Thesis title: The inhibition of the oncogenic Hedgehog signaling pathway through microRNA-driven modulation of KCASH2
GLI1 is a transcriptional factor that acts as the primary effector of the Hedgehog (Hh) signaling pathway, which regulates cell proliferation throughout embryonic development. Although GLI1 has been identified as the downstream effector of the Hh pathway, its transcriptional activity may also be increased by non-canonical signaling, and dysregulated activity is closely linked to cancer. GLI1 appears to be the most promising therapeutic target in hedgehog dependent tumors. As a result, understanding its regulatory mechanisms is becoming increasingly important.
In our research group a new tumor suppressor gene has been identified, KCASH2 (also known as KCTD21) which induces ubiquitination and degradation of the deacetylase HDAC1, thus increasing Gli acetylation and suppressing its transcriptional function. Indeed, the expression of KCASH2 in human sporadic medulloblastoma (MB) samples has been analyzed and we observed a significant reduction of KCASH2 expression in Hh-dependent tumors, indicating that loss of this gene contributes to MB tumorigenesis. As a consequence, a potential therapeutical approach in MB treatments may be to increase KCASH2 expression in these cells.
Reduction of KCASH2 expression in tumors may be associated to different causes: among them hypermethylation of the promoter, genetic deletion or mutations, but also upregulation of miRNAs targeting the KCASH2 mRNA. Indeed, microRNAs (miRNAs) are considered to have a major role in epigenetic modulation of several oncosuppressors. miRNAs act by binding to specific short sequences on the 3’ untranslated region (3’UTR) of target genes and lead to their degradation.
In order to explore the role of miRNA on KCASH2 regulation, we have analyzed in silico the putative miRNA’s binding sequences on the 3’ UTR of human KCASH2. By using three different software we have selected a group of miRNAs which were identified as more solid candidates, being confirmed in all three different systems. Among them, we have chosen, for a first analysis, the miRNAs who had been described to play a role in tumorigenesis. Following overexpression of the selected miRNA in cell lines, we have verified the level of reduction of KCASH2 proteins induced by the different miRNAs.
The miRNAs with more impact on downregulating our target gene have been selected for further studies (hsa-miR-24-3P, let-7f-5p, miR-125b-5P and hsa-miR-196b-5p). We observed that overexpressing these miRNA mimics leads to downregulation of KCASH2 protein level and subsequently reduction of HDAC1 degradation and increase in Gli1 activity, upregulating cell proliferation.
Through the analysis of basal miRNA expression levels, miR-24 emerged as the most highly expressed miRNA in MB cells. Inhibition of miR-24 resulted in a marked increase in KCASH2 protein levels and a corresponding decrease in GLI1 protein levels, highlighting a potential inverse regulatory relationship. At the protein level, these changes were significant, suggesting that miR-24 plays a critical role in modulating these key targets. Furthermore, functional assays demonstrated that miR-24 inhibition in MB cells led to a reduction in cell proliferation, supporting the hypothesis that miR-24 may contribute to the oncogenic phenotype of these cells by stabilizing GLI1 expression.
Collectively, these findings suggest that miR-24 could exert an oncogenic influence in MB by suppressing KCASH2 and thus increasing GLI1 activity. Our presented results may suggest new avenues for therapeutic intervention, highlighting the potential of targeting miR-24 to restore the tumor-suppressive activity of KCASH2 and promote GLI1 degradation. Our evidence supports the hypothesis that miR-24 may function as an oncomiR, offering novel insights into the role of miRNAs in MB progression and opening up promising strategies for future treatment approaches.