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The treatment of bladder cancers has improved with new immunotherapy such as PD-L1. However, not all bladder cancers respond to PD-L1 immunotherapy which is why new targets are being assessed. Melanoma-associated antigen gene A (MAGE-A) family proteins are expressed in a variety of tumors, with each MAGE-A protein having unique roles in cancer pathogenesis. One advantage of targeting MAGE-A family members is the lack of expression in normal tissues which makes them well-suited for targeted cancer immunotherapy. The challenge with screening the MAGE-A family is to find a specific antibody since the 12-member family has over 60% sequence in homology. In this study, we evaluate multiple MAGE-A3 and MAGE-A4 antibodies using CytoSections, a reproducible alternative to control tissue with an expression of target biomarkers. A series of antibodies specific to each MAGE-A3 and MAGE-A4 protein were discovered and used to screen twenty-five bladder cancer tissues. The results showed that MAGE-A3 was present in 9 of the 25 bladder cancers, while MAGE-A4 was present in 10 of the 25 bladder cancers evaluated; in the meantime, 7 bladder cancers were positive for both targets after double immunofluorescence staining with MAGE-A3 or A4 specific antibodies. The data presented here could be an important development in treatment protocols for pre-screening patients who are at risk. Our findings show that MAGE-A3 and MAGE-A4 can be both present in bladder cancer, suggesting that targeting both genes may be necessary for the success of immunotherapy treatment. Citation Format: Bailey Gilmore, Rachel Gonzalez, Aubrey Su, Dehe Kong, Eden Zewdu, Jina Yom, Tianli Qu, Zhaoying Guo, Xiaomin Hu, Qi Ren, Ranran Zhang, Eric Christenson, Yan Ma, Dezhong Yin, Patrick Yin, Wei Fu, Xuan Liu, Krishnan Allampallam. Colocalization of MAGE-A3 and MAGE-A4 in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 999.

Colorectal Cancer (CRC) is the second leading cause of death worldwide. Recent developments in cancer immunotherapy have obtained encouraging clinical responses, including PD-1/PD-L1 blockade therapy, although response rates vary depending on tumor type. For example, despite the lack of response by microsatellite-stable CRC, a subset of CRC with deficient mismatch repair (dMMR) and high microsatellite instability (MSI-H) responds especially well to the PD-1/PD-L1 blockade therapy. For other types of CRC which do not respond to PD-L1 therapy, new targets are being investigated. One family of targets is the cancer testis antigen MAGE-A whose expression is restricted to germline cells in normal tissue but is overexpressed in many cancer cells. In this study, immunohistochemical analysis of MAGE-A3 expression in colon cancer along with IHC screening of mismatch protein MSH6 was performed. Many of the cases in this study observed a co-expression in the tissue. In the tumors, MAGE-A3 was observed to have weak cytoplasmic expression while MSH6 had strong nuclear expression in the epithelial cells. The immune cells adjacent to the epithelial cells were also positive for MAGE-A3. This result suggests that MAGE-A3 may be a promising target for microsatellite-stable colorectal cancer immunotherapy. Citation Format: Jina Yom, Rachel Gonzalez, Eden Zewdu, Aubrey Su, Patrick Yin, Bailey Gilmore, Dehe Kong, Andy Han, Zhaoying Guo, Tianli Qu, Zoe Zhao, Eric Christenson, Yan Ma, Hailey Guo, Xiaomin Hu, Qi Ren, Zhaohui Wu, Xuan Liu, Wei Fu. MAGE-A3 expression with MMR-MSH6 screening suggests MAGE-A3 as a potential target for microsatellite-stable colorectal cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5948.

