NEW YORK— Researchers have unraveled the effects of mutations in the 3' untranslated regions (UTR) of prostate cancer-related genes on disease progression, mRNA stability, and translation.
In a study published in Cell Reports on Friday, the team, led by investigators at the Fred Hutchinson Cancer Center, studied somatic mutations in the 3' UTR of genes implicated in almost 200 advanced prostate cancer patients, followed by functional studies at the molecular and cellular level.
According to the authors, several previous studies had focused on how mutations in the coding sequence, changes in mRNA expression, and genomic structural variations affect prostate cancer. However, those findings did not explain all aspects of disease pathogenesis. "3' untranslated region (3' UTR) somatic mutations represent a largely unexplored avenue of alternative oncogenic gene dysregulation," they wrote.
The 3' UTR falls immediately downstream of the coding sequence and is transcribed but not translated. However, it plays a crucial role in post-transcriptional control by binding to microRNAs (miRNAs) and RNA-binding proteins (RBPs).
For their study, the researchers conducted whole-genome sequencing and, in some cases, exome sequencing on 185 castration-resistant prostate cancer (mCRPC) tumors. They found nearly 15,000 somatic single-nucleotide mutations in the 3' UTRs of 7,647 genes.
These 3' UTRs fell into genes without mutations in the coding regions but with known relevance to breast, gastric, and prostate cancer. This led them to hypothesize that these mutations may be essential cancer drivers.
The highest burden of 3' UTR mutations was found in FLRT2 and LPP, both cell adhesion proteins associated with cancer. While LPP regulates metastasis and treatment resistance, FLRT2 is known to act as a tumor suppressor in several cancers.
Next, the researchers wanted to understand how patient mutations change 3' UTR-mediated aspects of post-transcriptional gene regulation. For this, they developed two massively parallel reporter assays (MPRAs) – a polysome profiling-based assay that measures changes in translation efficiency and an assay that gauges mRNA stability by sequencing in vitro transcribed, transfected mRNA over time.
They found 180 3' UTR point mutations that significantly changed translation efficiency, mostly by changing RNA binding protein or miRNA binding motifs. Mutations affecting mRNA stability, meanwhile, were enriched in neuronal genes. "Together with our previous findings, this strengthens the hypothesis that 3' UTR mutations can be an alternative way neuroendocrine features are dysregulated in advanced prostate cancer," they wrote.
Their final experiment was focused on understanding how 3' UTR mutations affect cellular function. Using CRISPR-Cas9 base editing, they introduced two translation-enhancing 3'UTR mutations into the ZWILCH and IGF1R genes, which were top hits from the MPRA experiments.
The cell lines mutated to overexpress ZWILCH grew significantly faster under stress conditions such as nutrient deprivation or cisplatin than unmutated ones. The cisplatin resistance was striking, the authors said, as the drug is a direct challenge to the mitotic checkpoint and has clinical implications for chemotherapy use.
Meanwhile, cell lines mutated to overexpress IGF1R, a growth factor receptor involved in cancer progression, showed a growth advantage over unmutated cells when their access to IGF1R ligands was reduced.
Both findings confirmed that 3' UTR mutations could increase cancer cell oncogenicity by enhancing growth under stress, a quality particularly important within the harsh ecosystem of a tumor microenvironment, the authors noted.
Finally, they correlated 3' UTR mutations with patient prognosis and found that tumors harboring cancer-associated 3' UTR mutations progressed faster to bone metastasis and had poorer overall survival.
"Our work represents a comprehensive view of the extent to which disease-relevant 3' UTR mutations affect mRNA stability, [translational efficiency], and cancer phenotypes, expanding the boundaries of functional cancer genomics and potentially uncovering therapeutic targets in previously unexplored regulatory regions," the authors concluded.