Identification and Characterization of Alternatively Spliced Transcript Isoforms of IRX4 in Prostate Cancer

Abstract

Alternative splicing (AS) is tightly regulated to maintain genomic stability in humans. However, tumor growth, metastasis and therapy resistance benefit from aberrant RNA splicing. Iroquois-class homeodomain protein 4 (IRX4) is a TALE homeobox transcription factor which has been implicated in prostate cancer (PCa) as a tumor suppressor through genome-wide association studies (GWAS) and functional follow-up studies. In the current study, we characterized 12 IRX4 transcripts in PCa cell lines, including seven novel transcripts by RT-PCR and sequencing. They demonstrate unique expression profiles between androgen-responsive and nonresponsive cell lines. These transcripts were significantly overexpressed in PCa cell lines and the cancer genome atlas program (TCGA) PCa clinical specimens, suggesting their probable involvement in PCa progression. Moreover, a PCa risk-associated SNP rs12653946 genotype GG was corelated with lower IRX4 transcript levels. Using mass spectrometry analysis, we identified two IRX4 protein isoforms (54.4 kDa, 57 kDa) comprising all the functional domains and two novel isoforms (40 kDa, 8.7 kDa) lacking functional domains. These IRX4 isoforms might induce distinct functional programming that could contribute to PCa hallmarks, thus providing novel insights into diagnostic, prognostic and therapeutic significance in PCa management.

Identification of a Micropeptide Linked to Cancer Stem Cell Regulation and Chemoresistance

“…IRX4_PEP1 is 78 amino acids in length with an AUG canonical start codon and have a high CPPred-sORF coding potential of 91%. Our previous studies related to the IRX4 locus have identified a potential transcript variant that can encode IRX4_PEP1 and have validated its expression at the protein level in PCa cell lines (18).…”

“…Its expression is specific to PCa and increases with advanced PCa, suggesting its significance in this cancer type. In our previous study, a transcript variant of IRX4 that encode this miPEP was detected in both PCa cell lines and clinical samples (18). As a result, IRX4_PEP1 was chosen for further functional characterization.…”

“…We hypothesised, the IRX cluster can encode functional miPEPs and these miPEPs can have a role in cancer progression. We utilised a computational-proteomic workflow to identify IRX cluster encoded miPEPs by reprocessing SWATH-MS/MS PRIDE raw data of hormone-sensitive cancers, as IRXs seem to be hormone-regulated according to our previous findings (18). SWATH-MS/MS analysis is a robust, highly reproducible label-free quantification method that has been used for clinical sample analysis in many studies (11,12,45).…”

“…The IRX family includes six genes, IRX1, IRX2, and IRX4 (IRX cluster A) which are located in chromosome 5 and IRX3, IRX5, and IRX6 (IRX cluster B) which are located in chromosome 16 (13,16). Genome-Wide Association Studies (GWAS) have recently identified several cancer risk-associated single nucleotide polymorphisms within the IRX clusters in patient cohorts pointing to their emerging role in carcinogenesis (17)(18)(19)(20)(21)(22)(23)(24). The 5p15 locus bearing the IRX cluster A is associated with prostate, lung and gastric cancer risk (17,20,(25)(26)(27)(28)(29) while the 16q12 locus bearing IRX cluster B is associated with breast, endometrial, and retinoblastoma cancer risk (30)(31)(32).…”

Identification of Candidate mRNA Isoforms for Prostate Cancer-Risk SNPs Utilizing Iso-eQTL and sQTL Methods

“…Alternative splicing (AS) is a conserved biological process that allows the generation of several different mRNA isoforms from one gene locus [ 1 , 2 , 3 ]. This process occurs in close relation to transcription in eukaryotes [ 4 ].…”