The identification of small molecules that inhibit the sequence-specific binding of transcription factors to DNA is an attractive approach for regulation of gene expression. Hypoxia-inducible factor-1 (HIF-1) is a transcription factor that controls genes involved in glycolysis, angiogenesis, migration, and invasion, all of which are important for tumor progression and metastasis. To identify inhibitors of HIF-1 DNA-binding activity, we expressed truncated HIF-1α and HIF-1β proteins containing the basic-helix-loop-helix and PAS domains. Expressed recombinant HIF-1α and HIF-1β proteins induced a specific DNA-binding activity to a double-stranded oligonucleotide containing a canonical hypoxia-responsive element (HRE). One hundred twenty-eight compounds previously identified in a HIF-1–targeted cell-based high-throughput screen of the National Cancer Institute 140,000 small-molecule library were tested in a 96-well plate ELISA for inhibition of HIF-1 DNA-binding activity. One of the most potent compounds identified, echinomycin (NSC-13502), a small-molecule known to bind DNA in a sequence-specific fashion, was further investigated. Electrophoretic mobility shift assay experiments showed that NSC-13502 inhibited binding of HIF-1α and HIF-1β proteins to a HRE sequence but not binding of the corresponding proteins to activator protein-1 (AP-1) or nuclear factor-κB (NF-κB) consensus sequences. Interestingly, chromatin immunoprecipitation experiments showed that NSC-13502 specifically inhibited binding of HIF-1 to the HRE sequence contained in the vascular endothelial growth factor (VEGF) promoter but not binding of AP-1 or NF-κB to promoter regions of corresponding target genes. Accordingly, NSC-13502 inhibited hypoxic induction of luciferase in U251-HRE cells and VEGF mRNA expression in U251 cells. Our results indicate that it is possible to identify small molecules that inhibit HIF-1 DNA binding to endogenous promoters.

Mutations within microsatellite sequences, consisting of additions or deletions of repeat units, are known as the replication/repair error positive (RER+) phenotype or micorsatellite instability (MI). Microsatellite instability has been demonstrated in hereditary and sporadic colorectal carcinomas and is usually observed in noncoding regions of genomic DNA. However, relatively few coding region targets of MI have been identified thus far. Using PCR, we amplified regions encompassing (A)8 and (C)8 microsatellite tracts within hMSH3 and hMSH6 from 31 RER+ sporadic colorectal tumors, 8 hereditary colon cancers, 23 RER+ gastric carcinomas, and 32 RER- gastric tumors. Mutations were found in 11 (36%) of 31 sporadic colon carcinomas, 4 (50%) of 8 hereditary colorectal cancers, and 5 (22%) of 23 RER+ gastric carcinomas, but in only 2 (6%) of 32 RER- gastric carcinomas. These frameshift mutations cause premature stop codons downstream that are predicted to abolish normal protein function. Our results and those of others suggest that DNA mismatch repair genes, such as hMSH3 and hMSH6, are targets for the mutagenic activity of upstream mismatch repair gene mutations and that this enhanced genomic instability may accelerate the accumulation of mutations in RER+ tumors.

A significant portion of gastric cancers exhibit defective DNA mismatch repair, manifested as microsatellite instability (MSI). High-frequency MSI (MSI-H) is associated with hypermethylation of the human mut-L homologue 1 (hMLH1) mismatch repair gene promoter and diminished hMLH1 expression in advanced gastric cancers. However, the relationship between MSI and hMLH1 hypermethylation has not been studied in early gastric neoplasms. We therefore investigated hMLH1 hypermethylation, hMLH1 expression and MSI in a group of early gastric cancers and gastric adenomas. Sixty-four early gastric neoplasms were evaluated, comprising 28 adenomas, 18 mucosal carcinomas, and 18 carcinomas with superficial submucosal invasion but clear margins. MSI was evaluated using multiplex fluorescent PCR to amplify loci D2S123, D5S346, D17S250, BAT 25 and BAT 26. Methylation-specific PCR was performed to determine the methylation status of hMLH1. In two hypermethylated MSI-H cancers, hMLH1 protein expression was also evaluated by immunohistochemistry. Six of sixty-four early gastric lesions were MSI-H, comprising 1 adenoma, 4 mucosal carcinomas, and 1 carcinoma with superficial submucosal invasion. Two lesions (one adenoma and one mucosal carcinoma) demonstrated low-frequency MSI (MSI-L). The remaining 56 neoplasms were MSI-stable (MSI-S). Six of six MSI-H, one of two MSI-L, and none of thirty MSI-S lesions showed hMLH1 hypermethylation (P

