Investigation of Combined Effects of KRAS-G12V and KRAS- 2 LCS6 (rs61764370) Alterations on Tumor Progression and Me- 3 tastasis in HNSCC by Using Xenograft Mouse Model

Background Head and neck cancers (HNCs) are the seventh most common cancers worldwide, with KRAS mutations playing a key role for developing and progression of cancer. The G12V mutation is one of the most commonly identified KRAS mutation and has been associated with tumor progression and aggressive disease. Additionally, T > G alteration in the binding site of miRNA let-7 of the KRAS -3'UTR (rs61764370), let-7 complementary site 6 (LCS6), has been found to promote cancer development, metastasis, and treatment resistance. Material and Methods HEp2 laryngeal cancer cells, transfected with plasmid constructs carrying the KRAS -G12V mutation and the combined G12V + LCS6 mutations were transplanted subcutaneously (SC) or intraperitoneally (IP) into CD-1 nude mice with non-transfected control cells. The mice were euthanized on the 40th day post-transplantation and examined histopathologically. Results Primary tumor development was observed in all cell-transplanted mice. The G12V mutation significantly promoted tumor growth (p < 0.01), and LCS6 alteration further accelerated progression (p < 0.001). No metastases occurred in SC-injected mice, but IP-injected mice with KRAS mutations developed metastases. G12V alone led to only liver metastases (50%), whereas G12V + LCS6 resulted in metastases in the liver (100%), lung (75%), kidney (25%), and paraaortic lymph nodes (75%). No brain metastases were observed in any group. Conclusion This study shows that while the KRAS -G12V mutation enhances HNSCC tumor growth and metastasis at relatively low rates, its combination with the LCS6 alteration significantly accelerates both. These findings suggest that miRNAs and miRNAs binding site mutations in KRAS -3’UTR contribute to cancer aggressiveness and could serve as potential therapeutic targets.