Many studies on human colorectal cancer (hCRC) samples have documented a dysbiosis associated with the tumor at different levels. However a clear picture of the microorganisms whose abundance is altered during tumorigenesis and evidences of their functional implication in the tumorigenic process have not yet emerged. This study is aimed at dissecting the role of bacteria in CRC development by focusing on the intestinal mucus barrier as a key mediator in microbiota-host interaction. We used the ApcMin/+ murine model to demonstrate that in tumor-bearing mice, similarly to what happens in hCRC, the mucus barrier has altered properties. Mucin expression is altered at the level of dysplastic crypts, strictly relating mucus changes with neoplastic transformation. Being the mucus a fundamental component of the intestinal barrier we further addressed if bacterial penetrance was compromised in tumor-prone mice. As hypothesised upon oral administration we observe increased Salmonella penetration in the intestine and spreading to mesenteric lymph nodes of ApcMin/+ mice compared to wild types. To address the potential role of mucus alteration in the tumorigenic process we exogenously modified the mucus barrier. Upon treatment with Salmonella, a bacterium that is able to interact with the mucus, we observed an increase in colon tumorigenesis in ApcMin/+ mice. Moreover, when we coupled the AOM/DSS protocol of chemically induced colitis-associated colon carcinogenesis to mucolytic treatment we observed exacerbated colon tumorigenesis. We did also investigate whether there was a dysbiosis associated with tumor progression, and when it was arising. Metagenomic analysis in the faeces of mice at different ages highlighted a dysbiosis already at 4 weeks of age in ApcMin/+ mice when tumors are not yet established. In particular, Lactobacillus genus was expanded in ApcMin/+ mice concomitantly with a contraction in the Clostridium genus. Finally since epigenetic mechanisms have recently been hypothesised to contribute to the loss of heterozygosity of the normal apc allele and bacteria can alter miRNA expression we analysed miRNA profiles in WT and ApcMin/+ intestinal tissue observing alterations in ApcMin/+ ilei. We further tested if bacterial stimulation could drive alterations in the apc gene expression, possibly mediated by miRNA modulation, in the intestinal mucosa. Salmonella and E. coli, either invasive or not, did not produce alterations in apc gene expression in an ex-vivo organ culture model that allows polarized stimulation of the intestinal mucosa. In conclusion we found that the mucus layer and intestinal barrier properties are altered in tumor bearing mice. This could lead to the establishment of a dysbiosis, although the cause-effect relationships of this do not emerge clearly from the present work. Bacterial species that are underrepresented in tumor bearing subjects could be protective and their administration could delay tumor progression. Finally, dysbiosis could induce miRNA-mediated control of apc gene expression and this could be a mechanism involved in tumor initiation in the model used. However, even if bacterial stimuli can modulate miRNA expression patterns, the ones tested in this work are not efficient in modulating apc expression.

ROLE OF BACTERIA AND THE MUCUS SYSTEM IN INTESTINAL TUMORIGENESIS / E. Zagato ; internal supervisor: S. Casola ; external supervisor: F. Balkwill ; supervisor: M. Rescigno. - : . UNIVERSITA' DEGLI STUDI DI MILANO, 2015 Mar 18. ((26. ciclo, Anno Accademico 2014. [10.13130/zagato-elena_phd2015-03-18].

ROLE OF BACTERIA AND THE MUCUS SYSTEM IN INTESTINAL TUMORIGENESIS

E. Zagato
2015

Abstract

Many studies on human colorectal cancer (hCRC) samples have documented a dysbiosis associated with the tumor at different levels. However a clear picture of the microorganisms whose abundance is altered during tumorigenesis and evidences of their functional implication in the tumorigenic process have not yet emerged. This study is aimed at dissecting the role of bacteria in CRC development by focusing on the intestinal mucus barrier as a key mediator in microbiota-host interaction. We used the ApcMin/+ murine model to demonstrate that in tumor-bearing mice, similarly to what happens in hCRC, the mucus barrier has altered properties. Mucin expression is altered at the level of dysplastic crypts, strictly relating mucus changes with neoplastic transformation. Being the mucus a fundamental component of the intestinal barrier we further addressed if bacterial penetrance was compromised in tumor-prone mice. As hypothesised upon oral administration we observe increased Salmonella penetration in the intestine and spreading to mesenteric lymph nodes of ApcMin/+ mice compared to wild types. To address the potential role of mucus alteration in the tumorigenic process we exogenously modified the mucus barrier. Upon treatment with Salmonella, a bacterium that is able to interact with the mucus, we observed an increase in colon tumorigenesis in ApcMin/+ mice. Moreover, when we coupled the AOM/DSS protocol of chemically induced colitis-associated colon carcinogenesis to mucolytic treatment we observed exacerbated colon tumorigenesis. We did also investigate whether there was a dysbiosis associated with tumor progression, and when it was arising. Metagenomic analysis in the faeces of mice at different ages highlighted a dysbiosis already at 4 weeks of age in ApcMin/+ mice when tumors are not yet established. In particular, Lactobacillus genus was expanded in ApcMin/+ mice concomitantly with a contraction in the Clostridium genus. Finally since epigenetic mechanisms have recently been hypothesised to contribute to the loss of heterozygosity of the normal apc allele and bacteria can alter miRNA expression we analysed miRNA profiles in WT and ApcMin/+ intestinal tissue observing alterations in ApcMin/+ ilei. We further tested if bacterial stimulation could drive alterations in the apc gene expression, possibly mediated by miRNA modulation, in the intestinal mucosa. Salmonella and E. coli, either invasive or not, did not produce alterations in apc gene expression in an ex-vivo organ culture model that allows polarized stimulation of the intestinal mucosa. In conclusion we found that the mucus layer and intestinal barrier properties are altered in tumor bearing mice. This could lead to the establishment of a dysbiosis, although the cause-effect relationships of this do not emerge clearly from the present work. Bacterial species that are underrepresented in tumor bearing subjects could be protective and their administration could delay tumor progression. Finally, dysbiosis could induce miRNA-mediated control of apc gene expression and this could be a mechanism involved in tumor initiation in the model used. However, even if bacterial stimuli can modulate miRNA expression patterns, the ones tested in this work are not efficient in modulating apc expression.
RESCIGNO, MARIA
Colorectal cancer; mucus; microbiota
Settore MED/04 - Patologia Generale
ROLE OF BACTERIA AND THE MUCUS SYSTEM IN INTESTINAL TUMORIGENESIS / E. Zagato ; internal supervisor: S. Casola ; external supervisor: F. Balkwill ; supervisor: M. Rescigno. - : . UNIVERSITA' DEGLI STUDI DI MILANO, 2015 Mar 18. ((26. ciclo, Anno Accademico 2014. [10.13130/zagato-elena_phd2015-03-18].
Doctoral Thesis
File in questo prodotto:
File Dimensione Formato  
phd_unimi_R09401.pdf

Open Access dal 24/08/2016

Descrizione: Tesi Dottorato
Tipologia: Tesi di dottorato completa
Dimensione 53.18 MB
Formato Adobe PDF
53.18 MB Adobe PDF Visualizza/Apri
Pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/265380
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact