A safety-modified SV40 Tag developed for human cancer immunotherapy

Simian virus 40 (SV40)-like DNA sequences have been found in a variety of human tumors, raising the possibility that strategies targeting SV40 may provide a potential avenue for immunotherapy directed against SV40 large T Antigen (Tag)-expressing tumors. We generated a recombinant vaccinia (vac-mTag) expressing mTag and herein assessed the ability of mTag to transform cells and to interact with anti-oncoproteins, as well as screened for the presence of potential HLA-A2.1-restricted epitopes within mTag. We found that transfection of cells with mTag did not lead to their transformation. Also, we demonstrated that mTag protein is degraded rapidly in cells. In addition, our work revealed that mTag did not physically interact with certain anti-oncoproteins. Finally, two potential HLA-A2.1-restricted functional epitopes within mTag sequence were identified. Our results show that mTag lacks the oncogenecity of full-length Tag and harbors potential HLA-A2.1-restricted immunogenic epitopes, hence suggesting the safety of vac-mTag for use in cancer immunotherapy

EMAST is a Form of Microsatellite Instability That is Initiated by Inflammation and Modulates Colorectal Cancer Progression

DNA mismatch repair (MMR) function is critical for correcting errors coincident with polymerase-driven DNA replication, and its proteins are frequent targets for inactivation (germline or somatic), generating a hypermutable tumor that drives cancer progression. The biomarker for defective DNA MMR is microsatellite instability-high (MSI-H), observed in ~15% of colorectal cancers, and defined by mono- and dinucleotide microsatellite frameshift mutations. MSI-H is highly correlated with loss of MMR protein expression, is commonly diploid, is often located in the right side of the colon, prognosticates good patient outcome, and predicts poor efficacy with 5-fluorouracil treatment. Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is another form of MSI at tetranucleotide repeats that has been observed in multiple cancers, but its etiology and clinical relevance to patient care has only been recently illuminated. Specifically, EMAST is an acquired somatic defect observed in up to 60% of colorectal cancers and caused by unique dysfunction of the DNA MMR protein MSH3 (and its DNA MMR complex MutSβ, a heterodimer of MSH2-MSH3), and in particular a loss-of-function phenotype due to a reversible shift from its normal nuclear location into the cytosol in response to oxidative stress and the pro-inflammatory cytokine interleukin-6. Tumor hypoxia may also be a contributor. Patients with EMAST colorectal cancers show diminished prognosis compared to patients without the presence of EMAST in their cancer. In addition to defective DNA MMR recognized by tetranucleotide (and di- and tri-nucleotide) frameshifts, loss of MSH3 also contributes to homologous recombination-mediated repair of DNA double stranded breaks, indicating the MSH3 dysfunction is a complex defect for cancer cells that generates not only EMAST but also may contribute to chromosomal instability and aneuploidy. Areas for future investigation for this most common DNA MMR defect among colorectal cancers include relationships between EMAST and chemotherapy response, patient outcome with aneuploid changes in colorectal cancers, target gene mutation analysis, and mechanisms related to inflammation-induced compartmentalization and inactivation for MSH3

Functional conservation of Dhh1p, a cytoplasmic DExD/H-box protein present in large complexes

The DHH1 gene in the yeast Saccharomyces cerevisiae encodes a putative RNA helicase of remarkable sequence similarity to several other DExD/H-box proteins, including Xp54 in Xenopus laevis and Ste13p in Schizosaccharomyces pombe. We show here that over-expression of Xp54, an integral component of the stored messenger ribonucleoprotein (mRNP) particles, can rescue the loss of Dhh1p in yeast. Localization and sedimentation studies showed that Dhh1p exists predominantly in the cytoplasm and is present in large complexes whose sizes appear to vary according to the growth stage of the cell culture. In addition, deletion of dhh1, when placed in conjunction with the mutant dbp5 and ded1 alleles, resulted in a synergistically lethal effect, suggesting that Dhh1p may have a role in mRNA export and translation. Finally, similar to Ste13p, Dhh1p is required for sporulation in the budding yeast. Taken together, our data provide evidence that the functions of Dhh1p are conserved through evolution

Oxidative Stress Induces Nuclear-to-Cytosol Shift of hMSH3, a Potential Mechanism for EMAST in Colorectal Cancer Cells

<div><h3>Background</h3><p>Elevated microsatellite alterations at selected tetranucleotide repeats (EMAST) is a genetic signature observed in 60% of sporadic colorectal cancers (CRCs). Unlike microsatellite unstable CRCs where hypermethylation of the DNA mismatch repair (MMR) gene <em>hMLH1’s</em> promoter is causal, the precise cause of EMAST is not clearly defined but points towards <em>hMSH3</em> deficiency.</p> <h3>Aim</h3><p>To examine if <em>hMSH3</em> deficiency causes EMAST, and to explore mechanisms for its deficiency.</p> <h3>Methods</h3><p>We measured −4 bp framshifts at <em>D8S321</em> and <em>D20S82</em> loci within EGFP-containing constructs to determine EMAST formation in MMR-proficient, <em>hMLH1^−/−</em>, <em>hMSH6^−/−</em>, and <em>hMSH3^−/−</em> CRC cells. We observed the subcellular location of hMSH3 with oxidative stress.</p> <h3>Results</h3><p><em>D8S321</em> mutations occurred 31-and 40-fold higher and <em>D20S82</em> mutations occurred 82-and 49-fold higher in <em>hMLH1^−/−</em> and <em>hMSH3^−/−</em> cells, respectively, than in <em>hMSH6^−/−</em> or MMR-proficient cells. <em>hMSH3</em> knockdown in MMR-proficient cells caused higher <em>D8S321</em> mutation rates (18.14 and 11.14×10⁻⁴ mutations/cell/generation in two independent clones) than scrambled controls (0 and 0.26×10⁻⁴ mutations/cell/generation; <em>p</em><0.01). DNA sequencing confirmed the expected frameshift mutations with evidence for ongoing mutations of the constructs. Because EMAST-positive tumors are associated with inflammation, we subjected MMR-proficient cells to oxidative stress via H₂O₂ to examine its effect on <em>hMSH3</em>. A reversible nuclear-to-cytosol shift of hMSH3 was observed upon H₂O₂ treatment.</p> <h3>Conclusion</h3><p>EMAST is dependent upon the MMR background, with <em>hMSH3^−/−</em> more prone to frameshift mutations than <em>hMSH6^−/−</em>, opposite to frameshift mutations observed for mononucleotide repeats. <em>hMSH3^−/−</em> mimics complete MMR failure (<em>hMLH1^−/−</em>) in inducing EMAST. Given the observed heterogeneous expression of hMSH3 in CRCs with EMAST, <em>hMSH3</em>-deficiency appears to be the event that commences EMAST. Oxidative stress, which causes a shift of hMSH3’s subcellular location, may contribute to an hMSH3 loss-of-function phenotype by sequestering it to the cytosol.</p> </div

Calculated EMAST mutation rates for <i>D8S321</i> and <i>D20S82</i> in various MMR backgrounds.

<p>Data from the EGFP-positive population at weeks 4, 5, and 6 time points were used for -4 bp frameshift mutation rate analysis. Single mutation rates were calculated by combining and averaging time-specific mutation rates. Rates are expressed as mutations at microsatellite sequence per cell per generation. Data shown are mean±SEM.</p><p>a, b, c, and d represent significant difference in the mutation rate (<i>P</i><0.05).</p>a<p><i>hMLH1^−/−</i> vs. MMR-proficient (<i>D8S321 P</i> = 0.00012, <i>D20S82 P</i> = 0.01069);</p>b<p><i>hMLH1^−/−</i> vs. <i>hMSH6^−/−</i> (<i>D8S321 P</i> = 0.00012, <i>D20S82 P</i> = 0.01021);</p>c<p><i>hMSH3^−/−</i> vs. MMR-proficient (<i>D8S321 P</i> = 0.00008, <i>D20S82 P</i> = 0.01525);</p>d<p><i>hMSH3^−/−</i> vs. <i>hMSH6^−/−</i> (<i>D8S321 P</i> = 0.0001, <i>D20S82 P</i> = 0.01415). Mutations rates were calculated based on the “method of the mean” developed by Luria and Delbruck <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050616#pone.0050616-Luria1" target="_blank">[25]</a>.</p

EMAST mutation frequencies in <i>hMSH3</i> knockdown CRC single-clone cells.

<p>(<b>A</b>) SW480 cells (MMR proficient) carrying the EMAST construct (D8-OF or D8-MR-OF) were transfected with <i>hMSH3</i> shRNA construct (<i>hMSH3</i> KD) or scramble control (scramble) separately. Two independent <i>hMSH3</i> and scramble KD clones as well as the parental cells were included for this study. EMAST mutation frequencies were calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050616#pone-0050616-g001" target="_blank">Figure 1B</a> for parental, scramble, and/or <i>hMSH3</i> KD cells. There were significantly more EGFP-positive cells in <i>hMSH3</i> KD cells compared to parent and/or scramble control cells. *: p<0.001; **: p<0.05. (<b>B</b>) EGFP-negative and -positive cells from <i>hMSH3</i> KD cells carrying D8-OF were sorted for genomic DNA isolation to examine frameshift mutations by sequencing.</p

Efficacy of Adjuvant 5-Fluorouracil Therapy for Patients with EMAST-Positive Stage II/III Colorectal Cancer

