**3. Mechanism of carcinogenesis**

To ascertain the role of gut microbiota in the development of cancer, we need to look at the mechanisms responsible for carcinogenesis. The normal cells get altered into cancerous cells, by changes at the cellular, genetic or epigenetic levels. This process is known as "cell transformation" [41]. TP53 is a tumor suppressor gene that encodes the protein P53. P53 acts as a tumor suppressor which causes a transient cell cycle arrest, allowing the cells to repair the damage caused to the DNA before the cell divides. The cells that are unable to repair the damage undergo apoptosis. This ensures that the

potentially oncogenic mutations are not propagated [42]. Chronic inflammation causes alteration in the TP53 gene, leading to its inactivation. This results in an unregulated cell cycle and cell division, leading to the accumulation of mutations and uncontrolled cellular proliferation. TP53 alterations were seen in biliary epithelia of patients with gallstone disease with an increased frequency with the disease progression from metaplasia to carcinoma [43]. TP53 was the most commonly mutated gene, followed by PIK3CA, SMAD4, ARID1A, KRAS [44–46] and amplification of ERBB2 [47].

The study of these genetic alterations and mechanisms of carcinogenesis has been made possible with the development of various ex vivo and in vivo animal models. These models have been used extensively to decipher the etiopathogenesis of GBC and to develop and test the treatment protocols [48]. Ex vivo models use cell lines to study the tumor characteristics and cellular and genetic abnormalities. But it was seen that different cell lines yielded different tumor characteristics for the same type of tumor, thus complicating the interpretation [49]. In vivo models were superior to the cell lines as they used genetically engineered animals that could retain the genetic mutations and could undergo cellular differentiation. The tumor cells with mutated or amplified genes were inoculated in them and studied for the development of cancer [49–51]. The drawback of these models was the lack of innate immunity which led to altered results as the cancer was not strictly recapitulated [51].

Although these models indicate a causal relationship between the risk factors and carcinogenesis, the human body reacts quite differently as compared to an animal model, thus necessitating the need for the development of an ideal human model.
