When it comes to identifying cancer-driving mutations, research has focused almost exclusively on understanding how mutations in the coding region of genes may alter a protein’s function to promote cancer. However, mutations in the 5’ and 3’ untranslated regions (UTRs) have mostly slid under the radar, particularly those in the 3’ UTR, which generally mediates mRNA stability and translation. Results from a study recently published in Cell Reports by Dr. Andrew Hsieh’s Lab suggest that maybe we should shift the spotlight from the coding region towards the 3’UTR and appreciate how mutations in these sites could be critical in driving cancer progression as well. As Dr. Hsieh stated, this work was a true “tour de force” by Dr. Samantha Schuster, a recent PhD graduate from the Hsieh Lab that uncovered how mutations present in the 3’UTR of advanced prostate cancer tumors can contribute to cellular oncogenicity and correlate with poor prognosis in patients.
The cancer field broadly—as well as cancer mutation databases—have placed importance on oncogenic mutations in coding regions of genes, which seems to imply that the non-coding space is not really important. However, as Dr. Hsieh states: “Our data would say that’s not true. We’re just not looking at it in the right way,” So, what is the right way to look at whether mutations in non-coding regions functionally contribute to a cancer’s development or progression? It turns out that it’s not actually an easy feat to look at this correctly, and Dr. Schuster admitted that development of proper assays and validation systems was challenging and time-intensive. However, before things got complicated, she said that “the project started with a simple question: are there even mutations in the 3’UTR?” The authors started by analyzing whole-genome sequencing or UTR sequencing of tumors from patients with metastatic castration-resistant prostate cancer (mCRPC), a particularly aggressive and deadly cancer. Through analysis of 185 tumors, many collected via the University of Washington Rapid Autopsy Program, the research team identified nearly 15,000 mutations present in the 3’UTR of almost 8,000 genes, including known cancer-related genes. Next up was to functionally test whether any of these mutations had any functional effects that might help drive cancer. Dr. Schuster first focused on asking whether these patient-identified 3’UTR mutations affected gene-specific translation efficiency. Here she developed a polysome-profiling massively parallel reporter assay that would enable her to simultaneously measure how thousands of mutations change translation efficiency. Specifically, polysome profiling allowed the researchers to analyze how mRNAs associate with ribosomes, which could then help them understand if certain mutations might increase or decrease the protein levels of the genes harboring these mutations. Here, the researchers discovered 180 3’UTR point mutations that significantly altered the efficiency by which mRNAs were translated into protein. Notably, many functional 3’UTR mutations caused seemingly oncogenic changes in expression such as increasing translation efficiency of oncogenic mRNAs and decreasing translation of tumor suppressor genes. Since the 3’UTR is also an important regulator of mRNA stability, Dr. Schuster designed a second massively parallel reporter assay to test how mCRPC patient mutations affect mRNA degradation. Overall, they found 150 patient-based 3’UTR mutations that significantly altered mRNA stability, including increased stability of oncogenes which had an increased half-life of their respective mRNAs.
While these complimentary systems-based analyses demonstrated direct effects on mRNA translation and stability, this didn’t tell the researchers whether they impacted cellular function. To address this, the team turned to CRISPR-Cas9 base editing to explore the cellular consequences of 3’UTR mutations in cancer. Focusing on two genes that had been shown to increase mRNA translation of pro-proliferative genes in their reporter assay, the team introduced these specific 3’UTR mutations into their endogenous genomic loci in cultured cells and asked how this affected cell growth. Surprisingly, under normal cell culture conditions, cells harboring these mutations did not grow differently from unedited control cells. Instead, the true advantage that these mutations gave cancer cells was not revealed until the cells were cultured under stress conditions. In this context, the mutant cells showed off their increased tolerance to stress and were able to grow better than control cells, demonstrating that these mutations are “functional and super important for cells to overcome stress,” Dr. Hsieh stated. Schuster described these findings as being one of the most exciting parts of the project, definitively proving that single-nucleotide mutations in the 3’UTR can cause significant changes in cancer-relevant cellular phenotypes. Bringing this project full circle, Schuster et al. returned the focus back to the patients (where these mutations had initially been identified) and asked whether 3’UTR mutations had any effect on patient prognosis. The authors observed that patient tumors harboring any oncogenic 3’UTR mutations had significantly faster progression to androgen independence, shorter time until bone metastasis occurred and poorer overall survival than tumors without oncogenic 3’UTR mutations, thus highlighting that these mutations indeed correlate with poorer patient prognosis.
“One thing that sets this study apart from other studies is that we directly tested the effect of 3’UTR mutations [on mRNA and cellular cancer phenotypes],” Dr. Schuster explained. In addition to developing systems that enable researchers to functionally test the effect of 3’UTR mutations directly, this important study “opens up the door for more research and getting more attention on the topic, because we still don’t know that much about the 3’UTR,” Schuster noted. Together, this work uncovered alternative pathways of oncogenic dysregulation in 3’UTR-mediated post-transcriptional gene expression and demonstrated the importance of considering mutations in the untranslated regions of genes in addition to coding regions when working to find creative strategies to treat aggressive cancers like mCRPC.
This work was supported by the National Institutes of Health, a Fred Hutchinson Cancer Center CCEH Pilot Grant, the Emerson Collective, the Kleberg Foundation, the DOD Prostate Cancer Research Program, Pacific Northwest Prostate Cancer SPORE, the Institute for Prostate Cancer Research and the Richard M. Lucas Foundation.
UW/Fred Hutch/Seattle Children’s Cancer Consortium members Drs. Andrew Hsieh, Patrick Paddison and Gavin Ha contributed to this work.
Schuster SL, Arora S, Wladyka CL, Itagi P, Corey L, Young D, Stackhouse BL, Kollath L, Wu QV, Corey E, True LD, Ha G, Paddison PJ, Hsieh AC. Multi-level functional genomics reveals molecular and cellular oncogenicity of patient-based 3' untranslated region mutations. Cell Rep. 2023 Aug 29;42(8):112840. doi: 10.1016/j.celrep.2023.112840. Epub 2023 Jul 28. PMID: 37516102.