Functional interaction between the homeoprotein CDX1 and the transcriptional machinery containing the TATA-binding protein

We have previously reported that the CDX1 homeoprotein interacts with the TATA-box binding protein (TBP) on the promoter of the glucose-6-phosphatase (G6Pase) gene. We show here that CDX1 interacts with TBP via the homeodomain and that the transcriptional activity additionally requires the N-terminal domain upstream of the homeodomain. CDX1 interacting with TBP is connected to members of the TFIID and Mediator complexes, two major elements of the general transcriptional machinery. Transcription luciferase assays performed using an altered-specificity mutant of TBP provide evidence for the functionality of the interaction between CDX1 and TBP. Unlike CDX1, CDX2 does not interact with TBP nor does it transactivate the G6Pase promoter. Swapping experiments between the domains of CDX1 and CDX2 indicate that, despite opposite functional effects of the homeoproteins on the G6Pase promoter, the N-terminal domains and homeodomains of both CDX1 and CDX2 have the intrinsic ability to activate transcription and to interact with TBP. However, the carboxy domains define the specificity of CDX1 and CDX2. Thus, intra-molecular interactions control the activity and partner recruitment of CDX1 and CDX2, leading to different molecular functions

Multiple-contrast X-ray micro-CT visualization of colon malformations and tumours in situ in living mice

International audienc

Differences between CAFs and their paired NCF from adjacent colonic mucosa reveal functional heterogeneity of CAFs, providing prognostic information

Little is known about the difference in gene expression between carcinoma-associated fibroblasts (CAFs) and paired normal colonic fibroblasts (NCFs) in colorectal cancer. Paired CAFs and NCFs were isolated from eight primary human colorectal carcinoma specimens. In culture conditions, soluble factors secreted by CAFs in the conditioned media increased clonogenicity and migration of epithelial cancer cells lines to a greater extent than did NCF. In vivo, CAFs were more competent as tumour growth enhancers than paired NCFs when co-inoculated with colorectal cell lines. Gene expression analysis of microarrays of CAF and paired NCF populations enabled us to identify 108 deregulated genes (38 upregulated and 70 downregulated genes). Most of those genes are fibroblast-specific. This has been validated in silico in dataset GSE39396 and by qPCR in selected genes. GSEA analysis revealed a differential transcriptomic profile of CAFs, mainly involving the Wnt signallingsignalling pathway, focal adhesion and cell cycle. Both deregulated genes and biological processes involved depicted a considerable degree of overlap with deregulated genes reported in breast, lung, oesophagus and prostate CAFs. These observations suggest that similar transcriptomic programs may be active in the transition from normal fibroblast in adjacent tissues to CAFs, independently of their anatomic demarcation. Additionally NCF already depicted an activated pattern associated with inflammation. The deregulated genes signature score seemed to correlate with CAF tumour promoter abilities in vitro, suggesting a high degree of heterogeneity between CAFs, and it has also prognostic value in two independent datasets. Further characterization of the roles these biomarkers play in cancer will reveal how CAFs provide cancer cells with a suitable microenvironment and may help in the development of new therapeutic targets for cancer treatment

Immunostaining Protocol: P-Smad2 (Xenograft and Mice)

Metastasis depends on a gene program expressed by the tumor microenvironment upon TGF-beta stimulation. CRC (Colorectal cancer) cell lines did not induce robust stromal TGF- beta responses when injected into nude mice as shown by lack of p- SMAD2 accumulation in tumor-associated stromal cells. To enforce high TGF-beta signaling in xenografts, we engineered CRC cell lines to secrete active TGF-beta. Subcutaneous tumors obtained from HT29-M6TGF-β, KM12L4aTGF-β cells and SW48TGF-β cells contained abundant p-SMAD2+ stromal cells

Immunostaining Protocol: P-Stat3 (Xenograft and Mice)

