ADAM28: Another ambivalent protease in cancer

Emergence of novel therapeutic options in a perspective of personalized therapy of cancer relies on the discovery of precise molecular mechanisms involved in the switch from a localized tumor to invasive metastasis spread. Pro-tumor functions have been mostly ascribed to proteolytic enzymes from the metalloproteinase family including A Disintegrin And Metalloproteinases (ADAMs). Particularly, when expressed by cancer cells, ADAM28 protease supports cancer cell proliferation, survival and migration as well as metastatic progression. In sharp contrast, ADAM28 derived from the tumor microenvironment has shown to exert strong protective effects against deleterious metastasis dissemination. Indeed, depletion of host-derived ADAM28 (ADAM28 KO mice) accelerates colonization lung tissues, increases tumor foci implantation, and impairs T cell immune response. In this review, we outline specific ADAM28 functions when specifically expressed by carcinoma cells or by tumor microenvironment. Finally, we discuss about future research strategies that could be pursued to highlight new functions of this protease in cancer. © 2020 Elsevier B.V

ADAM28 - Generation of a conditional ADAM28 knock-out mouse Implication of ADAM28 in metastasis dissemination

Introduction: ADAMs (a disintegrin and metalloproteinase) and ADAMTS proteinases (a disintegrin and metalloproteinase with thrombospondin motifs) are MMP (matrix metalloproteinase) -related enzymes, bearing a multi-domain structure. ADAM28 is a multipotent membrane-bound proteinase and its expression in tissues derived from the foregut in embryonic tissues suggests its involvement in the organogenesis of the respiratory tract. Moreover, ADAM28 is highly overexpressed in non-small-cell lung cancer samples. Notably through a global proteomic approach, we recently identified an upregulation of ADAM28 after induction of lung inflammation in a mouse model of asthma. In addition to data published in the literature and to our findings about ADAM28 expression in various pathological tissues, intrinsic characteristics of this proteinase argue for considering it as a potential regulator of cellular signalling pathways leading to an inflammatory pulmonary microenvironment and, eventually, to carcinogenesis. Indeed, ADAM28 bears an active catalytic domain and interacts in a non-proteolytic manner with some integrins (α4β1) and some P-selectin ligands involved in inflammatory cell migration. ADAM28 was also reported to display the capacity to cleave key mediators such as Von Willebrand factor, IGFBP3 (Insulin-like growth factor-binding protein 3) and CTGF (Connective Tissue Growth Factor). Very recently, ADAM28 was shown to shed pro-TNF-alpha suggesting an important role in tumour control. The present project aims at characterizing molecular mechanisms leading to tumour development in lungs. In the first part of this work, we intend to characterize the effects of ADAM28 depletion on physiological and pathological processes such as tumor development and metastatic dissemination in ADAM28 conditional knock-out mice. Methods: To understand the implication of ADAM28 in lung tumour development we instillated lungs of mice displaying a full knock-out genotype for ADAM28 with Lewis Lung Carcinoma cells and B16K1 melanoma cells. ADAM28 KO mice were also injected intravenously and a subcutaneously with these tumour cells. Results: There is no spontaneous phenotype for ADAM28 full knock-out animals. Preliminary results show an impaired lung tumors engrafment in ADAM28 wild type animals when compared to heterozygous or knock out littermates. Conclusion: This unique mouse strain provides a very precious tool to further investigate ADAM28 implication in various disease models including tumour growth and dissemination. The role of ADAM28 as a pro-tumor factor is widely described in the literature whereas our results suggest that ADAM28 has an anti-tumor function

L'ADAM28 dérivée du microenvironnement influence l'apparition du cancer du poumon

Background ADAM28 expression is upregulated in non-small cell lung carcinoma and correlated with cell proliferation and metastatic dissemination. Moreover, in vivo studies have shown that knockdown of ADAM28 in tumor cells decreased the primary tumor growth and formation of lung metastasis. Besides, ADAM28 is thought to be an important regulator of inflammatory signaling pathways as it sheds the pro-inflammatory cytokine, pro-TNF-alpha. ADAM28 protease also interacts with integrins and the P-selectin glycoprotein ligand-1 leading to inflammatory cell migration. Altogether, these findings suggest that ADAM28 contributes to cellular mechanisms leading to cancer development and progression. Methods This study aims to characterize the effects of microenvironment-derived ADAM28 on lung metastasis formation. To achieve this purpose, we generated ADAM28-/- mice into two different mouse strains (C57BL/6 and BALB/c). Lung metastatic dissemination was assessed in both ADAM28-/- and wild-type (WT) mice after intravenous injection of Lewis Lung Carcinoma cells, B16K1 melanoma cells or 4T1 breast carcinoma cells. As ADAM28 promotes leukocyte transendothelial migration, lymphocyte subtypes implicated in tumor cytotoxicity or in regulation of immune response were studied by flow cytometry. Results An unexpected increased tumor burden was found in lungs of ADAM28-/- mice as compared to WT mice. Flow cytometry analysis revealed that less CD8+ T were infiltrated within lungs of ADAM28-/- tumor-bearing mice. Moreover, a reduced CD8+ T cell population was observed in the spleen of naïve ADAM28-/- mice that is not caused by an impaired T cell maturation in the thymus. Ex vivo assays demonstrated that intrinsic properties of CD8+ T cells from ADAM28-/- mice were not affected by ADAM28 deficiency as their proliferation, migration and activation was similar. Besides, we found no expression of ADAM28 in isolated CD8+ T cells from the spleen of ADAM28-/- and WT mice. Therefore, we hypothesized that ADAM28 indirectly modulates the anti-tumor cytotoxic immune response. We also found that ADAM28 depletion is associated with a reduced infiltration of NK cell within lungs of ADAM28-/- tumor-bearing mice. Conclusion Our results demonstrate a protective effect of microenvironment-derived ADAM28 by regulating the tumor-associated immune response

