AMCO

Mechanisms of MLH1 in SCs and CRCs as DPCs

MLH1 is one type of mismatch repair gene located on chromosome 3, associated with colorectal cancers (Lynch syndrome) [6]. Defects in MMR proteins give rise to genomic instability, which is characteristic of most cancers. Specifically, 8 – 21% of colorectal cancers are caused by the loss of MLH1 expression [7]. In sporadic CRCs, 10-20% of patients are dMMR, and approximately 95% were due to the promoter methylation of the MLH1 gene. Mutated MLH1 activates PI3/AKT rather than RAS/ MAPK signalling Pathways in CRCs, and it also stimulates other downstream genes such as RAF-MEK-ERK and PI3k/PTEN/AKT signalling Pathways, leading to an increase in the proliferation and progression of Cancerous cells [8].

Figure 2: The Immunohistochemistry (IHC) data identified the Major gene (MSI gene) expression between control and dual primary cancer patients.

In the STRING database, we found that MLH1 interacts with other repair genes such as EXO1 and ERCC2. MLH1 heterodimerizes with PMS2 to form MutL-alpha, a component of the postreplicative DNA mismatch repair system (MMR) [9]. DNA repair is initiated by the binding of MutS alpha (MSH2-MSH6) or MutS beta (MSH2-MSH3) to a DNA mismatch, followed by the recruitment of MutL alpha to the heteroduplex [10]. Assembly of the MutL-MutS-heteroduplex ternary complex in the presence of RFC and PCNA is sufficient to activate endonuclease activity of PMS2. It introduces single-strand breaks near the mismatch and thus generates new entry points for the exonuclease EXO1 to degrade the strand [11]. Whereas Exonuclease 1, 5’->3’ doublestranded DNA exonuclease, which may also possess a cryptic 3’>5’ double-stranded DNA exonuclease activity. Functions in DNA mismatch repair (MMR) to excise mismatch-containing DNA tracts directed by strand breaks located either 5’ or 3’ to the mismatch. Also exhibits endonuclease activity against 5’-overhanging flap structures similar to those generated by displacement synthesis when DNA polymerase encounters the 5’-end of a downstream Okazaki fragment [12]. Required for somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin genes [16]. The combined confidence of the functional interaction of MLH1 and EXO1 is 0.999, which denotes a strong correlation between these two genes according to the STRING database.

Figure 3: (a). The lollipop data of MLH1, which is a pathogenic gene mutated on Exon 19, and the location of the amino acid p. Trp714Ter. (b). STRING database observed normal MLH1 gene, strongly correlated with other genes like MSH2, PMS2, MSH6, EXO1, and ERCC2.

The role of ERCC2 is a general transcription and DNA repair factor IIH helicase subunit XPD; ATP-dependent 5’-3’ DNA helicase, component of the general transcription and DNA repair factor IIH (TFIIH) core complex, which is involved in general and transcription-coupled nucleotide excision repair (NER) of damaged DNA and, when complexed to CAK, in RNA transcription by RNA polymerase II [13]. In NER, TFIIH acts by opening DNA around the lesion to allow the excision of the damaged oligonucleotide and its replacement by a new DNA fragment [14]. The combined score of EXO1 and ERCC2 is 0.566. Another combined effect of MLH1 and ERCC2 is 0.681, which has a medium correlation between these genes.

Figure 4: (a). In DPCs, MLH1 mutation has an impact on two other genes, like EXO1 and ERCC2, because they have a strong correlation according to the STRING database. (b). In our study of DPC, we found all these mutated genes; the STRING database represents the role of different exon-specific genes that are strongly correlated with each other.

In this report, we find that when three (MLH1, EXO1, ERCC2) genes are mutated in the cells, it can help to activate the RAS/ MAPK/AKT pathways and stimulate other downstream genes, leading to the proliferation and progression of cancers. In Stomach and Colorectal Cancer, these three mutated genes have a crucial role in the progression of cancer cells and become a malignancy [15].

Chemotherapy for the Treatment of DPCs (SCs and CRCs)

The genome-wide profiling was used to identify common genetic alterations in primary cancers, and the treatment process was also given almost 14 days after surgery for stomach and colorectal carcinoma. The two major drugs, such as Oxaliplatin 180gm and Capecitabine 500gm, are suggested for this patient to recover from the patient’s cancer history and try to inhibit the recurrence rate of cancer progression and proliferation. Oxaliplatin 180 g is a type of cytotoxic Chemotherapeutic drug mainly used in colorectal carcinoma. It is a type of alkylating agent that contains platinum and is used to treat advanced levels of colon cancer [16]. It works by interfering with the DNA replication Process and cross-linking with DNA, stopping the division of Cancer cells and ultimately causing cell death. According to KEGG pathways, oxaliplatin targets genes involved in colon cancer progression, such as those related to the RAS/MAPK pathways, including PDGFR2, ANKRD26, NF1, ALK, and SPTPB. It acts as an inhibitor of the RAS/MAPK pathways, thereby stopping the progression and proliferation of cancer cells.

