Calcium Independent Effect of Orai1 and STIM1 in Non-Hodgkin B Cell Lymphoma Dissemination

International audienceCa 2+ release-activated Ca 2+ channels, composed of Orai1 and STIM1 (stromal interaction molecule 1) proteins, are the main Ca 2+ entry mechanism in lymphocytes. Their role in cell migration and metastasis is demonstrated in solid cancers but it remains elusive in malignant hemopathies. Diffuse large B cell lymphoma (DLBCL) is characterized by the dissemination of neoplastic B cells throughout the organism which is under the control of chemokines such as Stromal Derived Factor 1 (SDF-1) and its receptor CXCR4. CXCR4 activation triggers a complex intracellular signaling including an increase in intracellular Ca 2+ concentration whose role is still unclear. Using pharmacological and genetic approaches, we revealed that STIM1 and Orai1 were responsible for Ca 2+ influx induced by SDF-1. Furthermore, we provide in vitro and in vivo evidence that they are necessary for basal or SDF-1-induced DLBCL cell migration which is independent of Ca 2+ entry. We identify that they act as effectors coupling RhoA and ROCK dependent signaling pathway to MLC2 phosphorylation and actin polymerization. Finally, we revealed an alteration of Orai1 and STIM1 expression in extra-nodal DLBCL. Thus, we discovered a novel Ca 2+-independent but Orai1 and STIM1-dependent signaling pathway involved in basal and CXCR4 dependent cell migration, which could be relevant for DLBCL physiopathology

Abstract 1881: STIM1 and Orai1 control non-Hodgkin lymphoma cells migration

Abstract Non-Hodgkin lymphoma is one of the most common cancer in United States representing 4% of all cancer. Diffuse large B cell lymphoma (DLBCL) is the most common and aggressive non-Hodgkin B lymphoma. One of the characteristic features of this disease is the dissemination of the cancer cells, through the lymphatic system in the secondary lymphoid organs and extranodal sites, leading to the death of patients. Chemokines such as the Stromal Derived Factor 1 (SDF1) control the spread and the homing of the cancer cells. It is known that SDF1 activates various signalling pathways involved in cell proliferation, transcription or migration. Moreover, SDF1 induces an increase in intracellular calcium concentration whose role is still unknown in B cells. Store-operated Ca2+ entry (SOCE) is a major Ca2+ influx pathway in this type of cells. By definition, SOCE is activated by Ca2+ release from the endoplasmic reticulum. Two genes are responsible for SOCE activity: Stromal interaction molecule 1 (STIM1), an ER Ca2+ sensor that detects store depletion and ORAI1, the pore-forming subunit of Ca2+ release-activated Ca2+ (CRAC) channel. Several studies performed on adherent cells showed that Orai1/STIM1 proteins are involved in cancer cell migration. However, the molecular mechanisms involved in cell migration differ widely between adherent and non-adherent cells. We studied the role of both actors of calcium entry : Orai1 and STIM1 in DLBCL pathology and more precisely in basal and SDF1-induced of DLBCL cell migration. Using Tissue MicroArrays approach we revealed that both Orai1 and STIM1 are under-expressed in DLBCL tumoral tissue compared to normal lymphoid tissue. Next, using calcium imaging experiments we confirmed that SDF-1 triggered Ca2+ responses in two DLBCL cell lines (SUDHL4 and HLY1) involving intracellular Ca2+ store mobilization and extracellular Ca2+ influx. Based on these observations, we investigated the role of Orai1 and STIM1 on SDF1-induced Ca2+ influx using pharmacological and RNA interference approaches. The inhibition of Orai1 by BTP2 as well as the under-expression of Orai1 or STIM1 by shRNA, prevented Ca2+ influx induced by SDF1 suggesting the involvement of Orai1 and STIM1 in this process. Regarding this results , we studied the role of Orai1 and STIM1 on DLBCL cell migration in vitro and in vivo. Our results show that basal or SDF1-induced cell migration was significantly inhibited by underexpression of STIM1 or Orai1 in SUDHL4 and HLY1 cell lines. These results suggest that STIM1 and Orai1 play a key role in the DLBCL cell migration. The identification of STIM1 and Orai1 proteins as key players in the migration of DLBCL cells might provide new therapeutic targets for the treatment of this pathology. Citation Format: Simon Latour, Isabelle Mahouche, Floriane Cherrier, Jean-Philippe Merlio, Sandrine Poglio, Laurence Bresson Bepoldin. STIM1 and Orai1 control non-Hodgkin lymphoma cells migration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1881. doi:10.1158/1538-7445.AM2017-1881</jats:p

