Discovering the Mutational Profile of Early Colorectal Lesions: A Translational Impact.

Colorectal cancer (CRC) develops through a multi-step process characterized by the acquisition of multiple somatic mutations in oncogenes and tumor-suppressor genes, epigenetic alterations and genomic instability. These events lead to the progression from precancerous lesions to advanced carcinomas. This process requires several years in a sporadic setting, while occurring at an early age and or faster in patients affected by hereditary CRC-predisposing syndromes. Since advanced CRC is largely untreatable or unresponsive to standard or targeted therapies, the endoscopic treatment of colonic lesions remains the most efficient CRC-preventive strategy. In this review, we discuss recent studies that have assessed the genetic alterations in early colorectal lesions in both hereditary and sporadic settings. Establishing the genetic profile of early colorectal lesions is a critical goal in the development of risk-based preventive strategies.

Are Wnt/ß-Catenin and PI3K/AKT/mTORC1 Distinct Pathways in Colorectal Cancer?

Wnt/ß-catenin and phosphatidylinositol-3-kinase/protein kinase B/mammalian target of rapamycin complex 1 (PI3K/AKT/mTORC1) pathways both are critically involved in colorectal cancer (CRC) development, although they are implicated in the modulation of distinct oncogenic mechanisms. In homeostatic and pathologic conditions, these pathways show a fine regulation based mainly on feedback mechanisms, and are connected at multiple levels involving both upstream and downstream common effectors. The ability of the Wnt/ß-catenin and PI3K/AKT/mTORC1 pathways to reciprocally control themselves represents one of the main resistance mechanisms to selective inhibitors in CRC, leading to the hypothesis that in specific settings, particularly in cancer driven by genetic alterations in Wnt/ß-catenin signaling, the relationship between Wnt/ß-catenin and PI3K/AKT/mTORC1 pathways could be so close that they should be considered as a unique therapeutic target. This review provides an update on the Wnt/ß-catenin and PI3K/AKT/mTORC1 pathway interconnections in CRC, describing the main molecular players and the potential implications of combined inhibitors as an approach for CRC chemoprevention and treatment.

A Mediterranean Diet Mix Has Chemopreventive Effects in a Murine Model of Colorectal Cancer Modulating Apoptosis and the Gut Microbiota.

Objectives: Unhealthy dietary patterns have been associated with colorectal cancer (CRC) onset while Mediterranean Diet (MD) has been proposed for CRC prevention. This study evaluated the effect of a Mediterranean Diet Mix (MD-MIX) on colonic tumors development in A/J mice fed a low-fat (LFD) or a high-fat western diet (HFWD), and injected with the procarcinogen azoxymethane (AOM). Materials and Methods: Forty A/J male mice were randomly assigned into four feeding arms (10 mice/arm; LFD, LFD-MD-MIX, HFWD, HFWD-MD-MIX) to be treated with AOM. Ten mice were exposed to the diets alone (Healthy LFD and Healthy HFWD) to be used as control. Tumor incidence and multiplicity were evaluated at sacrifice. Mucosal fatty acid content and urinary phenolic compounds were assayed by mass spectrometry. Apoptosis was evaluated by TUNEL assay and gene expression markers. Cell proliferation was evaluated by Ki67 immunohistochemistry. Microbiota composition was assessed at different time points by 16S RNA sequencing. Results: A tumor incidence of 100% was obtained in AOM-treated mice. The MD-MIX supplementation was able to reduce the number of colonic lesions in both LFD and HFWD-fed mice and to induce apoptosis, in particular in the LFD-MD-MIX arm. Moreover, a preventive effect on low-grade dysplasia and macroscopical lesions (>1 mm) development was found in HFWD-fed mice together with a regulation of the AOM-driven intestinal dysbiosis. Conclusions: MD-MIX was able to counteract CRC development in mice under different dietary backgrounds through the regulation of apoptosis and gut microbiota.

miR-155 Is Downregulated in Familial Adenomatous Polyposis and Modulates WNT Signaling by Targeting AXIN1 and TCF4.

