Karline Guilloteau

Skin Inflammation Induced by the Synergistic Action of IL-17A, IL-22, Oncostatin M, IL-1α, and TNF-α Recapitulates Some Features of Psoriasis

Keratinocytes play a crucial role in the regulation of skin inflammation, responding to environmental and immune cells stimuli. They produce soluble factors that can act in an autocrine or paracrine manner on immune cells or directly on aggressors. A screening of the activities of 36 cytokines on keratinocyte gene expression identified IL-17A, IL-22, oncostatin M, TNF-α, and IL-1α as potent cytokines in inducing cutaneous inflammation. These five proinflammatory cytokines synergistically increased production of CXCL8 and β-defensin 2 (BD2). In addition, ex vivo studies on human skin explants demonstrated upregulation of BD2, S100A7, and CXCL8 expression in response to the same combination of cytokines. In vivo intradermal injection of these five cytokines in mouse increased CXCL1, CXCL2, CXCL3, S100A9, and BD3 expression, associated with neutrophil infiltration. We confirmed and extended this synergistic effect using quantitative real-time PCR analysis and observed increased expression of nine chemokines and 12 antimicrobial peptides. Production of CXCL, CXCL5, and CXCL8 by keratinocytes stimulated in the presence of this cytokine combination was associated with increased neutrophil chemotactic activity. Similarly, high production of BD2, BD3, and S100A7 was associated with an increased antimicrobial activity. Finally, the transcriptional profile observed in this in vitro model of inflammatory keratinocytes correlated with the one of lesional psoriatic skin. Our results demonstrate the important potentiating activities of IL-17A, IL-22, oncostatin M, TNF-α, and IL-1α on keratinocytes. This is particularly interesting in the context of psoriasis where these cytokines are overexpressed and could synergize to play an important role in upregulation of chemokines and antimicrobial peptides production.

Inhibition of keratinocyte differentiation by the synergistic effect of IL-17A, IL-22, IL-1α, TNFα and oncostatin M.

Keratinocyte differentiation program leading to an organized epidermis plays a key role in maintaining the first line of defense of the skin. Epidermal integrity is regulated by a tight communication between keratinocytes and leucocytes, particularly under cytokine control. Imbalance of the cytokine network leads to inflammatory diseases such as psoriasis. Our attempt to model skin inflammation showed that the combination of IL-17A, IL-22, IL-1α, OSM and TNFα (Mix M5) synergistically increases chemokine and antimicrobial-peptide expression, recapitulating some features of psoriasis. Other characteristics of psoriasis are acanthosis and down-regulation of keratinocyte differentiation markers. Our aim was to characterize the specific roles of these cytokines on keratinocyte differentiation, and to compare with psoriatic lesion features. All cytokines decrease keratinocyte differentiation markers, but IL-22 and OSM were the most powerful, and the M5 strongly synergized the effects. In addition, IL-22 and OSM induced epidermal hyperplasia in vitro and M5 induced epidermal thickening and decreased differentiation marker expression in a mouse model, as observed in human psoriatic skin lesions. This study highlights the precise role of cytokines in the skin inflammatory response. IL-22 and OSM more specifically drive epidermal hyperplasia and differentiation loss while IL-1α, IL-17A and TNFα were more involved in the activation of innate immunity.

A mesenchymal glioma stem cell profile is related to clinical outcome

Recent studies have demonstrated a relationship between the expression of stem cell-associated genes and relapses in glioblastoma (GBM), suggesting a key role for tumor stem cells in this process. Although there is increasing interest in this field, glioma stem cells (GSCs) are still poorly characterized, their ‘stemness’ state and factors maintaining these properties remain largely unknown. We performed an expression profiling analysis of pluripotency in gliomaspheres derived from 11 patients. Comparative analysis between GSCs and H1 and H9 human embryonic stem cells as well as H9-derived neural stem cells indicates major variations in gene expression of pluripotency factors Nanog and OCT4, but a stable pattern for SOX2 suggesting its important function in maintaining pluripotency in GSCs. Our results also showed that all GSC lines have the capacity to commit to neural differentiation and express mesenchymal or endothelial differentiation markers. In addition, hierarchical clustering analysis revealed two groups of GSCs reflecting their heterogeneity and identified COL1A1 and IFITM1 as the most discriminating genes. Similar patterns have been observed in tumors from which gliomaspheres have been established. To determine whether this heterogeneity could be clinically relevant, the expression of both genes was further analyzed in an independent cohort of 30 patients with GBM and revealed strong correlation with overall survival. In vitro silencing of COL1A1 and IFTM1 confirmed the effect of these mesenchymal-associated genes on cell invasion and gliomasphere initiation. Our results indicate that COL1A1 and IFITM1 genes could be considered for use in stratifying patients with GBM into subgroups for risk of recurrence at diagnosis, as well as for prognostic and therapeutic evolution.

