Identification and Selective Degradation of Neopeptide-Containing Truncated Mutant Proteins in the Tumors with High Microsatellite Instability

Abstract

Purpose: Frameshift mutations in coding mononucleotide repeats (cMNR) are common in tumors with high microsatellite instability (MSI-H). These mutations generate mRNAs containing abnormal coding sequences and premature termination codons (PTC). Normally, mRNAs containing PTCs are degraded by nonsense-mediated mRNA decay (NMD). However, mRNAs containing PTCs located in the last exon are not subject to degradation by NMD (NMD-irrelevant). This study aimed to discover whether genes with frameshift mutations in the last exon generate truncated mutant proteins.Experimental Design: We identified 66 genes containing cMNRs in the last exon by bioinformatic analysis. We found frequent insertion/deletion mutations in the cMNRs of 29 genes in 10 MSI-H cancer cell lines and in the cMNRs of 3 genes in 19 MSI-H cancer tissues. We selected 7 genes (TTK, TCF7L2, MARCKS, ASTE1, INO80E, CYHR1, and EBPL) for mutant mRNA expression analysis and 3 genes (TTK, TCF7L2, and MARCKS) for mutant protein expression analysis.Results: The PTC-containing NMD-irrelevant mRNAs from mutated genes were not degraded. However, only faint amounts of endogenous mutant TTK and TCF7L2 were detected, and we failed to detect endogenous mutant MARCKS. By polysome analysis, we showed that mRNAs from genomic mutant MARCKS constructs are normally translated. After inhibiting 3 protein degradation pathways, we found that only inhibition of the proteasomal pathway facilitated the rescue of endogenous mutant TTK, TCF7L2, and MARCKS.Conclusions: Our findings indicate that cancer cells scavenge potentially harmful neopeptide-containing mutant proteins derived from NMD-irrelevant abnormal mRNAs via the ubiquitin-proteasome system, and these mutant proteins may be important substrates for tumor-specific antigens.

Proteogenomic Analysis Reveals Unanticipated Adaptations of Colorectal Tumor Cells to Deficiencies in DNA Mismatch Repair

“…Evidence for increased protein folding activity came from the presence of FKBP2 and FKBP4, LMAN1, TRAP1 and WFS1, while a separate co-expression module indicated increased ubiquitin-dependent protein degradation: UBXN1, DPP7, TFRC, and ERLIN2. The involvement of ubiquitin-dependent proteasomal degradation is in agreement with a recent paper by Kim et al , demonstrating that aberrant mRNAs that escape NMD lead to mutant proteins that are degraded via the ubiquitin-proteasome system (34). …”

Correlation between Density of CD8+ T-cell Infiltrate in Microsatellite Unstable Colorectal Cancers and Frameshift Mutations: A Rationale for Personalized Immunotherapy

“…We observed that frameshift mutations in ASTE1, HNF1A, and TCF7L2 genes were robustly associated with an increased CD8 þ TIL density. Mutated TCF7L2 mRNA expression, in MSI colorectal cancers, had already been found correlated with a stronger peritumoral lymphoid reaction (33) and with CD3 þ infiltration (34), but we showed for the first time, to our knowledge, a correlation of these three mutated genes with an increased CD8 þ tumoral infiltration. Moreover, CD8 þ TIL densities were higher in tumors harboring ASTE1, HNF1A, or TCF7L2 mutation in all tumor cells.…”

PLS3 sequencing in childhood-onset primary osteoporosis identifies two novel disease-causing variants

“…In the case of patient 1 , the 30-year-old Finnish male was hemizygous for a novel nonsense variant located in the mid-part of the gene. Such a variant, based on what is known, leads to a complete loss of function of the mature protein and the patient will effectively be left without any functioning copy of PLS3 [ 26 , 27 ]. Both the inheritance pattern and the phenotype of previously described patients with premature stop codons in PLS3 support this conclusion [ 12 , 15 ] .…”