We sought to understand the mechanisms behind the potent effect of stromal TGF-beta program on the capacity of colorectal cancer (CRC) cells to initiate metastasis. We discovered that mice subcutaneous tumors and metastases generated in the context of a TGF-beta activated microenvironment displayed prominent accumulation of p-STAT3 in CRC cells compared with those derived from control cells. STAT3 signaling depended on GP130 as shown by strong reduction of epithelial p STAT3 levels upon GP130 shRNA-mediated knockdown in CRC cells

Long-term platinum-based drug accumulation in cancer-associated fibroblasts promotes colorectal cancer progression and resistance to therapy

A substantial proportion of cancer patients do not benefit from platinum-based chemotherapy (CT) due to the emergence of drug resistance. Here, we apply elemental imaging to the mapping of CT biodistribution after therapy in residual colorectal cancer and achieve a comprehensive analysis of the genetic program induced by oxaliplatin-based CT in the tumor microenvironment. We show that oxaliplatin is largely retained by cancer-associated fibroblasts (CAFs) long time after the treatment ceased. We determine that CT accumulation in CAFs intensifies TGF-beta activity, leading to the production of multiple factors enhancing cancer aggressiveness. We establish periostin as a stromal marker of chemotherapeutic activity intrinsically upregulated in consensus molecular subtype 4 (CMS4) tumors and highly expressed before and/or after treatment in patients unresponsive to therapy. Collectively, our study underscores the ability of CT-retaining CAFs to support cancer progression and resistance to treatment.This work has been supported by grants from Fundación científica AECC -Asociación Española contra el Cáncer- (GCAEC20030CERV) to A.Ce., from Instituto de Salud Carlos III (ISCIII) co-funded by the European Union (CP16/00151, PI17/00211, PI20/00011; Spanish Ministry of Economy and Competitiveness) to A.Ca. and PI20/00625 to P.N., from la Caixa Foundation (LCF/PR/HR19/52160018) and MICINN (PID2020- 119917RB-I00) to E.B., from Spanish Ministerio de Economia y Competitividad (MINECO) and FEDER funds (PID2019-104948RB-I00) to R.R.G. This work was supported by Grant PT20/00023, funded by Instituto de Salud Carlos III (ISCIII) and co-funded by the European Union, and the Xarxa de Bancs de tumors sponsored by Pla Director d’Oncologia de Catalunya (XBTC). A.Ca. is the recipient of funding from the Instituto de Salud Carlos III co-funded by the European Union (MS16/00151; CPII21/00012). J.L. is the recipient of a Junior Clinician fellowship from Fundación científica AECC (CLJUN19004LINA)

Les facteurs de transcription homéotiques CDX1 et CDX2 (implications dans les pathologies de l'intestin; partenaires moléculaires)

