Emodin Inhibits Inflammation, Carcinogenesis, and Cancer Progression in the AOM/DSS Model of Colitis-Associated Intestinal Tumorigenesis

Colorectal cancer (CRC) is one of the most common cancer worldwide. Chronic inflammation contributes to CRC development and progression. Emodin, is a natural anthraquinone derivative with anti-oxidant, anti-inflammatory, and anti-tumor activities. We used the AOM/DSS model of colitis-associated intestinal tumorigenesis to characterize the effect of Emodin on inflammation and tumorigenesis at weeks 3, 5, and 14 after initiation with AOM. At all three time points, Emodin (50 mg/kg) reduced inflammatory cell (i.e. CD11b(+) and F4/80(+)) recruitment, cytokine (i.e. TNF alpha, IL1 alpha/beta, IL6, CCL2, CXCL5) and pro-inflammatory enzymes (i.e. COX-2, NOS2) expression in the tumor microenvironment, while promoting recruitment of CD3(+) T lymphocytes at 14 weeks. Emodin decreased the incidence of premalignant lesions (adenoma) at week 3, the incidence of dysplastic lesions and carcinomas at week 5, and the incidence, size and the invasiveness of carcinomas at week 14. Emodin also reduced the acute clinical intestinal symptoms (i.e. bleeding and diarrhea) during DSS treatment. In vitro, Emodin inhibited the expression of pro-inflammatory mediators by LPS-stimulated RAW 264.7 macrophages, and reduced viability, adhesion, migration, and fibroblasts-induced invasion of SW620 and HCT116 colon cancer cells. In conclusion, this work demonstrates that Emodin suppresses carcinogenesis-associated intestinal inflammation and prevents AOM/DSS-induced intestinal tumorigenesis and progression. These results instigate further studies on Emodin as a natural agent for the prevention or treatment of colorectal cancer.

Gene expression analysis of metastases and IL-1R1 characterization in a NALP3 mouse model

Heidi Fournier

Tumor microenvironment contributes strongly to tumor evolution toward metastasis, a final stage in cancer progression. It provides essential clues to promote migration of cancer cells in metastatic sites. Moreover, inflammatory cells and immunomodulatory mediators present in the microenvironment are able to polarize the host immune response, thereby potentiating primary tumor development. The reciprocal and dynamic interactions between microenvironment and tumor cells orchestrate critical steps of evolution toward metastasis. To address the role of inflammation in tumor progression, a mouse model of downregulated inflammation was used: the NALP3 gene in these mice is knocked-out, causing a impaired processing of the two inflammatory cytokines Interleukin-1[beta] and IL-18 which are essential for the recruitment and activation of immune cells. In this case, a decrease in tumor growth and metastasis was observed in previous studies, leading us to investigate the molecular and cellular interactions of tumor and immune cells in inflammatory and non-inflammatory contexts. Gene expression analysis was performed on two steps of mouse tumor cells: the B16-F10 melanoma cells seem to be more dependent on the immune cells recruitment to metastasize in vivo, while the Lewis Lung Carcinomas cells showed a quite equivalent growth in both wild-type or NALP3 knock-out mice. Different techniques were used to do so, such as standard in vitro assays and mRNA expression analyses, and in vivo tests with extraction of tumoral mRNA by Laser Capture Microdissection. Gene expression analyses of tumor cells were performed to address the influence of the inflammatory background. Comparison of these two gene expression profiles may reveal genes related to the specific reactive inflammatory response surrounding the given metastases. Another point that was investigated was the importance of the IL-1R1, receptor of interleukin-1. Since it represents the counterpart of IL-1[beta] signaling, its expression by immune and tumor cells may correlate with degree of tumor growth and invasion. In vitro and in vivo assays allowed better understanding the importance of this receptor. While a downregulation of this receptor impairs proliferation of in vitro cell cultures, at the in vivo level, the number of metastases is decreased too. These results provide potential clues indicating the important role played by the inflammatory cells, and how they can support and promote tumor progression, in particular through the expression of NALP3. The adaptation mechanisms of tumor cells to their microenvironment were also assessed, opening different insights for targeting the tumor itself as well as the inflammatory microenvironment.