EGFR, KRAS, BRAF, and HER‐2 molecular status in brain metastases from 77 NSCLC patients

The aim of this study was to determine the frequency of EGFR, KRAS, BRAF, and HER‐2 mutations in brain metastases from non‐small cell lung carcinomas (BM‐NSCLC). A total of 77 samples of BM‐NSCLC were included and 19 samples of BM from breast, kidney, and colorectal tumors were also studied as controls. These samples were collected from patients followed between 2008 and 2011 at Poitiers and Nice University Hospitals in France. The frequencies of EGFR, KRAS, BRAF, and HER‐2 mutations in BM‐NSCLC were 2.6, 38.5, 0, and 0% respectively. The incidence of KRAS mutation was significantly higher in female and younger patients (P < 0.05). No mutations of the four genes were found in BM from breast or kidney. However, among six BM from colorectal tumors, we identified KRAS mutations in three cases and BRAF mutations in two other cases. This study is the largest analysis on genetic alterations in BM‐NSCLC performed to date. Our results suggest a low frequency of EGFR mutations in BM‐NSCLC whereas KRAS mutations are as frequent in BM‐NSCLC as in primitive NSCLC. These results raise the question of the variability of the brain metastatic potential of NSCLC cells in relation to the mutation pattern.

Selective Release of a Cyclopamine Glucuronide Prodrug toward Stem-like Cancer Cell Inhibition in Glioblastoma

Recent data suggest that inhibition of the Hedgehog pathway could be a therapeutic target for glioblastoma. Alkaloid cyclopamine inhibits Hedgehog signaling, depleting stem-like cancer cells derived from glioblastoma. However, this compound is toxic for somatic stem cells, preventing its use for clinical applications. In this study, we tested a derivatization product of cyclopamine in the form of cyclopamine glucuronide prodrug (CGP-2). This compound was used in vitro and in vivo toward glioblastoma-initiating cells (GIC). Results obtained in vitro indicate that CGP-2 is active only in the presence of β-glucuronidase, an enzyme detected in high levels in necrotic areas of glioblastomas. CGP-2 decreased proliferation and inhibited the self-renewal of all GIC lines tested. Hedgehog pathway blockade by 10 μmol/L of CGP-2 induced a 99% inhibition of clonogenicity on GICs, similar to cyclopamine treatment. Combination of CGP-2 with radiation decreased clonogenic survival in all GIC lines compared with CGP-2 alone. In a subcutaneous glioblastoma xenograft model, a two-week CGP-2 treatment prevented tumor growth with 75% inhibition at 8 weeks, and this inhibition was still significant after 14 weeks. Unlike cyclopamine, CGP-2 had no detectable toxic effects in intestinal crypts. Our study suggests that inhibition of the Hedgehog pathway with CGP-2 is more effective than conventional temozolomide adjuvant, with much lower concentrations, and seems to be an effective therapeutic strategy for targeting GICs. Mol Cancer Ther; 13(9); 2159–69. ©2014 AACR.

IL22/IL-22R pathway induces cell survival in human glioblastoma cells.

Interleukin-22 (IL-22) is a member of the IL-10 cytokine family that binds to a heterodimeric receptor consisting of IL-22 receptor 1 (IL-22R1) and IL-10R2. IL-22R expression was initially characterized on epithelial cells, and plays an essential role in a number of inflammatory diseases. Recently, a functional receptor was detected on cancer cells such as hepatocarcinoma and lung carcinoma, but its presence was not reported in glioblastoma (GBM). Two GBM cell lines and 10 primary cell lines established from patients undergoing surgery for malignant GBM were used to investigate the expression of IL-22 and IL-22R by using quantitative RT-PCR, western blotting and confocal microscopy studies. The role of IL-22 in proliferation and survival of GBM cell lines was investigated in vitro by BrdU and ELISA cell death assays. We report herein that the two subunits of the IL-22R complex are expressed on human GBM cells. Their activation, depending on exogenous IL-22, induced antiapoptotic effect and cell proliferation. IL-22 treatment of GBM cells resulted in increased levels of phosphorylated Akt, STAT3 signaling protein and its downstream antiapoptotic protein Bcl-xL and decreased level of phosphorylated ERK1/2. In addition, IL-22R subunits were expressed in all the 10 tested primary cell lines established from GBM tumors. Our results showed that IL-22R is expressed on GBM established and primary cell lines. Depending on STAT3, ERK1/2 and PI3K/Akt pathways, IL-22 induced GBM cell survival. These data are consistent with a potential role of IL-22R in tumorigenesis of GBM. Since endogenous IL-22 was not detected in all studied GBM cells, we hypothesize that IL-22R could be activated by immune microenvironmental IL-22 producing cells.

