Cisplatin-based chemotherapy is an established treatment for head and neck squamous cell carcinoma (HNSCC); however, treatment resistance remains a major challenge. Biomarkers predictive of cisplatin response may improve risk stratification and treatment selection. We retrospectively analyzed data from The Cancer Genome Atlas (TCGA)- HNSC dataset to investigate the association between LIM homeobox 1 (LHX1) expression and clinical outcomes, particularly among cisplatin-treated patients. Kaplan–Meier survival analysis and differential expression analyses were conducted based on disease-specific survival (DSS) status. LHX1 mRNA expression in tumor and non-tumor tissues was also assessed using TCGA-derived transcriptomic data. In addition, LHX1 mRNA and protein levels were examined in cisplatin-sensitive oral squamous cell carcinoma (OSCC) cell lines and their matched cisplatin-resistant counterparts. Functional relevance was evaluated by performing siRNA-mediated LHX1 knockdown followed by cisplatin treatment in resistant OSCC cells. LHX1 mRNA expression was significantly higher in HNSCC tumors compared with non-tumor tissues (p < 0.001). High LHX1 expression was associated with poor survival in the overall cohort and in the cisplatin-treated subgroup (log-rank p < 0.05 for both). In the cisplatin-treated subgroup, LHX1 expression was significantly associated with DSS status (p < 0.05, q < 0.05). LHX1 expression was also upregulated in cisplatin-resistant OSCC cells (p < 0.05), and its knockdown enhanced cisplatin sensitivity in resistant OSCC cells (p < 0.05). LHX1 may be associated with adverse outcomes and cisplatin resistance in HNSCC/OSCC, supporting its potential as a treatment-relevant biomarker.

Introduction
Materials and Methods
    1. Analysis of LHX1 mRNA expression in HNSCC andnon-tumor tissues
    2. Survival and differential expression analyses ofLHX1 in the TCGA-HNSC cohort
    3. Reagents
    4. Cell lines and patient-derived organoids
    5. Measurement of LHX1 mRNA and proteinexpression in OSCC cells
    6. Assessment of cisplatin sensitivity after LHX1knockdown in cisplatin-resistant OSCC cells
    7. Assessment of cisplatin efficacy after LHX1knockdown in OSCC organoids
    8. Statistical analysis
Results
    1. L HX1 mRNA expression is increased in HNSCCtissues
    2. Association of LHX1 expression with survival andclinical outcome in HNSCC
    3. mRNA and protein expression of LHX1 in cisplatinsensitiveand paired cisplatin-resistant OSCC cells
    4. Evaluation of cisplatin sensitivity incisplatin-resistant OSCC cells after LHX1knockdown
    5. Evaluation of cisplatin responsiveness incisplatin-sensitive OSCC organoids afterLHX1 knockdown
Discussion
Funding
Conflicts of Interest
References

• Su Young Oh(Department of Microbiology and Immunology, School of Dentistry, Kyungpook National University, Daegu 41940, Republic of Korea)
• Tae-Lyn Kim(Department of Microbiology and Immunology, School of Dentistry, Kyungpook National University, Daegu 41940, Republic of Korea)
• Han Byeol Shin(Department of Microbiology and Immunology, School of Dentistry, Kyungpook National University, Daegu 41940, Republic of Korea)
• Hye-Rin Choi(Department of Microbiology and Immunology, School of Dentistry, Kyungpook National University, Daegu 41940, Republic of Korea)
• Heon-Jin Lee(Department of Microbiology and Immunology, School of Dentistry, Kyungpook National University, Daegu 41940, Republic of Korea)
• Su-Hyung Hong(Department of Microbiology and Immunology, School of Dentistry, Kyungpook National University, Daegu 41940, Republic of Korea) Corresponding author
• Eun-Kyong Kim(Department of Preventative Dentistry, School of Dentistry, Kyungpook National University, Daegu 41940, Republic of Korea)
• So-Young Choi(Department of Oral and Maxillofacial Surgery, School of Dentistry, Kyungpook National University, Daegu 41940, Republic of Korea)