INTRODUCTION: Cancer cells typically show an increase in protein synthesis, with specific subsets of pro-tumourigenic mRNAs being more efficiently translated. tRNAs are RNA molecules that play a central role in protein synthesis by decoding mRNAs via direct base pairing between mRNA codons and tRNA anticodons at the ribosome. Modifications in the anticodon bases can affect the efficiency of decoding. We recently reported that key enzymes responsible for modifying uridine position 34 (U34) in the anticodon of specific tRNAs to generate mcm5s2-U34 are over-expressed in prostate and breast cancer cells (Lorent, Kusnadi et al., 2019, EMBO J.). Synthesis of mcm5s2-U34 requires three steps catalysed by the Elongator complex (ELP1-6), ALKBH8 and CTU1/2 in a sequential manner. Depletion of ELP3, the catalytic subunit of the Elongator complex, has been reported to diminish translational rates of mRNAs enriched in A-ending codons in different cancer types, including melanoma and breast cancer. We hypothesise that inhibition of the U34 modification pathway is a therapeutic vulnerability in various neoplasia, including prostate cancer (PCa).
METHODS: We applied CRISPR/Cas9 methodology to deplete ELP3 expression in PCa cell lines DU145 and LNCaP. Once the reduction in U34 modification has been validated by tRNA mass spectrometry, the resulting cells were analysed by polysome profiling and standard mass spectometry. Our current efforts are focused on identifying which transcripts and proteins were most affected by ELP3 depletion in PCa models.
RESULTS: Our data demonstrated that CRISPR/Cas9-mediated depletion of ELP3 impairs the proliferative potential of human PCa in vitro models, but has no effect on untransformed prostate epithelial cells. Moreover, ELP3 KO strongly reduced the clonogenic potential of both mouse-derived and human PCa cell lines. Mass spectrometry analysis of ELP3 KO DU145 cells also indicated a significant reshaping of the cellular proteome, driven by changes in proteins encoded by mRNAs that contain specific codons.
CONCLUSION: Most studies on PCa gene expression have focused on genomic aberrations, such as gene amplification or copy number variations, and changes at the level of transcription. We anticipate that our research will highlight the importance of post-transcriptional and epitranscriptomic regulations in the etiology of PCa.