Les protéines homéotiques CDX1 et CDX2 sont des facteurs de transcription spécifiques de l épithélium intestinal adulte. Ils jouent un rôle dans le contrôle de l homéostasie intestinale et leur expression est altérées dans la cancérogenèse colorectale. CDX1 et CDX2 exercent des fonctions distinctes et complémentaires dans la régulation de la prolifération et de la différenciation.J ai étudié d une part les conséquences de l altération de Cdx1 dans des modèles murins de cancérogenèse colorectale et d autre part les conséquences de l altération de Cdx2 dans l inflammation. J ai montré que:- l altération de l expression de Cdx1 n a d effet ni sur l initiation, ni sur la progression tumorale;- Cdx2 joue un rôle clé dans l inflammation aiguë et dans l inflammation chronique. Nos études suggèrent que la capacité de Cdx2 à réguler la migration cellulaire et l activité transcriptionnelle de SMAD3 pourraient expliquer le rôle de ce facteur dans l inflammation.J ai émis l hypothèse que les fonctions différentes de CDX1 et de CDX2 étaient liées à une interaction fonctionnelle avec des co-facteurs distincts.J ai montré que contrairement à CDX2, CDX1 interagit avec la TBP sur le promoteur de la G6Pase. Ce résultat associé à des études de mutagenèse nous a permis de proposer un modèle fonctionnel basé sur des interactions intra-moléculaires expliquant les effets différents de CDX1 et CDX2.En conclusion, la réalisation de ce travail a permis de caractériser les fonctions propres des gènes Cdx1 et Cdx2 dans deux modèles animaux de pathologies intestinales humaines, ainsi que les modes d action respectifs des deux facteurs de transcription codés par ces gènes.CDX1 and CDX2 homeotic proteins are transcription factors specifically expressed in the gut in adults. They are involved in the control of homeostasis and their expression is altered during colorectal carcinogenesis. CDX1 and CDX2 play distinct and complementary functions in the control of cell proliferation and differentiation.I have investigated the consequences of Cdx1 expression alterations in models of colorectal cancer and Cdx2 alteration in a model of inflamed bowel disease. I demonstrated that- Cdx1 alteration has no consequences on initiation and progression of colorectal cancer ;- Cdx2 plays key role during acute and chronic inflammation. Our studies suggest that the function of Cdx2 on migration and also on SMAD3 transcriptional activity regulation could explain the role of Cdx2 in inflammation. One mechanism to account for the different effects exerted by CDX1 and CDX2 could be that they may functionally interact with different partners.I demonstrated that CDX1 but not CDX2 interacts with the TBP, on the promoter of the Glucose-6-Phosphatase gene. This result combined to mutagenesis studies led us to propose a functional model for the differential effects of CDX1 and CDX2 based on intra-molecular interactions.Taken together, our results led us to characterize Cdx1 and Cdx2 functions in two murin models of human intestinal pathologies but also specific mechanisms of action related to each transcription factors encoded by these genes.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Determinants of metastatic competency in colorectal cancer

Colorectal cancer (CRC) is one of the most common cancer types and represents a major therapeutic challenge. Although initial events in colorectal carcinogenesis are relatively well characterized and treatment for early-stage disease has significantly improved over the last decades, the mechanisms underlying metastasis - the main cause of death - remain poorly understood. Correspondingly, no effective therapy is currently available for advanced or metastatic disease. There is increasing evidence that colorectal cancer is hierarchically organized and sustained by cancer stem cells, in concert with various stromal cell types. Here, we review the interplay between cancer stem cells and their microenvironment in promoting metastasis and discuss recent insights relating to both patient prognosis and novel targeted treatment strategies. A better understanding of these topics may aid the prevention or reduction of metastatic burdenWork by the authors has been supported by grant SAF2014-53784_R (EB) and Juan de la Cierva fellowships (DVFT and AC) from the Spanish Ministry of Economy and Competitiveness (MINECO), by fellowships from Fundacion Olga Torres (EL) and Asociacion Española contra el Cancer (EL) and by the Dr. Josef Steiner Foundation (EB). Work in the laboratory of EB is supported by Fundacion Botín and Banco Santander, through Santander Universities. IRB Barcelona is the recipient of a Severo Ochoa Award of Excellence from the MINECO. We would like to thank members of the laboratory of EB for fruitful discussion

LOBSTER: an environment to design bioimage analysis workflows for large and complex fluorescence microscopy data

© The Author(s) 2019.Open source software such as ImageJ and CellProfiler greatly simplified the quantitative analysis of microscopy images but their applicability is limited by the size, dimensionality and complexity of the images under study. In contrast, software optimized for the needs of specific research projects can overcome these limitations, but they may be harder to find, set up and customize to different needs. Overall, the analysis of large, complex, microscopy images is hence still a critical bottleneck for many Life Scientists. We introduce LOBSTER (Little Objects Segmentation and Tracking Environment), an environment designed to help scientists design and customize image analysis workflows to accurately characterize biological objects from a broad range of fluorescence microscopy images, including large images exceeding workstation main memory. LOBSTER comes with a starting set of over 75 sample image analysis workflows and associated images stemming from state-of-the-art image-based research projects