Figure 5: (a). The column Chart depicting the different genes involved in different signalling pathways and percentage of variation in allele frequency (VAF) changes in stomach (SC) and Colorectal Cancers (CRC) as DPCs. (b). Percentage of different signalling pathways specifically mutated genes in Stomach (SCs) and Colorectal cancers (CRCs).

Whereas, Capecitabine 500 mg is a type of antimetabolite chemotherapeutic drug, which is mainly used in stomach carcinoma. when it enters the body, it is converted into fluorouracil, which can directly interfere with the DNA of cancerous cells and inhibit Thymidylate synthase to stop their growth and proliferation [17]. Some clinical observation shows that Capecitabine has immunosuppressive effects, it can induce T-cell apoptosis and produce some anti-inflammatory cytokines, and act as an anticancer drug. Capecitabine increases the stress response in the endoplasmic reticulum and increases the amount of ROS, finally activating mitochondrial-mediated intrinsic apoptotic pathways. However, Capecitabine drugs produce both immunosuppressive and anti-cancer effects.

Discussion

In the present study, common genetic alternation in multiple primary Cancers (SCs & CRC) was observed by genomewide Profiling, providing insight into potential biomarkers. Recent treatments for DPCs remain limited to cisplatin-based chemotherapy, BCG therapy, and surgery, with a median survival of 14-15 months and no recognised second-line therapy [18]. The highest frequency of genetic alternation occurred on chromosomes 3, 16 and 19. Younger patients showed less dependence on genetic factors compared to older patients, suggesting that environmental influences play a role in cancer progression.

Major mutations were detected in DNA repair genes (MLH1, EXO1, ERCC2) and signalling pathways, including RAS/MAPK, apoptotic, cytoskeletal, and immunomodulatory pathways, particularly involving MLH1, EXO1, and ERCC2. MLH1 mutations, frequent in high-grade SCs and CRCs, are closely associated with other repair genes and activate RAS/MAPK signalling, promoting proliferation [19]. Targeted therapies, such as Oxaliplatin and capecitabine, may inhibit the RAS/MAPK Pathways and enhance T-cell-mediated immune responses, respectively.

Epigenetic alterations also suggest novel treatment options. DNA mismatch repair deficiency, especially MLH1 loss, increases EXO1 activity, causing DNA breaks and neoantigen formation. These neoantigens stimulate the dendritic cells and activate tumour-specific T cells, which can recognise and eliminate cancer cells [20]. Although neoantigens have not been reported in DPCs, their discovery could provide new immunostherapeutic targets.

Conclusion

In this report, we highlighted the molecular mechanisms of the major pathogenic gene MLH1 in stomach (SC) and colorectal cancers (CRC). The whole exome sequencing data identified the total number of genes mutated in dual primary cancers. Oxaliplatin and capecitabine are two major drugs that have anticancer properties in these DPCs. The epigenetic alteration of the DNA sequence produces neoantigens, leading to the activation of T cells driven by tumor antigen and killing cancer cells by binding with a specific cell surface receptor. The development of neoantigenspecific drugs will help target treatment options for stomach and colorectal cancers in the future, utilising DPCs.

Acknowledgements

We are grateful to the patient and to his family, who contributed to this study, as well as Dr. C. Mandal, Senior Scientist, who helped to design this study. Additional personnel and funding sources are acknowledged in the supplementary information.

Disclaimer

The author (s) hereby declare that NO generative AI technologies such as Large Language Models (ChatGPT, COPILOT, etc) and text-to-image generators have been used during the writing or editing of this manuscript.

Ethics Approval and Consent to Participate

The Institutional Ethics Committee (IEC) was conducted in Hybrid mode on 14 February 2025 in CNCI.

IEC Ref: CNCI-IEC-JC-2025-14. Finally, the committee approved the ethics. In this ethical approval committee meeting, the consent of the participant member was obtained.

Competing Interests

The authors have declared that no competing interests exist.

List of Authors Contribution

Rabindra Bera: Written and designed the whole manuscript; Sangita Dan: Collected the sample and maintained the entire data of the patient; Soma Sett: Data Curation, conceptualisation; Sparsha Dey: data identification and sample preparation; Palash Dhara: Sample handling and preparation; Krishna Prasad Sahoo: Data Curation, Formal Analysis; Parvez Alam: data interpretation and handling of sample; Jayanta Chakrabarty: Investigation, Maintain a patient’s routine checkup and provide information on treatment options; Subhranshu Mandal: Finalise all the experiments and methods; Sankar Sengupta: Finalise all the experiments and methods; Chandan Mandal: conceived and designed the experiments.

Availability of data and materials

All Project data is available on the Chittaranjan National Cancer Institute (CNCI) website, accessible through the Department of Medical Science’s web browser. https://www.cnci.ac.in

Funding Declaration

Provided by the Chittaranjan National Cancer Institute Core funding agency.

Consent for publication

The authors hereby confirm that all authors have read and approved the final version of the manuscript titled. All authors consent to the publication of this work in the Journal of Scientific Reports, in print and/or electronic form. The authors agree that the work may be edited or formatted to the journal’s requirements without changing the content

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