Development of pyrosequencing methods for the rapid detection of RAS mutations in clinical samples

In advanced colorectal carcinoma (CRC) patients, extended RAS mutations testing (KRAS exons 2 to 4 and NRAS exons 2 to 4) is a prerequisite for patient stratification to anti-EGFr therapy. Accurately distinguishing mutant patients from potential responders has a clinically critical impact, and thus effective and low cost methods are needed for identification of the mutation status. We have developed quantitative pyrosequencing assays for sensitive and rapid detection of mutant RAS alleles in formalin-fixed, paraffin-embedded tissues. Exons 2 to 4 of KRAS and NRAS genes were PCR amplified and analyzed by pyrosequencing. For validation, PCR products were sequenced by conventional Sanger sequencing. Analytical sensitivity of these assays was determined by calculating the limit of detection. The results showed that low levels of mutant RAS alleles (2-13%) can be detected with pyrosequencing assays.

Crizotinib targets in glioblastoma stem cells

Glioblastoma stem cells (GSCs) are believed to be involved in the mechanisms of tumor resistance, therapeutic failures, and recurrences after conventional glioblastoma therapy. Therefore, elimination of GSCs might be a prerequisite for the development of successful therapeutic strategies. ALK, ROS1, and MET are targeted by Crizotinib, a tyrosine kinase inhibitor which has been approved for treatment of ALK‐rearranged non–small‐cell lung cancer. In this study we investigated ALK, ROS1, and MET status in nine glioblastoma stem cell lines and tumors from which they arise. Fluorescent in situ hybridization (FISH), Sangers direct sequencing, and immunohistochemistry were used to screen genomic rearrangements (or amplifications), genomic mutations, and protein expression, respectively. The immunohistochemical and FISH studies revealed no significant dysregulation of ROS1 in GSCs and associated tumors. Neither amplification nor polysomy of ALK was observed in GSC, but weak overexpression was detected by IHC in three of nine GSCs. Similarly, no MET amplification was found by FISH but three GSCs presented significant immunohistochemical staining. No ALK or MET mutation was found by Sangers direct sequencing. In this study, we show no molecular rearrangement of ALK, ROS1, and MET that would lead us not to propose, as a valid strategy, the use of crizotinib to eradicate GSCs. However, MET was overexpressed in all GSCs with mesenchymal subtype and three GSCs presented an overexpression of ALK. Therefore, our study corroborates the idea that MET and ALK may assume a role in the tumorigenicity of GSC.

Absence of IDH mutation in colorectal cancers with microsatellite instability

BACKGROUND Colorectal cancers (CRCs) with deficient DNA mismatch repair system (dMMR) represents approximately 12% of all CRCs. Sporadic dMMR CRCs are due to hypermethylation MLH1 gene promoter and associated with the CpG island methylator phenotype (CIMP). Isocitrate dehydrogenase (IDH) mutant tumors are associated with DNA hypermethylation in various cancers. AIMS Look if dMMR CRC are associated with IDH mutations. METHODS All consecutive dMMR CRCs between 2005 and 2014 were included in this study. Tumoral DNA used for dMMR analysis was also used to determine IDH1 and IDH2 mutations using pyrosequencing method. RESULTS A total of 152 dMMR CRCs patients were analyzed. No IDH mutation was observed in dMMR CRC, nor in IDH1 or IDH2. Especially, no mutation was found in sporadic dMMR CRC which is known to be associated with CIMP. CONCLUSIONS In contrast with others types of hypermethylated tumors, dMMR CRCs are not associated with IDH mutations.

Additional file 4: Figure S1. of A radiosensitizing effect of RAD51 inhibition in glioblastoma stem-like cells

Analysis of ten glioblastoma-derived GSCs. LOH, Loss Of Heterozygosity; IDH, Isocitrate Dehydrogenase; WT, Wildtype; Mut, Mutant; PTEN, Phosphatase and TENsin homolog; EGFR, Epidermal Growth Factor Receptor; MGMT, O-6-methylguanine-DNA methyltransferase; PFS, Progression-free survival, IQ, insufficient quantity. Group 1 and group 2 are described in the results section. (DOCX 16 kb)