Elevated Microsatellite Alterations at Selected Tetranucleotide repeats (EMAST) is a genetic signature found in up to 60% of colorectal cancers (CRCs) that is caused by somatic dysfunction of the DNA mismatch repair (MMR) protein hMSH3. We have previously shown in vitro that recognition of 5-fluorouracil (5-FU) within DNA and subsequent cytotoxicity was most effective when both hMutSα (hMSH2-hMSH6 heterodimer) and hMutSβ (hMSH2-hMSH3 heterodimer) MMR complexes were present, compared to hMutSα > hMutSβ alone. We tested if patients with EMAST CRCs (hMutSβ defective) had diminished response to adjuvant 5-FU chemotherapy, paralleling in vitro findings. We analyzed 230 patients with stage II/III sporadic colorectal cancers for which we had 5-FU treatment and survival data. Archival DNA was analyzed for EMAST (>2 of 5 markers mutated among UT5037, D8S321, D9S242, D20S82, D20S85 tetranucleotide loci). Kaplan-Meier survival curves were generated and multivariate analysis was used to determine contribution to risk. We identified 102 (44%) EMAST cancers. Ninety-four patients (41%) received adjuvant 5-FU chemotherapy, and median follow-up for all patients was 51 months. Patients with EMAST CRCs demonstrated improved survival with adjuvant 5FU to the same extent as patients with non-EMAST CRCs (P<0.05). We observed no difference in survival between patients with stage II/III EMAST and non-EMAST cancers (P = 0.36). There is improved survival for stage II/III CRC patients after adjuvant 5-FU-based chemotherapy regardless of EMAST status. The loss of contribution of hMSH3 for 5-FU cytotoxicity may not adversely affect patient outcome, contrasting patients whose tumors completely lack DNA MMR function (MSI-H).This work was supported by the United States Public Health Service (DK067287 and CA162147) and the A. Alfred Taubman Medical Research Institute of the University of Michigan

DNA sequencing results and EMAST mutation frequencies in CRC cells with different MMR backgrounds.

<p>(<b>A</b>) Genomic DNA isolated from EGFP-negative and -positive cells carrying experimental constructs (D8-OF and/or D20-OF) was used to amplify DNA fragments containing EMAST loci to examine if the frameshift mutations had occurred. (<b>B</b>) Mutation frequencies were calculated by dividing the percentage of EGFP positive population in the experimental group (OF) by the percentage of EGFP positive in its negative control (MR-OF) to compute the increase of the EGFP-positive population in folds. There were significantly more EGFP-positive cells in <i>hMLH1</i>- and <i>hMSH3</i>-deficient cells compared to MMR-proficient and/or <i>hMSH6</i>-deficient cells. *: p<0.05.</p

Efficacy of Adjuvant 5-Fluorouracil Therapy for Patients with EMAST-Positive Stage II/III Colorectal Cancer

Elevated Microsatellite Alterations at Selected Tetranucleotide repeats (EMAST) is a genetic signature found in up to 60% of colorectal cancers (CRCs) that is caused by somatic dysfunction of the DNA mismatch repair (MMR) protein hMSH3. We have previously shown in vitro that recognition of 5-fluorouracil (5-FU) within DNA and subsequent cytotoxicity was most effective when both hMutSα (hMSH2-hMSH6 heterodimer) and hMutSβ (hMSH2-hMSH3 heterodimer) MMR complexes were present, compared to hMutSα > hMutSβ alone. We tested if patients with EMAST CRCs (hMutSβ defective) had diminished response to adjuvant 5-FU chemotherapy, paralleling in vitro findings. We analyzed 230 patients with stage II/III sporadic colorectal cancers for which we had 5-FU treatment and survival data. Archival DNA was analyzed for EMAST (>2 of 5 markers mutated among UT5037, D8S321, D9S242, D20S82, D20S85 tetranucleotide loci). Kaplan-Meier survival curves were generated and multivariate analysis was used to determine contribution to risk. We identified 102 (44%) EMAST cancers. Ninety-four patients (41%) received adjuvant 5-FU chemotherapy, and median follow-up for all patients was 51 months. Patients with EMAST CRCs demonstrated improved survival with adjuvant 5FU to the same extent as patients with non-EMAST CRCs (P<0.05). We observed no difference in survival between patients with stage II/III EMAST and non-EMAST cancers (P = 0.36). There is improved survival for stage II/III CRC patients after adjuvant 5-FU-based chemotherapy regardless of EMAST status. The loss of contribution of hMSH3 for 5-FU cytotoxicity may not adversely affect patient outcome, contrasting patients whose tumors completely lack DNA MMR function (MSI-H).This work was supported by the United States Public Health Service (DK067287 and CA162147) and the A. Alfred Taubman Medical Research Institute of the University of Michigan