Introduction

The interplay between diet, host and intestinal microbiota plays a key role in colorectal cancer (CRC), with processed western diets (WD) of high fat and low fiber content as main contributors. The endoplasmic reticulum unfolded protein response effector activating transcription factor 6 (ATF6) is associated with early changes in CRC and colitis-associated cancer (CAC). We showed that murine intestinal epithelial cell specific expression of active ATF6 (nATF6) triggers spontaneous CRC (nATF6^IEC) and CAC (nATF6^IEC x IL10^-/-) in a microbiota-dependent manner.

Aims & Methods

To substantiate clinical relevance of ATF6 and validate nATF6-driven CRC and CAC mouse models, we performed ATF6 immunohistochemical staining in CRC patients (N = 972), and human-derived fecal microbiota transfer (FMT) into germfree (GF) nATF6^IEC and nATF6^IEC x IL10^-/- mice. Microbiota profiling together with RNA sequencing, metabolomics and lipidomics were used to elucidate ATF6 functional changes in the epithelium. To clarify the role of fat and fiber in tumorigenesis, mono- and biallelic nATF6^IEC, and monoallelic nATF6^IEC x IL10^-/- mice were fed unprocessed chow or processed polymeric diets for 6 weeks after weaning.

Results

ATF6 represents a clinically relevant putative oncogene, with 16 % of CRC patients showing high ATF6 expression in the epithelium. CRC patient FMT established tumorigenesis in GF biallelic nATF6^IEC and monoallelic nATF6^IEC x IL10^-/- mice that are otherwise tumor-free. Multi-omics analyses in specific pathogen-free and GF nATF6^IEC mice point towards ATF6-induced alterations in lipid metabolism. Compared to an unprocessed chow diet, the polymeric WD enriched in fat and depleted in fermentable fiber, increased tumor load in biallelic nATF6^IEC mice and tumor incidence in monoallelic nATF6^IEC x IL10^-/- mice. Unexpectedly, the highly processed WD induced de novo tumorigenesis in otherwise tumor-free monoallelic nATF6^IEC mice. To understand the impact of high fat in this context, we modified the WD by lowering fat content or by supplementing with fermentable fiber. The absence of fermentable fiber in the polymeric diet already accelerated tumor load as well as de novo tumorigenesis. Surprisingly, fiber supplementation failed to protect the WD-induced tumorigenic phenotype despite microbial and metabolic changes. High fermentable fiber content in the low-fat diet even exacerbated nATF6-driven tumorigenesis, suggesting a tumor promoting rather than tumor protective function of fermentable fiber in processed diets.

Conclusion

These findings highlight the ability of all processed diets to exacerbate nATF6-related tumorigenesis, with dietary fermentable fiber unexpectedly acting as a tumor driver. This implies that plant-derived metabolites of unprocessed chow diet may fulfill protective functions in our mouse models.

Introduction

The induction of endoplasmic reticulum unfolded protein responses (UPR^ER) contributes to cancer development and progression. We set out to identify the clinical relevance of signal transducer activating transcription factor 6 (ATF6) and related bacterial genera in colorectal cancer (CRC) patients, and to elucidate the consequence of ATF6 signaling on the murine host.

Aims & Methods

To determine the clinical relevance of ATF6 in CRC, we quantified ATF6 expression in two independent German CRC patient cohorts (n=959; n=256), and a third cohort for validation (n=50), using immunohistochemical staining and artificial intelligence. We used the publicly available The Cancer Genome Atlas (TCGA) Dataset to compare transcription factor activity and microbial profiles. To understand the impact of ATF6 signaling on CRC pathogenesis, we employed our transgenic murine model of intestinal epithelial cell (IEC)-specific activation of the active form of ATF6 (nATF6^IEC) that develops spontaneous and microbiota-dependent colon tumors. We assessed the transcriptional response to ATF6 signaling in colon tissue (bulk mRNA sequencing), and characterized metabolic (untargeted metabolomics) and microbial (16S rRNA sequencing) alterations in murine and CRC tissue. For mechanistic experiments we performed in vivo fatty acid synthase (FASN) inhibition in mice, germ-free mouse colonization, organoid cultures, bioorthogonal non-canonical amino acid tagging (BONCAT) and bacterial growth analysis.