Prognostic Role of Inflammasome Components in Human Colorectal Cancer

(1) We wanted to assess the prognostic impact of inflammasomes involved in gut epithelial homeostasis and the development of human colorectal cancer (CRC). (2) We investigated the expression of inflammasome components in colonic epithelial cells at the protein level in patient tissues, through an immunofluorescence assay. (3) In a cohort of 104 patients, we found that all inflammasome components were downregulated in CRC. Loss of epithelial (but not stromal) expression of NLRP6, caspase-1 and IL-18 was associated with an increased mortality of 72%, 58% and 68% respectively and to disease progression into metastasis. The loss of epithelial and stromal IL-18 but not NLRP6, was associated to lower tumor immune infiltrates in the lymphoid compartment and higher Programmed cell Death receptor 1 (PD-1) expression. Finally, we found that combined downregulation of IL-18 and NLRP6 was associated with a worse outcome. Indeed, 5-year survival rates were 26% for the NLRP6low/IL-18low tumors, compared to 64.4% for the entire cohort. This downregulation was associated with a more advanced disease (p &lt; 0.0001) and a trend to lower lymphoid cell infiltration. (4) We identified critical inflammasome markers that may help in better stratifying patients for prognosis in CRC and could help clinicians to determine which patients may benefit from immunotherapies.</jats:p

Prognostic Role of Inflammasome Components in Human Colorectal Cancer.

International audience(1) We wanted to assess the prognostic impact of inflammasomes involved in gut epithelial homeostasis and the development of human colorectal cancer (CRC). (2) We investigated the expression of inflammasome components in colonic epithelial cells at the protein level in patient tissues, through an immunofluorescence assay. (3) In a cohort of 104 patients, we found that all inflammasome components were downregulated in CRC. Loss of epithelial (but not stromal) expression of NLRP6, caspase-1 and IL-18 was associated with an increased mortality of 72%, 58% and 68% respectively and to disease progression into metastasis. The loss of epithelial and stromal IL-18 but not NLRP6, was associated to lower tumor immune infiltrates in the lymphoid compartment and higher Programmed cell Death receptor 1 (PD-1) expression. Finally, we found that combined downregulation of IL-18 and NLRP6 was associated with a worse outcome. Indeed, 5-year survival rates were 26% for the NLRP6low/IL-18low tumors, compared to 64.4% for the entire cohort. This downregulation was associated with a more advanced disease ( < 0.0001) and a trend to lower lymphoid cell infiltration. (4) We identified critical inflammasome markers that may help in better stratifying patients for prognosis in CRC and could help clinicians to determine which patients may benefit from immunotherapies

Anterior Gradient-2 (AGR2) is overexpressed in colon cancer and is a potential biomarker of microsatellite instability (MSI) tumors

AbstractBackgroundColon cancer is one of the most common leading causes of death worldwide. Prognostic at an early stage is an efficient way to decrease mortality. The Endoplasmic Reticulum (ER)-resident protein anterior gradient-2 (AGR2), a Protein Disulfide Isomerase (PDI) is highly expressed in various tumours and is involved in tumour-associated processes. This study aims at examining the expression of AGR2 protein in colon cancer.MethodsAGR2 protein expression was determined using immunohistochemistry on tissue samples issued from a cohort of 82 colorectal carcinomas.ResultsAGR2 protein expression was significantly higher in tumours than in adjacent nontumour controls. AGR2 expression subgroup analyses indicated that AGR2 low expression in colon cancer patients was significantly associated with worse overall survival. Mucinous colon cancers exhibited higher AGR2 expression levels than non-mucinous cancers. Additionally, tumours with microsatellite instability (MSI) were characterised by a strong upregulation of AGR2 mRNA and protein expression despite an absence of MLH1/MSH2 mutations.ConclusionsOur findings indicate that high AGR2 protein expression is correlated with longer patient survival and that AGR2 overexpression is associated with MSI tumours and could represent an MSI biomarker. Overall, AGR2 might serve as a biomarker to stratify colon tumours and to contribute to the prognosis of colon cancer patients.</jats:sec

