The laminin response in inflammatory bowel disease: Protection or malignancy?

Laminins (LM), basement membrane molecules and mediators of epithelial-stromal communication, are crucial in tissue homeostasis. Inflammatory Bowel Diseases (IBD) are multifactorial pathologies where the microenvironment and in particular LM play an important yet poorly understood role in tissue maintenance, and in cancer progression which represents an inherent risk of IBD. Here we showed first that in human IBD colonic samples and in murine colitis the LMα1 and LMα5 chains are specifically and ectopically overexpressed with a concomitant nuclear p53 accumulation. Linked to this observation, we provided a mechanism showing that p53 induces LMα1 expression at the promoter level by ChIP analysis and this was confirmed by knockdown in cell transfection experiments. To mimic the human disease, we induced colitis and colitis-associated cancer by chemical treatment (DSS) combined or not with a carcinogen (AOM) in transgenic mice overexpressing LMα1 or LMα5 specifically in the intestine. We demonstrated that high LMα1 or LMα5 expression decreased susceptibility towards experimentally DSS-induced colon inflammation as assessed by histological scoring and decrease of pro-inflammatory cytokines. Yet in a pro-oncogenic context, we showed that LM would favor tumorigenesis as revealed by enhanced tumor lesion formation in both LM transgenic mice. Altogether, our results showed that nuclear p53 and associated overexpression of LMα1 and LMα5 protect tissue from inflammation. But in a mutation setting, the same LM molecules favor progression of IBD into colitis-associated cancer. Our transgenic mice represent attractive new models to acquire knowledge about the paradoxical effect of LM that mediate either tissue reparation or cancer according to the microenvironment. In the early phases of IBD, reinforcing basement membrane stability/organization could be a promising therapeutic approach

Ploidy variations between secondary resistant and parental soft-tissue sarcoma cell lines.

<p><i>only sensitive clone</i></p><p><b>only resistant clone</b></p><p>-: absence of allelic imbalance</p><p>Ploidy variations between secondary resistant and parental soft-tissue sarcoma cell lines.</p

Heterogeneous Mechanisms of Secondary Resistance and Clonal Selection in Sarcoma during Treatment with Nutlin

<div><p>Nutlin inhibits TP53-MDM2 interaction and is under investigation in soft-tissue sarcomas (STS) and other malignancies. Molecular mechanisms of secondary resistance to nutlin in STS are unknown. We performed whole-transcriptome sequencing (RNA-seq) on three pretreatment and secondary resistant STS cell lines selected based on their high primary sensitivity to nutlin. Our data identified a subset of cancer gene mutations and ploidy variations that were positively selected following treatment, including TP53 mutations in 2 out of 3 resistant cell lines. Further, secondary resistance to nutlin was associated with deregulation of apoptosis-related genes and marked productive autophagy, the inhibition of which resulted in significant restoration of nutlin-induced cell death. Collectively, our findings argue that secondary resistance to nutlin in STS involved heterogeneous mechanisms resulting from clonal evolution and several biological pathways. Alternative dosing regimens and combination with other targeted agents are needed to achieve successful development of nutlin in the clinical setting.</p></div

Heterogeneous Mechanisms of Secondary Resistance and Clonal Selection in Sarcoma during Treatment with Nutlin - Fig 3

<p>(A) Antiproliferative activity and impact on loss of potential mitochondrial membrane with TMRM fluorescent assay (cell viability) (B) of RG7388 in parental and secondary resistant IB111, IB115 and IB128 STS cell lines. (C) Apoptosis induction analysis using FITC annexin-V and propidium iodide assay. Sensitive (IB111 and IB115) and resistant cells (IB111P4 and IB115P4) were treated 72h by RG-7388 at 1μM.</p

Ploidy variations between secondary resistant and parental soft-tissue sarcoma cell lines.

<p><i>only sensitive clone</i></p><p><b>only resistant clone</b></p><p>-: absence of allelic imbalance</p><p>Ploidy variations between secondary resistant and parental soft-tissue sarcoma cell lines.</p

Effect of RG7388 on cell cycle progression and cell viability in human STS cell lines.

<p>(A) Cell cycle profile before and after treatment with 1μM of RG7388 analyzed by PI incorporation and flow cytometry in the IB111, IB115 and IB128 cell lines. (B) Cell-cycle distribution was calculated from the flow cytogram. (C) Effect of RG7388 on loss of potential mitochondrial membrane with TMRM fluorescent assay in the IB111, IB115 and IB128 cell lines.</p

Secondary resistance to RG7388 is associated with autophagy induction.

<p>(A) parental and secondary resistant cells were incubated with 20μM of chloroquine for 6h, or RG7388 alone for 72h or RG7388 (over 72h) and chloroquine for 6h before protein extraction, and immunoblot. (B) Densitometry of the immunoblot, the graph represents the percentage of LC3-II /GAPDH relative to the untreated control (C) IB111 and IB111P4 were treated with 1μM and 10 μM of RG-7388 respectively with or without chloroquine and fixed for immuno-staining with LC3 antibody (D) Quantification of autophagy induction (number of GFP-LC3 punctuae) in IB111 and IB111P4 treated with RG7388, chloroquine and RG7388+chloroquine (E) IC50 fold-change related to antiproliferative activity of RG7388+chloroquine versus RG7388 alone in secondary resistant IB111, and sensitive cells IB111.</p