Mechanistic characterization and functional evaluation of new multi-targeted small molecules as therapeutic agents for malignant gastrointestinal tumors

Pancreatic adenocarcinoma (PAAD) is one of the most aggressive and deadliest malignant neoplasms; this solid tumor, with the complex tumor microenvironment (TME), consists of a myriad of different types of cells, such as inflammatory, satellite, activated fibroblast, immune cells and numerous growth factors embedded in the extracellular matrix (ECM). Cancer-associated fibroblast (CAFs) was reported to remodel the ECM, along with CAFs in ECM hyaluronan or hyaluronic acid (HA) and hyaluronidases (HA-enzyme) also present, and these enzymes catalyze the degradation of HA, accumulation of HA plays an important role in the pathogenesis and metastasis of many cancers. However, the link between CAFs and HA-enzyme is not yet studied; therefore, to decode the correlation between CAFs and HA-enzyme in PAAD pathogenesis, we applied this in-silico study. Data from the publicly available Gene Expression Omnibus (GEO) database (GSE172096) of CAFs and NFs samples and in-house sequencing of NSC777201 treatment on Panc1 cells were analyzed for differential gene expression (DEGs), gene ontology (GO), and pathway analysis using R packages. Overlapped DEGs between CAFs-DEGs and NSC777201 were validated using TCGA-PAAD and the Human Protein Atlas database to identify survival (KM plot) and diagnostic and prognostic (ROC) values of overlapped genes. Furthermore, Ingenuity pathway (IPA) and in-silico docking were performed to display the therapeutic potential of NSC777201 for survival, diagnostic, and predictive values, along with molecular docking of NSC777201, in-vitro and preclinical in-vivo validation. A total of 416 DEGs (CAFs-DEGs) were obtained between CAFs and NFs samples and 570 DEGs (NSC777201 -DEGs) from NSC777201 treatment. A total of 9-overlapped DEGs were obtained, WDHD1, ATP9A, CBLB, CCNE2, DIDO1, HMBOX1, TGFBR2, TIPARP, and TMEM2 after intersection analysis. Interestingly CEMIP2 (TMEM2) is one of the important overlapped DEGs, significantly overexpressed in CAFs and PAAD tumors, and is strongly correlated with the FAP marker (r = 0.480, p = 9.83E-12), a classical CAFs marker and has remained associated with the higher risk group of PAAD. Noticeable TMEM2 gene inhibition after NSC777201 treatment was observed. Additionally, IPA and molecular docking demonstrated NSC777201 may be an important novel small molecule target the HA-enzyme TMEM2 in TME and regulate the CAFs cells senescence with a resultant anti-PAAD effect, which is, interestingly, validated in-vitro and in-vivo, explained the inhibitory ability of NSC777201, which targets TMEM2 expression, reinforcing its potential to target PAAD. In conclusion, TMEM2 may serve as an important prognostic, diagnostic, and therapeutic biomarker and promotes the development of PAAD through CAFs modulation, and HA accumulation in TME. NSC777201 treatment may target the TMEM2 and reverse the process mentioned above. Therefore, NSC777201 may serve as an important small molecule drug worthy of therapeutic effects and warrants additional investigation to be therapeutically used in PAAD treatment.