Results

We demonstrate the relevance of ATF6 expression in CRC patients across multiple independent German cohorts by identifying a clear sub-population (19-38% late onset CRC; 11% early onset CRC) of patients that present with high ATF6 expression, underlining the importance of ATF6 in CRC. Transcriptional analysis of nATF6^IEC mice identified bacteria-specific changes in cellular metabolism enriched for fatty acid biosynthesis in IECs. Untargeted metabolomics and isotype-labeling confirmed ATF6-related enrichment of long chain fatty acids (LCFAs) in colonic tissue of CRC patients, nATF6^IEC mice and murine organoid cultures. FASN inhibition and microbiota transfer into germ-free nATF6^IEC mice confirmed the causal involvement of ATF6-induced lipid alterations in tumorigenesis. Multi-omics integration associated tumor-associated LCFAs with tumor-associated bacteria in nATF6^IEC mice. BONCAT analysis reveals the impact of LCFAs on the expansion of specific taxa of the intestinal microbiota. Specifically, the selective expansion of tumor-relevant microbial taxa, including Desulfovibrio fairfieldensis, was mechanistically linked to LCFA exposure. Analyses of the TCGA Dataset identified ATF6 activity to correlate positively with CRC-enriched genera, including Desulfovibrio fairfieldensis, supporting a causal link between ATF6-mediated LCFA synthesis and tumor-relevant changes in the microbiota.

Conclusion

Our study highlights the intricate interplay between UPR^ER, lipid metabolism, and the gut microbiota in CRC. We postulate that chronic ATF6 signaling in the intestinal epithelium promotes the selection of tumor-promoting microbiota by altering lipid metabolism. ATF6 signaling emerges as a key driver that reshapes lipid metabolism and creates a microbiota environment conducive to tumor development, indicating new targetable vulnerabilities in CRC.

Disclosure

no conflict of interest

Introduction

The Mediterranean Diet (MedDiet) has proven to be highly beneficial to human health thanks to its balanced combination of anti-inflammatory and antioxidants nutrients. Several studies^1,2,3 have highlighted the inverse relationship between MedDiet, visceral adiposity, and chronic conditions, namely cardiovascular disease, diabetes, and cancer. The Chrono Med Diet Score (CMDS)⁴ is a recently validated, easy-to-use adherence questionnaire to the MedDiet that, differently from previously validated scores⁵, considers food, lifestyle habits and time of food consumption. Here, we evaluated the CMDS ability to predict the increased risk of gastrointestinal (GI) cancer (colorectal, liver, and stomach) development in a cohort of metabolic subjects.

Aims & Methods

We enrolled 261 patients who accessed our outpatients’ clinic to assess fatty liver disease and Metabolic Syndrome and followed them for a period of 3.2 years. At first evaluation, CMDS was calculated for each patient. Fatty Liver Index (FLI) was calculated as= (e 0.953 × loge (triglycerides) + 0.139 × BMI + 0.718 × loge (GGT) + 0.053 × WC − 15.745)/(1 + e 0.953 × loge (TG) + 0.139 × BMI + 0.718 × loge (GGT) + 0.053 × WC − 15.745) × 100. ROC curves were used to identify the capability of each score to predict cancer development. T-test comparisons for CMDS values were performed for cancer (prostate, bladder, ovarian, and endometrium) and GI cancer diagnosis. P-values lower than 0.05 were considered significant. Chi-square test was used to calculate the associated Odds Ratio (OR) with 95% confidence interval (CI).

Results

A total of 47 out of 261 patients developed cancer. CMDS showed a significant discriminative power (AUC 0.7428) to predict cancer development with a cut-off of 11 (77% sensitivity, 70% specificity, p-value <0.001), while in the 26 subjects with a diagnosis of GI cancer (AUC 0.7961) the cut-off was 13 (67% sensitivity, 85% specificity). Among subjects with CMDS ≤13, those who developed GI cancer showed significantly altered metabolic markers, namely waist circumference, triglycerides, fasting glucose, HDL cholesterol, and FLI compared to patients who did not develop cancer during the follow-up period (see Table). Values of CMDS ≤13 significantly increased the risk of GI cancer with an OR of 3.41 (95% CI: 1.99-6.12). Intriguingly, CMDS was significantly lower (7.1±0.4) in subjects who were diagnosed with gastrointestinal cancer, compared to patients that developed non – gastrointestinal cancer (9.5±0.7).

