Hemidesmosome integrity protects the colon against colitis and colorectal cancer

OBJECTIVE: Epidemiological and clinical data indicate that patients suffering from IBD with long-standing colitis display a higher risk to develop colorectal high-grade dysplasia. Whereas carcinoma invasion and metastasis rely on basement membrane (BM) disruption, experimental evidence is lacking regarding the potential contribution of epithelial cell/BM anchorage on inflammation onset and subsequent neoplastic transformation of inflammatory lesions. Herein, we analyse the role of the alpha6beta4 integrin receptor found in hemidesmosomes that attach intestinal epithelial cells (IECs) to the laminin-containing BM. DESIGN: We developed new mouse models inducing IEC-specific ablation of alpha6 integrin either during development (alpha6DeltaIEC) or in adults (alpha6DeltaIEC-TAM). RESULTS: Strikingly, all alpha6DeltaIEC mutant mice spontaneously developed long-standing colitis, which degenerated overtime into infiltrating adenocarcinoma. The sequence of events leading to disease onset entails hemidesmosome disruption, BM detachment, IL-18 overproduction by IECs, hyperplasia and enhanced intestinal permeability. Likewise, IEC-specific ablation of alpha6 integrin induced in adult mice (alpha6DeltaIEC-TAM) resulted in fully penetrant colitis and tumour progression. Whereas broad-spectrum antibiotic treatment lowered tissue pathology and IL-1beta secretion from infiltrating myeloid cells, it failed to reduce Th1 and Th17 response. Interestingly, while the initial intestinal inflammation occurred independently of the adaptive immune system, tumourigenesis required B and T lymphocyte activation. CONCLUSIONS: We provide for the first time evidence that loss of IECs/BM interactions triggered by hemidesmosome disruption initiates the development of inflammatory lesions that progress into high-grade dysplasia and carcinoma. Colorectal neoplasia in our mouse models resemble that seen in patients with IBD, making them highly attractive for discovering more efficient therapies.PMC559510

Abnormal Wnt and PI3Kinase Signaling in the Malformed Intestine of lama5 Deficient Mice

Laminins are major constituents of basement membranes and are essential for tissue homeostasis. Laminin-511 is highly expressed in the intestine and its absence causes severe malformation of the intestine and embryonic lethality. To understand the mechanistic role of laminin-511 in tissue homeostasis, we used RNA profiling of embryonic intestinal tissue of lama5 knockout mice and identified a lama5 specific gene expression signature. By combining cell culture experiments with mediated knockdown approaches, we provide a mechanistic link between laminin α5 gene deficiency and the physiological phenotype. We show that laminin α5 plays a crucial role in both epithelial and mesenchymal cell behavior by inhibiting Wnt and activating PI3K signaling. We conclude that conflicting signals are elicited in the absence of lama5, which alter cell adhesion, migration as well as epithelial and muscle differentiation. Conversely, adhesion to laminin-511 may serve as a potent regulator of known interconnected PI3K/Akt and Wnt signaling pathways. Thus deregulated adhesion to laminin-511 may be instrumental in diseases such as human pathologies of the gut where laminin-511 is abnormally expressed as it is shown here

Stimulation of the intestinal Cdx2 homeobox gene by butyrate in colon cancer cells

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Inflammation response triggers expression of LMα1/α5 and nuclear p53 accumulation in human and murine colitis.

<p>(<b>A</b>) Expression of the LMα5 and LMα1 chains in normal mucosa (control), in mild inflamed glands and around UACL from colon of patients with IBD. Immunostaining for LMα5 in IBD samples is extended along the colonic glands with a strong staining at the deeper crypt region and is highly expressed in UACL (arrows). LMα1 is detected selectively at the BM at the crypt bottom in IBD samples and in UACL while it is absent from uninflamed regions; unspecific cytoplasmic immunoreactivity is seen in goblet cells (g). Inset: higher magnification of the deeper crypt region. (<b>B</b>) Expression of LMα5 and LMα1 in cryosections of colon from control and DSS-treated mice. Note, as in human IBD, high expression of LMα5 and LMα1 was observed in mouse colitis at the bottom of the colonic glands. (<b>C</b>) Nuclei of epithelial cells from inflamed colonic segments and in UACL of IBD patients were positive for p53 while nuclei of the adjacent normal crypts showed rare p53-positive cells scattered within the glands. (<b>D</b>) Expression of p53 (green) and LMα1 (red) in colon samples from DSS-treated mice. At day 3 after treatment, p53 immunoreactivity was present in numerous nuclei within epithelial cells while LMα1 co-staining was weak; inset: enlarged area with nuclear p53 expression. At day 5, intense LMα1 staining was observed surrounding weaker p53-positive glands (inset). (<b>E</b>) The diagram summarizes the distribution of the main LM chains found in normal colonic mucosa (control), in mild inflamed glands and around UACL from colon of patients with IBD. Note that only the staining at the epithelial BM is schematically represented for clarity. e: epithelial cells; lp: <i>lamina propria</i>; mm: <i>muscularis mucosae</i>; arrows: ectopic staining at crypt bottom and staining around UACL. Scale bars: 50 µm (human), 25 µm (mouse).</p

Wild-type p53 induces LMα1 expression in epithelial cells via binding to the <i>LAMA1</i> promoter.