Adenomatous Polyposis Coli (APC) gene mutations are responsible for the onset of familial adenomatous polyposis (FAP) and sporadic colorectal cancer and have been associated with miRNAs dysregulation. The capacity of miR-155, a cancer-related miRNA, to target components of the WNT/ß-CATENIN pathway suggests that APC gene mutations, controlling miRNAs expression, may critically regulate WNT/ß-CATENIN signaling. To this end, APC gene target sequencing was performed on colonic adenomatous polyps and paired normal mucosa clinical specimens from FAP patients (n = 9) to elucidate the role of miR-155-5p in APC-mutant setting. The expression of selected miRNAs and WNT/ß-CATENIN signaling components was characterized in FAP patients and non-FAP control subjects (n = 5). miR-155-5p expression and functional effects on WNT cascade, cell survival, growth, and apoptosis were investigated in different colorectal cancer cell lines. A somatic second hit in the APC gene was found in adenomatous polyps from 6 of 9 FAP patients. Heterozygous APC gene mutations in FAP patients were associated with altered expression of candidate miRNAs and increased levels of AXIN1 and AXIN2 mRNAs. miR-155-5p was downregulated in FAP patients and in the APC and ß-CATENIN-mutant colorectal cancer cell lines, and critically regulates WNT/ß-CATENIN cascade by targeting both AXIN1 and TCF4. Importantly, miR-155-5p may sustain long-term WNT/ß-CATENIN activation in colorectal cancer cells upon WNT3A stimulation. IMPLICATIONS: This study supports a key role of miR-155-5p in modulating WNT/ß-CATENIN signaling in colorectal cancer and unravels a new mechanism for AXIN1 regulation which represents a potential therapeutic target in specific tumor subtypes.

Nucleotide-Binding Domain Leucine-Rich Repeat Containing Proteins and Intestinal Microbiota: Pivotal Players in Colitis and Colitis-Associated Cancer Development.

The nucleotide-binding domain leucine-rich repeat containing (NLR) proteins play a fundamental role in innate immunity and intestinal tissue repair. A dysbiotic intestinal microbiota, developed as a consequence of alterations in NLR proteins, has recently emerged as a crucial hit for the development of ulcerative colitis (UC) and colitis-associated cancer (CAC). The concept of the existence of functional axes interconnecting bacteria with NLR proteins in a causal role in intestinal inflammation and CAC aroused a great interest for the potential development of preventive and therapeutic strategies against UC and CAC. However, the most recent scientific evidence, which highlights many confounding factors in studies based on microbiota characterization, underlines the need for an in-depth reconsideration of the data obtained until now. The purpose of this review is to discuss the recent findings concerning the cross talk between the NLR signaling and the intestinal microbiota in UC and CAC development, and to highlight the open issues that should be explored and addressed in future studies.

Somatic APC mosaicism and oligogenic inheritance in genetically unsolved colorectal adenomatous polyposis patients.

Germline variants in the APC gene cause familial adenomatous polyposis. Inherited variants in MutYH, POLE, POLD1, NTHL1, and MSH3 genes and somatic APC mosaicism have been reported as alternative causes of polyposis. However, ~30-50% of cases of polyposis remain genetically unsolved. Thus, the aim of this study was to investigate the genetic causes of unexplained adenomatous polyposis. Eight sporadic cases with >20 adenomatous polyps by 35 years of age or >50 adenomatous polyps by 55 years of age, and no causative germline variants in APC and/or MutYH, were enrolled from a cohort of 56 subjects with adenomatous colorectal polyposis. APC gene mosaicism was investigated on DNA from colonic adenomas by Sanger sequencing or Whole Exome Sequencing (WES). Mosaicism extension to other tissues (peripheral blood, saliva, hair follicles) was evaluated using Sanger sequencing and/or digital PCR. APC second hit was investigated in adenomas from mosaic patients. WES was performed on DNA from peripheral blood to identify additional polyposis candidate variants. We identified APC mosaicism in 50% of patients. In three cases mosaicism was restricted to the colon, while in one it also extended to the duodenum and saliva. One patient without APC mosaicism, carrying an APC in-frame deletion of uncertain significance, was found to harbor rare germline variants in OGG1, POLQ, and EXO1 genes. In conclusion, our restrictive selection criteria improved the detection of mosaic APC patients. In addition, we showed for the first time that an oligogenic inheritance of rare variants might have a cooperative role in sporadic colorectal polyposis onset.