Emerging role of oncogenic ß-catenin in exosome biogenesis as a driver of immune escape in hepatocellular carcinoma

Immune checkpoint inhibitors have produced encouraging results in cancer patients. However, the majority of ß-catenin-mutated tumors have been described as lacking immune infiltrates and resistant to immunotherapy. The mechanisms by which oncogenic ß-catenin affects immune surveillance remain unclear. Herein, we highlighted the involvement of ß-catenin in the regulation of the exosomal pathway and, by extension, in immune/cancer cell communication in hepatocellular carcinoma (HCC). We showed that mutated ß-catenin represses expression of SDC4 and RAB27A, two main actors in exosome biogenesis, in both liver cancer cell lines and HCC patient samples. Using nanoparticle tracking analysis and live-cell imaging, we further demonstrated that activated ß-catenin represses exosome release. Then, we demonstrated in 3D spheroid models that activation of β-catenin promotes a decrease in immune cell infiltration through a defect in exosome secretion. Taken together, our results provide the first evidence that oncogenic ß-catenin plays a key role in exosome biogenesis. Our study gives new insight into the impact of ß-catenin mutations on tumor microenvironment remodeling, which could lead to the development of new strategies to enhance immunotherapeutic response

Human γδ T cell sensing of AMPK-dependent metabolic tumor reprogramming through TCR recognition of EphA2

International audienceHuman γδ T cells contribute to tissue homeostasis and participate in epithelial stress surveillance through mechanisms that are not well understood. Here, we identified ephrin type-A receptor 2 (EphA2) as a stress antigen recognized by a human Vγ9Vδ1 TCR. EphA2 is recognized coordinately by ephrin A to enable γδ TCR activation. We identified a putative TCR binding site on the ligand-binding domain of EphA2 that was distinct from the ephrin A binding site. Expression of EphA2 was up-regulated upon AMP-activated protein kinase (AMPK)-dependent metabolic reprogramming of cancer cells, and coexpression of EphA2 and active AMPK in tumors was associated with higher CD3 T cell infiltration in human colorectal cancer tissue. These results highlight the potential of the human γδ TCR to cooperate with a co-receptor to recognize non-MHC-encoded proteins as signals of cellular dysregulation, potentially allowing γδ T cells to sense metabolic energy changes associated with either viral infection or cancer

Stromal cells regulate malignant B-cell spatial organization, survival, and drug response in a new 3D model mimicking lymphoma tumor niche

Non-Hodgkin B-cell lymphomas (B-NHL) mainly develop within lymph nodes as densely packed aggregates of tumor cells and their surrounding microenvironment, creating a tumor niche specific to each lymphoma subtypes. Until now, in vitro preclinical models mimicking biomechanical forces, cellular microenvironment, and 3D organization of B lymphomas remain scarce while all these parameters constitute key determinants of lymphomagenesis and drug resistance. Using a microfluidic method based on the encapsulation of cells inside permeable, elastic, and hollow alginate microspheres, we developed a new tunable 3D-model incorporating extracellular matrix and/or stromal cells. Lymphoma B cells and stromal cells dynamically formed self-organized 3D spheroids, thus initiating a coevolution of these two cell types, reflecting their bidirectional crosstalk, and recapitulating the heterogeneity of B-NHL subtypes. In addition, this approach makes it suitable to assess in a relevant in vitro model the activity of new therapeutic agents in B-NHL

Human γδ T cell sensing of AMPK-dependent metabolic tumor reprogramming through TCR recognition of EphA2

A γδ TCR senses metabolic tumor reprogramming through recognition of EphA2.</jats:p