2011

Identification of MAGI1 as a tumor-suppressor protein induced by cyclooxygenase-2 inhibitors in colorectal cancer cells

Cyclooxyganase-2 (COX-2), a rate-limiting enzyme in the prostaglandin synthesis pathway, is overexpressed in many cancers and contributes to cancer progression through tumor cell-autonomous and paracrine effects. Regular use of non-steroidal anti-inflammatory drugs or selective COX-2 inhibitors (COXIBs) reduces the risk of cancer development and progression, in particular of the colon. The COXIB celecoxib is approved for adjunct therapy in patients with Familial adenomatous polyposis at high risk for colorectal cancer (CRC) formation. Long-term use of COXIBs, however, is associated with potentially severe cardiovascular complications, which hampers their broader use as preventive anticancer agents. In an effort to better understand the tumor-suppressive mechanisms of COXIBs, we identified MAGUK with Inverted domain structure-1 (MAGI1), a scaffolding protein implicated in the stabilization of adherens junctions, as a gene upregulated by COXIB in CRC cells and acting as tumor suppressor. Overexpression of MAGI1 in CRC cell lines SW480 and HCT116 induced an epithelial-like morphology; stabilized E-cadherin and beta-catenin localization at cell-cell junctions; enhanced actin stress fiber and focal adhesion formation; increased cell adhesion to matrix proteins and suppressed Wnt signaling, anchorage-independent growth, migration and invasion in vitro. Conversely, MAGI1 silencing decreased E-cadherin and beta-catenin localization at cell-cell junctions; disrupted actin stress fiber and focal adhesion formation; and enhanced Wnt signaling, anchorage-independent growth, migration and invasion in vitro. MAGI1 overexpression suppressed SW480 and HCT116 subcutaneous primary tumor growth, attenuated primary tumor growth and spontaneous lung metastasis in an orthotopic model of CRC, and decreased the number and size of metastatic nodules in an experimental model of lung metastasis. Collectively, these results identify MAG1 as a COXIB-induced inhibitor of the Wnt/beta-catenin signaling pathway, with tumor-suppressive and anti-metastatic activity in experimental colon cancer. Oncogene (2012) 31, 48-59; doi: 10.1038/onc.2011.218; published online 13 June 2011

2012

Intestinal stem cells differentiation and its application in cancer therapy

Maxim Norkin

Colorectal cancer (CRC) is one of the deadliest cancers worldwide. Despite extensive research in the field, new therapies targeting CRC are in urgent need. CRC is a highly heterogeneous disease with high levels of intra- and inter-patient heterogeneity. Cancer stem cells are the driving force of tumor development and metastasis. One of the approaches to treat CRC is differentiation therapy thereby induction of cancer stem cell differentiation may lead to tumor regression. Intestinal normal and cancer organoids represent an ideal model for studying the differentiation process ex vivo. Gene expression profiling of drug-treated organoids can be used for quantification of the differentiation process with high precision. RNA-seq based drug-screening platforms in organoids are not yet established. Here we developed a high-throughput high-content targeted RNA-seq-based platform (TORNADO-seq) for monitoring gene expression of large gene signatures in intestinal organoids which allowed us to quantify and evaluate complex cellular phenotypes and cellular alterations upon drug treatment. We applied TORNADO-seq in drug screening in wt and cancer intestinal organoids. We found many differentiation-inducing drugs in wt organoids, including some drugs specifically enriching for certain cell phenotypes, e.g. enteroendocrine cells. Further, we applied identified differentiation-inducing drugs to cancer organoids (APClof;KRASG12D;TP53lof) and discovered that the majority of those differentiation-inducing drugs also targeted cancer organoids. Differentiation induction seems to be a common mode of action of cancer-targeting drugs. Based on gene expression profiles of treated cancer organoids, we were able to propose and confirm the mechanism of action of several drugs indicating that cancer cells may to be sensitive to alterations in the cholesterol biosynthesis pathway. In general, utilization of TORNADO-seq allows predicting connections between cellular phenotypes and signaling pathways in organoids which can be used for hypothesis generation in a variety of biological systems. The second part of the thesis is devoted to the investigation of mechanisms underlying the accumulation of genetic alterations in CRC tumors. It is not known whether cancer driver genes affect the rate of mutation acquisition. Here we performed exome sequencing of single cell-derived organoids obtained from tumors with either APClof, APClof;KRASG12D or APClof;KRASG12D;TP53lof mutation phenotypes that reflect early stages of CRC development. We discovered that presence of KRAS and P53 mutations didn't affect the number of single nucleotide substitutions (SNSs) in tumors, while the latter was increased in APC mutated tumors compared to wt cells and was highly correlated with the tumor age. P53 inactivation was highly correlated with the presence of large-scale (>10 Mb) copy number alterations (CNAs).

EPFL2021