Impact de la déplétion en ADAM28 sur la dissémination métastatique et la progression du cancer pulmonaire

ADAM28 is highly overexpressed in non-small-cell lung cancer samples. Notably through a global proteomic approach, we recently identified an upregulation of ADAM28 after induction of lung inflammation in a mouse model of asthma. In addition to data published in the literature and to our findings about ADAM28 expression in various pathological tissues, intrinsic characteristics of this proteinase argue for considering it as a potential regulator of cellular signalling pathways leading to an inflammatory pulmonary microenvironment and to carcinogenesis. Indeed, ADAM28 possesses an active catalytic domain and interacts in a non-proteolytic manner with some integrins (α4β1) and some P-selectin ligands involved in inflammatory cell migration. Recently, ADAM28 was shown to shed pro-TNF-alpha suggesting an important role in tumour control. This project aims to characterize molecular mechanisms involving host-derived ADAM28 in tumour development and metastatic dissemination to lung tissues. We intend to determine the effects of ADAM28 depletion on physiological and pathological processes in ADAM28 conditional knockout mice, which have been developed in our laboratory (in collaboration with the Max Planck Institute, Munich). This unique mouse strain provides a precious tool to investigate ADAM28 implication in disease models including tumour growth and dissemination. There is no spontaneous phenotype for ADAM28 full knockout animals, which are fertile and do not display any significant abnormality or defect. To understand the implication of ADAM28 in lung tumour cell dissemination, ADAM28 KO mice were intravenously injected with Lewis Lung Carcinoma cells and B16K1 melanoma cells. Our results show an increased tumour development in lungs of KO mice. To explain these surprising data, the vessel structure and permeability will be observed in our KO and WT littermates. Moreover, ADAM28 is described to be associated with lymphocyte transendothelial migration and is known to be expressed by lymphocytes. Consequently, we analysed the presence of lymphocyte subtypes implicated in tumour cytotoxicity or in the regulation of immune response. We observed that CD8 positive T-cells are less expressed in the spleen of naive KO mice and in the early stages after tumour cells injection. We also observed less CD8 T-cells in the lung of KO mice at a later stage in our model of tumour dissemination. These preliminary data suggest a role of ADAM28 in maturation and function of T cells. Hence, we will investigate the antigen presentation to immune cells and the link between tumour cells, cytokines production and B and T-cells stimulation. The role of ADAM28 derived from tumour cell as a pro-tumour factor is widely described in the literature. However, our results demonstrate an anti-tumour function of ADAM28 expressed in tumour microenvironment suggesting a dual role of ADAM28, depending on the origin of the protease. More investigations are required to explain this contradictory effect before drawing further conclusions

Effet de la déplétion en ADAM28 sur les processus d'angiogenèse et de lymphangiogenèse