Background E (epithelial)-cadherin, the cell adhesion molecule also considered a potential invasion/metastasis suppressor, is mutationally inactivated in nearly half of all undifferentiated-scattered (diffuse-type) gastric carcinomas. In addition, silencing of E-cadherin by CpG methylation within its promoter region has been reported in several gastric carcinoma cell lines. We investigated the methylation status of the E-cadherin promoter region in 53 primary human gastric carcinomas. Methods Hypermethylation of the E-cadherin promoter was determined by utilizing methylation-specific polymerase chain reaction (PCR)-single-strand conformation polymorphism (MSP-SSCP) analysis followed by direct sequencing of PCR products. Expression of E-cadherin was studied by western blot analysis. All statistical tests were two-sided. Results Hypermethylation of the E-cadherin promoter was evident in 27 (51%) of 53 primary gastric carcinomas examined by MSP-SSCP. It occurred more frequently in carcinomas of the undifferentiated-scattered type (in 15 [83%] of 18) than in other histologic subtypes (in 12 [34%] of 35) (P =.0011, Fisher's exact test), and it was present at similar rates in early (in six [60%] of 10) versus advanced (in 21 [49%] of 43) carcinomas (P =.73, Fisher's exact test). Methylation occurring at all cytosine-guanosine sequences (CpGs) near the transcriptional start site was confirmed in six of six tumors examined by bisulfite-DNA sequencing, including two early gastric carcinomas. In addition, loss or diminished expression of E-cadherin was confirmed by western blotting in four of the six tumor tissues demonstrating hypermethylation. Conclusions The E-cadherin promoter frequently undergoes hypermethylation in human gastric cancers, particularly those of the undifferentiated-scattered histologic subtype. E-cadherin promoter hypermethylation is associated with decreased expression and may occur early in gastric carcinogenesis.

A germline sequence alteration at codon 1307 of the APC gene (I1307K) has been reported in 6 – 7% of the Ashkenazi Jewish population in the United States. This alteration is believed to predispose the APC gene to a secondary mutation at the same locus, resulting in an increased risk of colorectal carcinoma. There is an increased risk of colorectal carcinoma in patients with inflammatory bowel disease (IBD), a relatively large proportion of whom are Ashkenazi Jews. We therefore sought to determine whether the I1307K sequence variant occurred in the germline DNA of IBD patients. To our surprise, we found this sequence in only two of 267 patients with IBD (0.7%), occurring in only 1.5% of Jewish IBD patients. The I1307K sequence variant was not found in 67 patients with esophageal cancer, 53 patients with gastric carcinoma (13 MSI-H and 44 MSI-negative), or ten patients with sporadic MSI-H colon cancer. These findings suggest that the I1307K sequence is relatively rare in the germline of Jewish as well as non-Jewish IBD patients. It does not appear to contribute to the increased colorectal cancer risk present in these patients.

The insulin-like growth factor II receptor (IGFIIR) has been implicated as a tumor suppressor gene in human malignancy. Frequent mutation, loss of heterozygosity, and microsatellite instability (MSI) directly affecting the IGFIIR gene have been reported in several primary human tumor types. However, to our knowledge, dynamic functional evidence of a growth-suppressive role for IGFIIR has not yet been provided. We identified one MSI-positive colorectal carcinoma cell line, SW48, with monoallelic mutation in IGFIIR identical to that seen in primary colorectal carcinomas. A zinc-inducible construct containing the wild-type IGFIIR cDNA was stably transfected into SW48 cells. Growth rate and apoptosis were compared between zinc-treated, untreated, and untransfected cells. A twofold increase in IGFIIR protein expression was detected after zinc treatment in discrete clonal isolates of transfected SW48 cells. Moreover, zinc induction of exogenous wild-type IGFIIR expression reproducibly decreased growth rate and increased apoptosis. These data prove that wild-type IGFIIR functions as a growth suppressor gene in colorectal cancer cells and provide dynamic in vitro functional support for the hypothesis that IGFIIR is a human growth suppressor gene.

