In vitro and in vivo Apc-mutated models to study colon carcinogenesis and nutraceutical chemopreventive products

Colorectal cancer (CRC) is the third most common cancer in Western Countries and one of the leading causes of mortality (WHO). CRC is a multistep process involving hyperproliferation of normal epithelial cells, due to an Apc gene mutation, and proceeding through adenomas and adenocarcinomas development due to progressive accumulation of mutations on oncogenes and oncosuppressor genes. These mutations can be due, among others, to increased genomic instability, oxidative stress, and epigenetic changes. Among this last one, DNA methylation changes in CpG islands are involved in 18% of CRC cases (Gallois et al. 2016). Moreover, progress and invasion of CRC are also stimulated by the microenvironment: Cancer Associated Fibroblasts (CAFs) are its main component, responsible for the epithelialmesenchymal transition and inflammation promotion (Wang et al. 2017). A wide range of data are present in literature about CAFs role in colon carcinogenesis: nevertheless, the link between Apc mutation and colon tissue microenvironment has not been clearly defined yet. Three aims have characterized this PhD project: first, the study of natural chemopreventive strategies capable of interrupting CRC carcinogenesis with associated very low toxicity. Indeed, the use of NSAIDs, showed to be a promising chemopreventive strategy in the past decades, is limited by their possible side effects: instead, the use of natural products could allow to overtake this limitation and be useful for secondary and tertiary prevention in individuals at high risk of CRC development. Three different natural compounds/products (a bergamot juice extract from endocarp (BJe), morin, and a pomegranate mesocarp decoction (PMD)) were tested in vivo in the Pirc rat model (F344/NTac-Apcam1137), bearing an Apc gene mutation and spontaneously developing colorectal polyps (Amos-Landgraf et al. 2007) and microscopic preneoplastic lesions (MDFs, Femia et al. 2015) in the normal mucosa (NM): MDFs are also considered useful endpoint for short-term chemopreventive studies. In addition, these in vivo experiments were supported by in vitro tests, aimed to explain the molecular mechanisms of the 3 tested compounds. The second aim was the study of the link between Apc gene mutation and tissue microenvironment at very early stages of colon carcinogenesis, addressing the role of colon fibroblasts within tissue microenvironment, at a stage in which colon carcinogenesis is already ongoing but no macroscopic lesions can be found. We evaluated the effects of this mutation on colon fibroblasts phenotype in established primary cultures from the colon of Pirc and F344-Wt age-matched (one month) rats. The third aim was to add knowledge on the role of Apc mutation on DNA stability at early stages of colon carcinogenesis, with the use of the COMET assay to evaluate DNA strand breaks and oxidative damage in both cultured colon fibroblasts of Pirc and Wt rats and in apparently morphological normal mucosa samples of Pirc and Wt rats aged one month. In addition, we set up a modification of the method, aiming at evaluating the global DNA methylation status. The effects of the treatments tested in Pirc rats were evaluated assessing the number of MDFs, apoptosis and proliferation both histologically and measuring relevant genes and/or proteins expression. Established Pirc and Wt colon fibroblasts primary cultures were characterized for their proliferative and phenotypic profile: both inflammatory and senescence-associated markers were evaluated with immunochemical and cytochemical assays. Concerning BJe, morin and PMD tested in vivo, each of them demonstrated to be capable of perturbing colon carcinogenesis as suggested by the reduction in MDFs number and size, possibly through a combination of proapoptotic and pro-inflammatory actions as suggested by in vitro experiments and also in an ex-vivo model of adenoma (for PMD study) from Pirc rats (Tortora et al. 2018). The novelty of these studies was represented by either the use of non-canonical sources of natural compounds (fruit by-products) or by the possibility to target an oncoprotein (LMW-PTP, low molecular weight phospho-tyrosine phosphatase) consequently enhancing the therapeutic response (study on morin). These data support the idea that a combination of natural compounds acting synergistically with each other, and possibly with drugs, can represent a promising secondary and tertiary chemopreventive strategy. The data obtained on colon fibroblasts mutated in Apc suggest that this mutation determines the development of a proinflammatory phenotype in this tissue microenvironment component, which might be involved in the creation of a protumorigenic environment favoring micro and macro pre-neoplastic lesion development. Moreover, data obtained with the COMET assay on colon fibroblasts and NMs from one month-old Pirc and Wt rats showed a lower level of oxidative damage in Pirc compared to Wt animals: also based on previous data from this lab (Femia et al. 2015), we speculate that Apc mutation could account for a selective advantage for carcinogenesis development through an increase in antioxidants defenses, as reported in the litterature (Ogasawara and Zhang 2008). Finally, we succeeded in the development of a methyl sensitive COMET assay, as proven by the reliability of the methylation changes observed after hypo- and hypermethylating stimuli in two different kinds of normal colon cell lines (epithelial and fibroblasts): this method will be used in the future to address the link between Apc gene and methylation.