Introduction ADAM28, a protease of the adamalysin family, is overexpressed by carcinoma cells in various cancers including non-small cell lung carcinoma (NSCLC) and breast cancer. In vivo studies reported that knockdown of ADAM28 reduced primary tumor growth and lung metastatic formation suggesting a potential pro-tumoral role of ADAM28. Although cellular mechanism remain poorly described, ADAM28 is thought to stimulate angiogenesis and thus, promoting tumor progression. Indeed, ADAM28 can cleave CTGF from the CTGF/VEGF165 complex leading to the release of VEGF165. Another known substrate of ADAM28 is von Willebrand factor (vWF) which deficiency is associated with an increased angiogenesis and vasculature density. Therefore, we could hypothesize that ADAM28 induces angiogenesis through the cleavage of vWF. All these findings suggest that ADAM28 contributes to cancer development and progression by stimulating angiogenesis. Aim This project aims to investigate ADAM28 functions in the lung tumor microenvironment especially in the angiogenesis using an ADAM28 conditional knockout mouse model. Results Angiogenesis and lymphangiogenesis were assessed in lung metastasis of both ADAM28 KO and WT mice after intravenous injection of LLC cells. Immunofluorescence staining indicated that blood vessel density was increased within lung metastasis of WT mice as compared to ADAM28 KO mice. In contrast, lymphatic vessel density was similar between both groups. In addition, endothelial cell migration was evaluated in tumor-free mice using the ex vivo aortic ring assay. Result indicated that endothelial cells from ADAM28 KO mice migrated more than endothelial cells from WT mice. We also showed that blood vessel permeability was similar in WT and ADAM28 KO mice suggesting that ADAM28 depletion don’t affect blood vessel structure. Moreover, we assessed the expression of adhesion markers (ICAM-1, VCAM-1) and found no difference between both groups. Perspectives All these data need to be confirmed before drawing further conclusions. Indeed, we will confirm the increased blood vessel density in lung metastasis of WT mice using other tumor mouse models. It could also be interesting to evaluate angiogenesis in primary tumor of WT and ADAM28 KO mice. We will repeat the aortic ring experiment and perform immunostaining of pericytes and endothelial cells to study in more details the structure of the newly formed blood vessels. Angiogenesis and lymphangiogenesis will be investigated using different in vivo models such as the ear sponge assay and the corneal neovascularization model which are well-established in the laboratory. Furthermore, we would like to isolate endothelial cells from the lung of WT and ADAM28 KO mice to determine ADAM28 expression level in these cells and then, perform different in vitro assays (proliferation, migration, activation)

La délétion en ADAM28 a un impact sur la formation de métastases dans le poumon

ADAM28 expression is upregulated in non-small cell lung carcinoma and correlated with cell proliferation and metastatic dissemination. In addition, ADAM28 is thought to be an important regulator of inflammatory signaling pathways as this protease shed the pro-inflammatory cytokine, pro-TNF-alpha. ADAM28 also interacts with integrins and a P-selectin ligand (PSGL-1) involved in inflammatory cell migration. All these findings suggest that ADAM28 contributes to cancer development and progression. This project aims to characterize the effects of host-derived ADAM28 on lung metastasis formation using an ADAM28-/- mouse model. Lewis Lung Carcinoma cells and B16K1 melanoma cells were intravenously injected in ADAM28-/- and wild-type (WT) mice. An unexpected increased tumor development was found in lungs of ADAM28-/- mice. As ADAM28 is associated with lymphocyte transendothelial migration, lymphocyte subtypes implicated in tumor cytotoxicity or in regulation of immune response were studied by flow cytometry. Results showed that less CD8+ T and NK cells were infiltrated within lungs of ADAM28-/- tumor-bearing mice as compared to WT mice. Moreover, less CD8+ T cells were counted within the spleen of naïve ADAM28-/- mice. These data were confirmed in a mouse model where 4T1 breast carcinoma cells were intravenously injected in ADAM28-/- BALB/c mice. Intrinsic properties of CD8+ T cells from ADAM28-/- mice were not affected by ADAM28 depletion as they were able to proliferate, to be activated and to migrate. Further investigations are required to explain the CD8+ T cell phenotype in ADAM28-deficient mice. Our results suggest a protective effect of ADAM28 derived from the host microenvironment by indirectly regulate the immune response

Role for the metalloproteinase ADAM28 in the control of airway inflammation, remodelling and responsiveness in asthma

peer reviewedBackgroundAsthma is characterized by morphological modifications of the airways (inflammation and remodelling) and bronchial hyperresponsiveness. Mechanisms linking these two key features of asthma are still poorly understood. ADAM28 (a disintegrin and metalloproteinase 28) might play a role in asthma pathophysiology. ADAM28 exists as membrane-bound and soluble forms and is mainly expressed by lymphocytes and epithelial cells.MethodsADAM28-/- mice and ADAM28+/+ counterparts were sensitized and exposed to ovalbumin (OVA). Airway responsiveness was measured using the flexiVent® system. After sacrifice, bronchoalveolar lavage (BAL) was performed and lungs were collected for analysis of airway inflammation and remodelling.ResultsThe expression of the soluble form of ADAM28 was lower in the lungs of OVA-exposed mice (as compared to PBS-exposed mice) and progressively increased in correlation with the duration of allergen exposure. In lungs of ADAM28-/- mice exposed to allergens, the proportion of Th2 cells among CD4+ cells and the number of B cells were decreased. Bronchial responsiveness was lower in ADAM28-/- mice exposed to allergens and similar to the responsiveness of sham-challenged mice. Similarly, features of airway remodelling (collagen deposition, smooth muscle hyperplasia, mucous hyperplasia) were significantly less developed in OVA-exposed ADAM28-/- animals in sharp contrasts to ADAM28+/+. In addition, we report the first evidence of ADAM28 RNA expression by lung fibroblasts and we unveil a decreased capacity of lung fibroblasts extracted from OVA-exposed ADAM28-/- mice to proliferate as compared to those extracted from OVA-exposed ADAM28+/+ suggesting a direct contribution of this enzyme to the modulation of airway remodelling.ConclusionThese results suggest that ADAM28 might be a key contributor to the pathophysiology of asthma