FHIT (fragile histidine triad gene), a candidate tumor suppressor gene, was recently identified and cloned at chromosome 3p14.2. Alterations of this gene have been reported in a number of primary human tumors, including colorectal, esophageal, gastric and lung carcinomas. However, some reports have found no abnormalities in this gene. We investigated a total of 63 primary esophageal tumors, nine esophageal cancer cell lines and 17 ulcerative colitis-associated neoplasms (UCANs) for alterations of FHIT. In 13 esophageal tumors, we employed overlapping reverse transcriptase-PCRs (RT-PCRs) to amplify and sequence the complete open reading frame of FHIT. One of 13 primary esophageal tumors analysed by RT-PCR expressed no detectable FHIT transcript; the remaining 12 expressed normal-sized transcripts with wild-type open reading frame sequences. In an additional 50 esophageal tumors, the polymorphic microsatellite loci D3S1300 and D3S1313 were used to evaluate loss of heterozygosity (LOH) at 3p14.2. Eleven of these 50 tumors showed LOH at one or both loci. In all these 11 tumors, genomic PCR and direct sequencing of FHIT exons 5-9 was performed. This analysis revealed that none of these 11 primary esophageal tumors contained any alterations in the FHIT open reading frame or adjacent intron sequences. Finally, among 17 UCANs, the in vitro synthesized protein (IVSP) assay detected no truncated protein products, nor were there any abnormalities in size or DNA sequence of FHIT RT-PCR products. However, in six of nine esophageal carcinoma cell lines, no FHIT RT-PCR product was detectable using either of the overlapping primer sets. Genomic PCR and direct sequencing of exons 5-9, also performed in these nine cell lines, revealed wild-type sequence in eight cell lines; however, one cell line contained no exon 5 PCR product. This cell line also lacked detectable FHIT transcript. These data suggest that the open reading frame of FHIT is not important in the development or progression of most primary esophageal carcinomas or UCANs, although lack of expression of the FHIT transcript may be common in esophageal cancer-derived cell lines. The possibility of an additional tumor suppressor gene at chromosome 3p14.2 remains to be evaluated.

The adenomatous polyposis coli (APC) tumor suppressor gene is mutationally inactivated in both familial and sporadic forms of colorectal cancers. In addition, hypermethylation of CpG islands in the upstream portion of APC, a potential alternative mechanism of tumor suppressor gene inactivation, has been described in colorectal cancer. Because a subset of both gastric and colorectal cancers display the CpG island methylator phenotype, we hypothesized that epigenetic inactivation of APC was likely to occur in at least some gastric cancers. APC exhibits two forms of transcripts from exons 1A and 1B in the stomach. Therefore, we investigated CpG island methylation in the sequences upstream of exons 1A and 1B, i.e., promoters 1A and 1B, respectively. We evaluated DNAs from 10 gastric cancer cell lines, 40 primary gastric cancers, and 40 matching non-cancerous gastric mucosae. Methylated alleles of promoter 1A were present in 10 (100%) of 10 gastric cancer cell lines, 33 (82.5%) of 40 primary gastric cancers, and 39 (97.5%) of 40 noncancerous gastric mucosae. In contrast, promoter 1B was unmethylated in all of these same samples. APC transcripts from exon 1A were not expressed in nine of the 10 methylated gastric cancer cell lines, whereas APC transcripts were expressed from exon 1B. Thus, expression from a given promoter correlated well with its methylation status. We conclude that in contrast to the colon, methylation of promoter 1A is a normal event in the stomach; moreover, promoter 1B is protected from methylation in the stomach and thus probably does not participate in this form of epigenetic APC inactivation.