Conclusion

In our cohort, adherence to the MedDiet is strongly associated to an increased risk of cancer development in metabolic subjects. This association is even stronger in patients who developed GI cancer, as demonstrated by the significant correlation between the CMDS and specific parameters of dysmetabolism, such as visceral adiposity, dyslipidemia, and liver fibrosis. Taken together, our data indicates not only that the risk of GI cancer can be identified using the CMDS as a non-invasive reference method, but also emphasize that dietary and lifestyle changes, promoting a higher adherence to the MedDiet, are significantly recommended for people affected by dysmetabolic conditions.

References

1. Bamia, C.; Lagiou, P.; Buckland, G.; Grioni, S.; Agnoli, C.; Taylor, A.J.; Dahm, C.C.; Overvad, K.; Olsen, A.; Tjønneland, A., et al. Mediterranean diet and colorectal cancer risk: results from a European cohort. European Journal of Epidemiology 2013, 28, 317-328, doi:10.1007/s10654-013-9795-x
2. Mentella, M.C.; Scaldaferri, F.; Ricci, C.; Gasbarrini, A.; Miggiano, G.A.D. Cancer and Mediterranean Diet: A Review. Nutrients 2019, 11, 2059. https://doi.org/10.3390/nu11092059
3. Crudele, L.; Piccinin, E.; Moschetta, A. Visceral Adiposity and Cancer: Role in Pathogenesis and Prognosis. Nutrients 2021, 13, 2101. https://doi.org/10.3390/nu13062101
4. De Matteis, C.; Crudele, L.; Battaglia, S.; Loconte, T.; Rotondo, A.; Ferrulli, R.; Gadaleta, R.M.; Piazzolla, G.; Suppressa, P.; Sabbà, C.; Cariello, M.; Moschetta, A. Identification of a Novel Score for Adherence to the Mediterranean Diet That Is Inversely Associated with Visceral Adiposity and Cardiovascular Risk: The Chrono Med Diet Score (CMDS). Nutrients 2023, 15, 1910. https://doi.org/10.3390/nu15081910.
5. Panagiotakos, D.B.; Pitsavos, C.; Arvaniti, F.; Stefanadis, C. Adherence to the Mediterranean food pattern predicts the prevalence of hypertension, hypercholesterolemia, diabetes and obesity, among healthy adults; the accuracy of the MedDietScore. Prev. Med. 2007, 44, 335–340. https://doi.org/10.1016/j.ypmed.2006.12.009.

Introduction

The incidence of early-onset colorectal cancer (eoCRC), defined as colorectal cancer occurring in young adults under the age of 50, is increasing globally. Questionable eoCRCs' exogenous risk factors are represented by diet and lifestyle, even though a scant literature is available to date. The DEMETRA study is being conducted to assess diet and lifestyle as potential risk factors for eoCRC in various European and non-European countries with populations characterized by large variations in dietary/drinking habits and eoCRC incidence.

Aims & Methods

Therefore, our aims were to (i) develop a unique and shared semi-quantitative food frequency questionnaire (SQFFQ) able to accurately describe dietary and drinking habits of eoCRCs and healthy age-matched controls of different countries at global level that will be involved in the DEMETRA study; (ii) validate the SQFFQ.
We designed an ad-hoc, shared, online SQFFQ to investigate the usual consumption of 329 foods over the past year. These foods were classified in groups and subgroups using the same criteria as the EPIC Italy study [1]. In addition to frequency of consumption, the tool investigates the portions habitually consumed using validated photographs, household measures, and standard units, as well as types of seasoning and methods of cooking and storage. A special software was then developed to analyze responses and link them to food composition tables in order to provide a nutritional breakdown of individual and collective diets. The SQFFQ was then validated for repeatability by administering it twice, 3 weeks apart, to a sample of 30 young adults under 50 years (mean age 35.1 y ± sd 7.71; 63,3% females, 33.3% males, 3% preferred not to answer). To evaluate repeatability, the measurement error for each food group was estimated as the percentage change between the estimates of food consumption for the same individual. Afterwards, the agreement between the two measurements for each food group was measured with Cohen's kappa coefficient.

Results

For 40 out of 65 food groups, the difference between the two SFFQs was less than 20%. The greatest percentage differences were found, as expected, for food groups with sporadic consumption (snacks, barbecue sauces, ketchup, mayonnaise) or consumed in minimal quantities (spices). Agreement levels, represented by the calculation of Cohen's kappa coefficient, were as follows: 12 food groups showed fair/sufficient agreement (Cohen's kappa 20-40), 23 foods exhibited good/moderate agreement (Cohen's kappa >40-60), and 22 food groups demonstrated high substantial agreement (Cohen's kappa >60). Insufficient agreement, with Cohen's Kappa values < 0.21, was observed for only three food groups (snacks, sauces, mayonnaises).

