Molecular features of chemoresistance in Gastric Adenocarcinoma

Gastric Cancer (GC) is one of the main malignancies in the world, characterized by high incidence (the fourth, according to GLOBOCAN, 2020) and high lethality (the fifth for cancer-related death). The survival of patients with GC is directly related to therapeutic choices and surgery; indeed, the only resolutive approach is the surgical resection accompanied by chemo- or radiotherapy. Recently, the available therapeutic options have been increased thanks to the use of new drugs: the introduction of biological therapy with the use of Trastuzumab (a monoclonal antibody used for the HER2-positive GC) and the integration of new chemotherapeutics agents such as taxanes (paclitaxel and docetaxel) have improved the therapeutic outcomes. Furthermore, a new approach to therapy in combination with surgery has been developed using FLOT (based on 5-Fluorouracil, Oxaliplatin and Docetaxel) or FOLFOX (without docetaxel) regimens. Despite their effectiveness, these therapies are not free from critical issues, such as side effects (due to the high toxicity of the drugs) and the acquisition of chemoresistance by tumor cells, which is also one of the main steps for the worsening of the disease. This event has been closely correlated with the environmental peculiarity of cancers: hypoxia (low O2 concentration), acidosis and the presence of a stem compartment (the so-called Cancer Stem Cells, CSCs). In this work we characterized different aspects of chemoresistance in GC, using two cell lines of gastric adenocarcinoma (AGS and ACC-201) from which we established four distinct resistant cell lines towards the current therapeutic treatments. Moreover, the metabolic characterization of these cancer cell lines highlighted differences between the wild-type (WT) and resistant cells: the latter presented a more glycolytic metabolism, characterized by acidification of the extracellular milieu; this data is also accompanied by an increased expression of CAIX, an enzyme emerged in recent years for its role in cancer progression. The correlation between CAIX expression and chemoresistance was identified by evaluating the effect of a selective CAIX inhibitor, the SLC-0111, determining a decrease in vitality following combined treatment with chemotherapy drugs; as further confirmation of the role of CAIX in tumor cell survival, we observed increased sensitivity to FLOT treatment after silencing CAIX expression. To overcome chemoresistance we also investigated the activity of the polyphenols Oleocanthal and Genistein on our WT and chemoresistant cell lines. In both cases, we observed a general reduction in cell viability with some differences between the various chemoresistant lines. The compounds' toxicity was evaluated through the characterization of the expression of transcription factors (p53, p21, pRb) and the production of ROS following treatment. We also found that the simultaneous administration of Oleocanthal or Genistein with synthetic drugs resulted in an enhancement of the response to treatment in resistant cells. Finally, we characterized a subpopulation of cells with a peculiar stem profile on the ACC- 201 cell line. Indeed, many articles have recently been published regarding the correlation between chemoresistance and tumor progression, demonstrating the involvement of the microenvironment and the presence of a tumor stem niche. We identified, in two chemoresistant cell lines, an increased Aldehyde Dehydrogenase activity (commonly considered a marker of stemness) compared to the WT control; furthermore, following limit dilution assays, we identified a greater capacity for tumor regeneration in the same two resistant lines compared to the others. This first characterization will be further expanded and is currently subject to study. In conclusion, this project thanks to the evaluation of the efficacy of three different compounds on chemoresistant GC cells, and the characterization of an important marker of tumor progression (CAIX), lays the foundations for future research into new targets for overcoming chemoresistance.