Sana K. Baba ¹ Shahabuddin Usmani ² Saira Hamid ¹ Tariq Masoodi ² Hana Q. Sadida ² IKHLAK AHMED ² Mohd S. Khan ³ Inara Abeer ⁴ IBRAHIM ALTEDLAWI ALBALAWI ⁵ Ruqaiah I. Bedaiwi ⁵ Ammira S. Akil ² Ajaz A. Bhat ^2* Dr. MUZAFAR A. MACHA ¹

• ¹ Islamic University of Science and Technology, Awantipora, Jammu and Kashmir, India
• ² Sidra Medicine, Doha, Qatar
• ³ King Saud University, Riyadh, Riyadh, Saudi Arabia
• ⁴ Sher-I-Kashmir Institute of Medical Sciences, Srinagar, Jammu and Kashmir, India
• ⁵ University of Tabuk, Tabuk, Tabuk, Saudi Arabia

Esophageal squamous cell carcinoma (ESCC) accounts for over 90% of all esophageal tumors. However, the molecular mechanism underlying ESCC development and prognosis remains unclear, and there are still no effective molecular biomarkers for diagnosing or predicting the clinical outcome of patients with ESCC. Here, we used bioinformatics analysis to identify potential biomarkers and therapeutic targets for ESCC. Differentially expressed genes (DEGs) between ESCC and normal esophageal tissue samples were obtained by comprehensively analyzing publicly available RNA-seq datasets from the TCGA and GTEX. Gene Ontology (GO) annotation and Reactome pathway analysis identified the biological roles of the DEGs. Moreover, the Cytoscape 3.10.1 platform and subsidiary tools such as CytoHubba were used to visualize the DEGs' protein-protein interaction (PPI) network and identify hub genes. Identification of 2524 genes exhibiting altered expression enriched in pathways including keratinization, epidermal cell differentiation, G alpha(s) signaling events, and biological process of cell proliferation and division, extracellular matrix (ECM) disassembly, and muscle function. Moreover, upregulation of hallmarks E2F targets, G2M checkpoints, and TNF signaling. CytoHubba revealed 20 hub genes that had a valuable influence on the progression of ESCC in these patients. Among these, the high expression levels of four genes, CDK1 MAD2L1, PLK1, and TOP2A, were associated with critical dependence for cell survival in ESCC cell lines, as indicated by CRISPR dependency scores, gene expression data, and cell line metadata. We also identify the molecules targeting these essential hub genes, among which GSK461364 is a promising inhibitor of PLK1, BMS265246, and Valrubicin inhibitors of CDK1 and TOP2A, respectively. Moreover, we identified that elevated expression of MMP9 is associated with worse overall survival in ESCC patients, which may serve as potential prognostic biomarkers or therapeutic targets for ESCC. The single-cell RNA analysis showed that MMP9 is highly expressed in myeloid, fibroblast, and epithelial cells but low in T, endothelial, and B cells. This suggests MMP9's role in tumor progression and matrix remodeling, highlighting its potential as a prognostic marker and therapeutic target.