Conclusion

The ad hoc designed SQFFQ provides a reasonably repeatable measure of dietary intake and can be used to assess the dietary and drinking habits of volunteers in this age group. Indeed, most staple foods in the Italian diet, including pasta, fruit, vegetables, legumes, eggs, meat, coffee, and tea, are well estimated by the SQFFQ. However, as yet described in literature, challenges persist in estimating the consumption of foods assumed sporadically (such as snacks) or in small quantities such as spices. Definitive conclusions will be drawn after the completion of the external validation, in which the SQFFQ will be validated against the gold standard, represented by the completion of a 4-day food diary followed by a dietary recall.

References

1. Pala V, Sieri S, Palli D, Salvini S, Berrino F, Bellegotti M, et al. Diet in the Italian EPIC cohorts: presentation of data and methodological issues. Tumori. 2003;89: 594–607.

Introduction

The molecular and clinicopathological features of primary small bowel adenocarcinoma (SBA) are less well understood than those of colorectal and gastric cancers, primarily due to its rarity. Cancer stem cells, a small subpopulation of tumor cells, are capable of initiating tumors and contributing to relapse. However, the clinicopathological significance of cancer stem cell marker expression in SBA remains unclear.

Aims & Methods

To investigate the localization and clinicopathological relevance of stem cell markers in SBA, we conducted immunohistochemical analysis of CD44, CD44v, CD44v9, CD133, Lgr5, β-catenin and E-cadherin using formalin-fixed paraffin-embedded tissue samples from 57 SBA cases. DNA mismatch repair (MMR) status was assessed through immunostaining for MLH1, MSH2, MLH6, and PMS2. We then compared the expression patterns with clinicopathological data, including sex, age, localization, histological type, depth of invasion, lymph node metastasis, distant metastasis, clinical stage, disease-free survival and cancer-specific survival. Correlations between protein expression and prognosis were evaluated using the Kaplan-Meier method and Cox proportional hazards regression analysis.

Results

The mean follow-up period was 52 months (median: 46; range: 4–110 months), during which 19 patients died. Tumor localization was as follows: duodenum (n=24), jejunum (n=28), and ileum (n=5). Histologically, 47 tumors were well differentiated, 6 poorly differentiated, and 4 were mucinous adenocarcinoma. According to the TNM classification, 15 cases were stage I, 18 stage II, 10 stage III, and 14 stage IV. Positive expression rates for each protein were CD44, 54%; CD44v6, 46%; CD44v9, 49%; CD133, 39%; and Lgr5, 37%. Lgr5 expression was significantly correlated with lymph node metastasis, distant metastasis, and advanced TNM stage. It was also associated with reduced membranous β-catenin staining, indicative of Wnt/β-catenin pathway activation. CD44 and its variants were significantly associated with both lymph node metastasis and distant metastasis, as well as abnormal reduced membranous β-catenin staining and E-cadherin staining. CD133 was expressed in 39% of cases but did not correlate with any clinicopathological factors. Notably, its expression was higher in MMR-proficient tumors compared to MMR-deficiency ones. Univariate analysis identified histological type, depth of invasion, lymph node metastasis, CD44, CD44v6, CD44v9, CD133 and Lgr5 expression as significantly associated with cancer-specific survival. In multivariate analysis, both lymph node status (hazard ratio: 16.4) and Lgr5 expression (hazard ratio: 3.7) retained independent prognostic significance. For disease-free survival, univariate analysis using the Cox proportional hazards model revealed that lymph node metastasis, CD44v6 and Lgr5 had significant prognostic value. Multivariate analysis confirmed lymph node metastasis (hazard ratio: 31.2) and Lgr5 expression (hazard ratio: 28.7) as independent predictors of poorer disease-free survival.

Conclusion

Our findings suggest that all analyzed cancer stem cell markers had prognostic relevance in SBA. CD44 and its variant isoforms expression are associated with epithelial-mesenchymal transition. Lgr5 expression is linked to Wnt/β-catenin pathway and is an independent predictor of poor cancer-specific survival and disease-free survival, regardless of lymph node metastasis.