<p>(<b>A</b>) HCT116 cells were seeded on LM-111 or LM-511-enriched matrix. (Left) Endogenous p53 mRNA was quantified by RT-qPCR (normalized to <i>GAPDH</i>) and expressed as ratio relative to the control (plastic dishes). The values are given as a mean +/− SEM of 9 independent experiments which were pooled (n = 3 at 2 days; n = 3 at 3 days; n = 3 at 4 days). (Right) Representative immunoblot of p53 and actin from HCT116 cells in the different conditions. Data show that LM substrata do not activate endogenous expression of p53. (<b>B</b>) Relative mRNA expression of LM chains in HCT116 cells upon transfection with a <i>TP53</i> vector assessed at different time points. Transcript levels were determined by RT-qPCR and normalized to <i>GAPDH</i> and are presented relative to the control vector (n = 3 to 4 experiments, except for α1 at 6 h n = 6 and at 48 h n = 8). Data show that wild-type (wt) p53 induces selectively and progressively LMα1 mRNA levels. (<b>C</b>) Semiquantitative analysis of intracellular LMα1 after immunofluorescence staining of HCT116 cells (48 h of irinotecan treatment). Note a 2.3-fold intracellular deposition of LMα1 in irinotecan-treated cells as compared to untreated cells (n = 15). (<b>D</b>) Expression of LMα1 mRNA from two independent stable sh-<i>TP53</i> HCT116 cell lines and in a sh-RNA control cell line, upon treatment with irinotecan. After 48 h, relative mRNA expression of LMα1 was assessed by RT-qPCR and normalized to <i>GAPDH;</i> values are given as ratios relative to those found in the corresponding untreated cells (n = 3). In p53-deprived cells, irinotecan was unable to stimulate LMα1 expression. (<b>E</b>) Relative <i>LAMA1</i> mRNA expression in HCT116 cells following transfection with wt or mutants p53 (ratios calculated as stated above; n = 6). LMα1 mRNA were only activated by wt p53. (<b>F</b>) Diagram showing the location of putative p53 binding motifs (sites 1 to 6) in the <i>LAMA1</i> promoter 7 kb upstream of the transcription site and in the 5 kb region of intron 1 (i). (<b>G</b>) Chromatin immunoprecipitation experiments. Chromatin was prepared from HCT116 cells transfected with either the control (pCMV) or <i>TP53</i> vector (p53). Cross-linked p53-DNA complexes were immunoprecipitated by either IgG (negative control) or anti-p53 antibodies (DO1 or 1801) followed by PCR amplification using primers that flank the putative p53 binding sites. Input represents chromatin before immunoprecipitation. Note that p53 binds to 7 candidate p53 binding sites. Bars represent mean +/−SEM; *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

Important role of p53 in regulating LM expression in IBD and colitis-associated cancer.

<p>We propose the following scenario where inflammation triggers nuclear accumulation of p53 that transactivates expression of LMα1 and its deposition to the BM concomitantly to that of LMα5. Overexpression of LMα5 in response to inflammation is a p53 independent mechanism. In a chronically inflamed microenvironment highly expressed LMα1 and LMα5 may provide physical barrier function resulting in attenuated inflammation as demonstrated in transgenic mice. Yet, high LMα1 and LMα5 in a carcinogenic setting subsequent to chronic colitis may contribute to a pro-tumorigenic microenvironment.</p

Laminins protect from inflammation.

<p>(<b>A–B</b>) Histological views of colon tissue (stained with PAS) and expression of LMα5 and LMα1 on cryosections of colon from parental, Tg-<i>lama5</i> or Tg-<i>lama1</i> mice untreated or treated with DSS. This showed that both chains were ectopically expressed in the glandular crypt region in the transgenic animals (arrows) and that LMα1 expression was further extended by DSS treatment. (<b>C</b>) Inflammatory scores (mean +/− SEM; n = 5) assessed on the Swiss-roll of the colon and rectum of transgenic <i>lama5</i> and <i>lama1</i> animals (black columns) as compared to controls (grey columns), all treated with DSS. (<b>D</b>) Levels of pro-inflammatory cytokines (mean +/− SEM; n = 6) measured by ELISA in protein extracts from distal colon of DSS-treated parental (grey columns) and DSS-Tg-<i>lama1</i> or DSS-Tg-<i>lama5</i> (black columns) mice. Data were normalized to the mean of parental values as levels of cytokines turned out to be mouse strain-dependent. Statistical differences were compared to parental mice. e: epithelial cells; lp: <i>lamina propria</i>; mm: <i>muscularis mucosae</i>. Nuclei are visualized with DAPI. *<i>p</i><0.05, **<i>p</i><0.01. Scale bars: 50 µm.</p

Colitis-associated tumor development is increased in transgenic mice overexpressing LMα1.

<p>(<b>A</b>) Schematic overview of the AOM/DSS and of the cyclic DSS protocols. (<b>B</b>) Control and transgenic mice treated with AOM/DSS or with cyclic DSS develop different types of lesions among them dysplasia and <i>in situ</i> carcinomas that were quantified. Tg-<i>lama1</i> animals develop more tumors than controls when submitted to the treatments (n = 7, *p<0.05 for AOM/DSS; n = 7, *p<0.05 for cyclic DSS). (<b>C</b>) Dysplasia and an <i>in situ</i> carcinoma are shown by hematoxylin-eosin staining (HE). Glands in these lesions are strongly positive for LMα1 (red), LMα5 (green, lower panels) and present nuclear p53 (green, middle panels). Nuclei were visualized with DAPI. e: epithelial cells; m: muscle; c: cancer cells; s: stroma; arrows: BM area. Scale bars: 50 µm.</p