkers as well as effective therapeutic targets. In this sense, there is still a limited knowledge about alternative splicing, a key process emerging as a transversal hallmark of cancer, as its frequent dysregulation influences most tumor cell features. In a pilot study, we discovered that CELF4, — a member of the CELF/BRUNOL family, which is involved in the regulation of RNA splicing, editing, and translation in the central nervous system —, may be altered in PanNETs. Therefore, we aimed at evaluating the role of CELF4 as a biomarker and/or therapeutic target in PanNETs. To this end, CELF4 expression levels were determined using a microfluidic based technology, comparing tumor and adjacent non-tumor tissue, in a cohort of 20 PanNETs patients. This revealed a clear overexpression of CELF4 in tumor tissue compared to adjacent non-tumoral tissue. Then, an RNA-seq dataset was used to investigate the associations between CELF4 expression, patient clinical parameters, and splicing event patterns. We observed that CELF4 is linked to critical features of malignancy, the expression of key genes in tumors (TP53 or CDKN2B) and different splicing events profiles. Likewise, the functional relevance of this factor was determined in vitro with several functional assays (cell proliferation and drug response) in two PanNETs cell models (BON-1 and QGP-1), including an mTOR phospho-antibody array to determine the mechanism of action of CELF4. Remarkably, the modulation of CELF4 expression levels in the cell lines resulted in a significant change in proliferation as well as in the response of these cells to the mTOR inhibitor everolimus. In particular, CELF4 silencing resulted in a disruption of several crucial intermediaries in the mTOR signaling pathway. Finally, we carried out in vivo studies using BON-1-xenografted mice, observing a significant reduction of tumor growth by silencing CELF4. These results demonstrate that the splicing factor CELF4 is dysregulated in PanNETs, and its alteration can contribute to tumor development and a more aggressive phenotype, impacting the mTOR signaling pathway. Altogether, these findings provide original evidence that encourage further study of this factor as a novel potential target in PanNETs.