Gene specific alternative polyadenylation can reduce tumor growth
Source: Fred Hutch Cancer Center, March 2024
Cells regulate RNA and protein abundance through a multi-layered process. Excitingly, peeling back one layer has revealed additional layers below, highlighting the complex nature of this process. For example, continued study of mRNA—the template sequence for making proteins—has revealed that alternative cleavage and polyadenylation (APA) can regulate RNA and protein abundance. Dr. Rob Bradley, a Professor in the Basic and Public Health Sciences Divisions, is interested in determining if APA events are simply passengers or functionally relevant to cancer development and progression. In a recent Nature Communications publication, Austin Gabel, an MD/PhD student in the Bradley lab, investigated if APA events that occur in melanoma are involved in the regulation of tumor growth.
The process of mRNA polyadenylation occurs by adding adenosine residues to create a poly(A) tail at the 3’-terminal end of the mRNA. This RNA modification plays an important role in mRNA stability and protein production. APA occurs when the poly(A) tail is added at an alternative site—either distal or proximal to the preferred site—resulting in a shorter or longer 3’ untranslated region (UTR). Since only untranslated regions are modified, the mRNA 3’UTR isoforms still express identical proteins. “APA has long been known to be dysregulated in a number of disease states including cancer; however, the functional relevance of that dysregulation has been debated,” shared Gabel. “Some experts in the field even go so far as to speculate that APA harbors no functional effects and is merely a consequence of a polyadenylation error rate.” To connect the dots between APA and cancer, Gabel explained that they first cataloged a large number of APA events in human melanoma and other cancers to identify associations between the two. The researchers uncovered variable associations across several cancer types. Specifically, global 3’UTR shortening was associated with worse patient survival for ovarian, kidney, breast, and lung carcinomas, while the opposite—global lengthening of 3’UTRs—correlated with poor patient survival outcomes for head and neck carcinomas, gliomas and melanomas. Among these, the strongest positive correlation was observed between lengthened 3’UTRs and poor prognosis for melanoma patients. The researchers turned to a mouse model of melanoma to dissect the role of APA dysregulation in cancer. Their initial studies revealed that melanoma tumors from these mice had more than 600 genes with different poly(A) site selections as compared to control, immortalized melanocytes from the same mouse background. Intriguingly, the poly(A) sites selected for genes in the mouse melanoma model correlated with those in human melanoma tumors. This continuity between the mouse and human systems suggested that the mouse model of melanoma could be useful for dissecting the role of APA events in cancer.
Next, using this melanoma mouse model, the researchers conducted the first ever functional screen of APA events to identify 3’UTR isoforms that enhance or restrict tumor growth. The screening approach utilized a “CRISPR-Cas9 paired-guide RNA library designed to knock-out proximal poly(A) sites, which will then force the use of distal poly(A) sites,” explained Gabel. “We identified several genes that when forced to use a distal poly(A) site either reduced or enhanced mouse melanoma growth in vitro and in vivo.” One of these, the long 3’UTR isoform of the Atg7 autophagy factor, resulted in less Atg7 protein and “caused slowed mouse melanoma growth. Excitingly, we observed that human patients with melanoma that preferentially utilize a longer Atg7 3’ UTR mRNA isoform also had significantly better outcomes.” Gabel emphasized that these findings suggest that the melanoma mouse model may exhibit APA events relevant to human disease. Additionally, these data demonstrate that changes in 3’UTR length for specific genes can influence tumor growth.