Short-term treatment with eicosapentaenoic acid improves inflammation and affects colonic differentiation markers and microbiota in patients with ulcerative colitis.

Patients with long-standing ulcerative colitis (UC) have an increased colorectal cancer (CRC) risk. In this pilot study we evaluated the effect of Eicosapentaenoic acid as free fatty acid (EPA-FFA) supplementation on mucosal disease activity, colonic differentiation markers and microbiota composition in UC patients. Twenty long-standing UC patients in stable clinical remission and with fecal calprotectin (FC) > 150 µg/g were enrolled (T0) and supplemented with EPA-FFA 2 g/daily for 90 days (T3). Endoscopic and histologic disease activities were measured by Mayo and Geboes scores, respectively. HES1, KLF4, STAT3, IL-10 and SOCS3 levels were determined using western blotting and qRT-PCR, while phospho-STAT3 levels were assessed by western blotting. Goblet cells were stained by Alcian blue. Microbiota analyses were performed on both fecal and colonic samples. Nineteen patients completed the study; seventeen (89.5%) were compliant. EPA-FFA treatment reduced FC levels at T3. Patients with FC > 150 µg/g at T3 (n = 2) were assumed as non-responders. EPA-FFA improved endoscopic and histological inflammation and induced IL-10, SOCS3, HES1 and KLF4 in compliant and responder patients. Importantly, long-term UC-driven microbiota composition was partially redressed by EPA-FFA. In conclusion, EPA-FFA supplementation reduced mucosal inflammation, promoted goblet cells differentiation and modulated intestinal microbiota composition in long-standing UC patients.

Inflammation increases NOTCH1 activity via MMP9 and is counteracted by Eicosapentaenoic Acid-free fatty acid in colon cancer cells.

Aberrant NOTCH1 signalling is critically involved in multiple models of colorectal cancer (CRC) and a prominent role of NOTCH1 activity during inflammation has emerged. Epithelial to Mesenchymal Transition (EMT), a crucial event promoting malignant transformation, is regulated by inflammation and Metalloproteinase-9 (MMP9) plays an important role in this process. Eicosapentaenoic Acid (EPA), an omega-3 polyunsaturated fatty acid, was shown to prevent colonic tumors in different settings. We recently found that an extra-pure formulation of EPA as Free Fatty Acid (EPA-FFA) protects from colon cancer development in a mouse model of Colitis-Associated Cancer (CAC) through modulation of NOTCH1 signalling. In this study, we exposed colon cancer cells to an inflammatory stimulus represented by a cytokine-enriched Conditioned Medium (CM), obtained from THP1-differentiated macrophages. We found, for the first time, that CM strongly up-regulated NOTCH1 signalling and EMT markers, leading to increased invasiveness. Importantly, NOTCH1 signalling was dependent on MMP9 activity, upon CM exposure. We show that a non-cytotoxic pre-treatment with EPA-FFA antagonizes the effect of inflammation on NOTCH1 signalling, with reduction of MMP9 activity and invasiveness. In conclusion, our data suggest that, in CRC cells, inflammation induces NOTCH1 activity through MMP9 up-regulation and that this mechanism can be counteracted by EPA-FFA.

High-throughput genotyping of high-risk Human Papillomavirus by MALDI-TOF Mass Spectrometry-based method.

