Influence of p53 on melatonin oncostatic effects in colorectal cancer

Resumen

Colorectal cancer (CRC) is the third most common cancer and the fourth most common cause of cancer-related death. Therefore, it is very important to find useful biomarkers that help us identified which patients have a worse prognosis. In this sense, cancer stem cells (CSCs) have attracted much attention of the research community in recent years. CSCs have very special functions including long-term self-renewal capacity, multi-lineage differentiation, and resistance to chemotherapy and radiotherapy. Thus, a better understanding of the molecular mechanism involved in its regulation could help us achieve more effective therapies. P53 plays a critical role in the adenoma-carcinoma transition and the frequency of gene alterations increases with the progression of the lesion. Different types of p53 mutations play a pivotal role in determining the biologic behavior of CRC, such as invasive depth, metastatic site and even the prognosis of patients. It has been reported that p53 gain-of-function mutations increase the expression of CSC markers in CRC patients. MicroRNAs (miRNAs) are endogenous non-coding RNAs that regulate self-renewal, differentiation, and division of cells via post-transcriptional gene silencing. These molecules have been suggested to involved in cancer signaling pathways as tumor suppressors or oncogenes. Recent studies have demonstrated that p53 and its network are regulated by miRNAs at multiple levels. miRNAs are involved in complex p53 signaling pathways while p53 can regulate the transcription expression and the maturation of a group of miRNAs. miRNAs can regulate the levels, activity, and function of p53 either through direct repression of p53 or indirectly by interacting with p53-regulating factors. The dysregulation of miRNAs has also been confirmed to engage in CRC development and progression. The altered expression of miRNAs has frequently been correlated with tumor staging, diagnosis, and prognosis. MicroRNAs are critical for both stem cell development and cancer pathogenesis, therefore, they have been examined for their regulatory roles in self-renewal, proliferation, and resistance to therapy. miRNA regulate both self-renewal and differentiation pathways of embryonic stem cells (ESC) by regulating various factors that mediate these processes. Melatonin, the major product of the pineal gland, is synthesized from tryptophan under the control of arylalkylamine N-acetyltransferase (AA-NAT) and N-Acetylserotonin O-methyltransferase (ASMT). Some of its functions are mediated through membrane (MT1 and MT2) or nuclear receptors (RZR/RORa). Melatonin plays an important role as an oncostatic molecule in cancer by inhibiting tumor growth and metastasis through different pathways. Recently, some reports suggest that the oncostatic effects of melatonin could be related to its ability to regulate the phenotype of CSCs. Although there are no data linking TP53 mutations and melatonin synthesis or signaling in cancer, melatonin does activate the p53 tumor-suppressor pathway in CRC. Therefore, the objectives of our study are: (1) To analyze the expression of CSCs markers and melatonin synthesis and signaling genes in tumor samples from CRC patients in Andalusia; (2) To investigate p53 gene mutations in CRC samples; (3) To correlate these results with other tumor and patient pathological parameters; (4) To search for additional information from public databases to find molecular connections between p53, stemnes,s and melatonin in CRC, mainly through miRNAs. The results of our study showed: The frequency of p53 mutations was higher in advanced stages of the disease (stage III and IV). The expression of AA-NAT, MT1, and MT2 decreased in tumor samples versus normal mucosa. Considering the p53 status, the expression of AA-NAT, MT1, and MT2 was lower in patients with mtp53. Protein expression of AA-NAT, MT1, and MT2 was higher in normal mucosa than in tumor samples in wtp53 tumors. When stratifying by tumor stage and p53 status, the expression of AA-NAT decreased in advanced-stage tumors. In wtp53 tumors, differences in AA-NAT and MT2 and MT2 expression levels were found between early and advanced tumors. The expression of CD44 and CD66c was significantly higher in wtp53 than in mtp53 tumors. The expression of CD44 increased in advanced tumors versus those at early stages and independently of the p53 status. However, CD66 expression was similar in both stages of the disease, also independently of the p53 status. the expression of AA-NAT, MT1, MT2, CD44, and CD66c mRNA according to tumor stage and p53 status revealed a significant negative correlation between CD44 and AA-NAT in advanced-stage tumors regardless of the p53 status. A negative correlation was also found between CD66c and AA-NAT in both early and advanced tumors, but only in wtp53, and between CD66c and MT2 in advanced and wtp53 tumors. In our cohort of patients, 32.2% of tumors showed high expression of both CD44 and CD66c markers, while 21.9% showed low expression of both markers. CD44highCD66chigh tumors correlated with low expression of both AA-NAT and MT2 in wtp53 tumors and advanced staged of the disease. In our analysis of public databases, we found several miRNAs whose expression correlates at the same time with CMC marks and melatonin synthesis and signaling genes. It should be noted that, at least in this database, we have not found any miRNAs related to MT2. Conclusions: (1) In our cohort of patients, p53 mutations correlated with advanced stages of the disease; (2) The presence of p53 mutations leads to a decrease of the expression levels of melatonin synthesis and signaling genes (AA-NAT, MT1, and MT2) in early stages of the disease. (3) In wtp53 tumors, the progression of colorectal cancer leads to a significant decrease in the expression of melatonin synthesis and signaling genes; (4) Colorectal tumors bearing p53 mutations showed a decrease in the expression of CSCs markers. CD66c expression was independent of the stage of the disease, whilst expression of CD44 increased in advanced tumors; (5) The progression of the disease results in an increase of the expression of CD44, a CSCs marker, independently of the status (wild type or mutated) of p53 (6) Tumors with high expression of CD44 and CD66c correlated with low expression of AA-NAT and MT2, though only in tumors with wtp53. Therefore, this could be used as a therapeutic approach (7) The analysis of public databases demonstrates common molecular pathways of CSCs markers and melatonin synthesis and signaling genes through miRNAs. Although, this would require further in vitro and in vivo analysis to be able to demonstrate. This, likewise, would open a new field of study in relation to the regulation of the subpopulation of CSCs in the CRC.