A high-throughput matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS)-based method was here developed to genotype 16 high-risk human papillomavirus (HPV) types in cervical cytology specimens. This method was compared to a commercial kit, the Inno-LiPA HPV genotyping assay, which detects a broad spectrum of HPV types. HPV DNA was assessed by the two methods in a total of 325 cervical cytology specimens collected in PreservCyt® solution. The overall agreement was almost perfect (Cohen's k=0.86) in term of positive and negative cases. Indeed, HPV types 16, 35, 56 and 66 showed the highest agreement values (>0.80). The highest agreement values (K >0.80) were found for all 16 HPV types in single infections, but only for HPV 16, 35, 45 and 56 in multiple infections. In conclusion, the high-throughput MS-based method developed here is well-suited for broad spectrum HPV genotyping in large-scale epidemiological studies.

A combination of eicosapentaenoic acid-free fatty acid, epigallocatechin-3-gallate and proanthocyanidins has a strong effect on mTOR signaling in colorectal cancer cells.

Colorectal cancer (CRC) is one of the major causes of cancer death worldwide. The development of novel anti-CRC agents able to overcome drug resistance and/or off-target toxicity is of pivotal importance. The mammalian target of rapamycin (mTOR) plays a critical role in CRC, regulating protein translation and controlling cell growth, proliferation, metabolism and survival. The aim of this study was to explore the effect of a combination of three natural compounds, eicosapentaenoic acid-free fatty acid (EPA-FFA), epigallocatechin-3-gallate (EGCG) and proanthocyanidins (grape seed [GS] extract) at low cytotoxic concentrations on CRC cells and test their activity on mTOR and translational regulation. The CRC cell lines HCT116 and SW480 were treated for 24h with combinations of EPA-FFA (0-150 µM), EGCG (0-175 µM) and GS extract (0-15 µM) to evaluate the effect on cell viability. The low cytotoxic combination of EPA-FFA 150 µM, EGCG 175 µM and GS extract 15 µM completely inhibited the mTOR signaling in HCT116 and SW480 cells, reaching an effect stronger than or comparable to that of the mTOR inhibitor Rapamycin in HCT116 or SW480 cells, respectively. Moreover, the treatment led to changes of protein translation of ribosomal proteins, c-Myc and cyclin D1. In addition, we found a reduction of clonal capability in both cell lines, with block of cell cycle in G0G1 and induction of apoptosis. Our data suggest that the low cytotoxic combination of EPA-FFA, EGCG and GS extract, tested for the first time here, inhibits mTOR signaling and thus could be considered for CRC treatment.

Eicosapentaenoic acid free fatty acid prevents and suppresses colonic neoplasia in colitis-associated colorectal cancer acting on Notch signaling and gut microbiota.

Inflammatory bowel diseases are associated with increased risk of developing colitis-associated colorectal cancer (CAC). Epidemiological data show that the consumption of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) decreases the risk of sporadic colorectal cancer (CRC). Importantly, recent data have shown that eicosapentaenoic acid-free fatty acid (EPA-FFA) reduces polyp formation and growth in models of familial adenomatous polyposis. However, the effects of dietary EPA-FFA are unknown in CAC. We tested the effectiveness of substituting EPA-FFA, for other dietary fats, in preventing inflammation and cancer in the AOM-DSS model of CAC. The AOM-DSS protocols were designed to evaluate the effect of EPA-FFA on both initiation and promotion of carcinogenesis. We found that EPA-FFA diet strongly decreased tumor multiplicity, incidence and maximum tumor size in the promotion and initiation arms. Moreover EPA-FFA, in particular in the initiation arm, led to reduced cell proliferation and nuclear ß-catenin expression, whilst it increased apoptosis. In both arms, EPA-FFA treatment led to increased membrane switch from ω-6 to ω-3 PUFAs and a concomitant reduction in PGE2 production. We observed no significant changes in intestinal inflammation between EPA-FFA treated arms and AOM-DSS controls. Importantly, we found that EPA-FFA treatment restored the loss of Notch signaling found in the AOM-DSS control and resulted in the enrichment of Lactobacillus species in the gut microbiota. Taken together, our data suggest that EPA-FFA is an excellent candidate for CRC chemoprevention in CAC.