Fawzi Aoudjit

Integrin signaling inhibits paclitaxel-induced apoptosis in breast cancer cells

Inherent or acquired drug resistance is one of the major problems in chemotherapy. The mechanisms by which cancer cells survive and escape the cytotoxic effects of chemotherapeutic agents are essentially unknown. In the present study, we demonstrate that in the MDA-MB-231 and MDA-MB-435 breast cancer cells, ligation of β1 integrins by their extracellular matrix ligands inhibits significantly apoptosis induced by paclitaxel and vincristine, two microtubule-directed chemotherapeutic agents that are widely used in the therapy of breast cancer. We show that β1 integrin signaling inhibits drug-induced apoptosis by inhibiting the release of cytochrome c from the mitochondria in response to drug treatment. Further, integrin-mediated protection from drug-induced apoptosis and inhibition of cytochrome c release are dependent on the activation of the PI 3-kinase/Akt pathway. Our results identify β1 integrin signaling as an important survival pathway in drug-induced apoptosis in breast cancer cells and suggest that activation of this pathway may contribute to the generation of drug resistance.

Integrin Signaling in Cancer Cell Survival and Chemoresistance

Resistance to apoptosis and chemotherapy is a hallmark of cancer cells, and it is a critical factor in cancer recurrence and patient relapse. Extracellular matrix (ECM) via its receptors, the integrins, has emerged as a major pathway contributing to cancer cell survival and resistance to chemotherapy. Several studies over the last decade have demonstrated that ECM/integrin signaling provides a survival advantage to various cancer cell types against numerous chemotherapeutic drugs and against antibody therapy. In this paper, we will discuss the major findings on how ECM/integrin signaling protects tumor cells from drug-induced apoptosis. We will also discuss the potential role of ECM in malignant T-cell survival and in cancer stem cell resistance. Understanding how integrins and their signaling partners promote tumor cell survival and chemoresistance will likely lead to the development of new therapeutic strategies and agents for cancer treatment.

Down-regulation of mcl-1 by small interfering RNA sensitizes resistant melanoma cells to fas-mediated apoptosis.

Resistance of malignant melanoma cells to Fas-mediated apoptosis is among the mechanisms by which they escape immune surveillance. However, the mechanisms contributing to their resistance are not completely understood, and it is still unclear whether antiapoptotic Bcl-2–related family proteins play a role in this resistance. In this study, we report that treatment of Fas-resistant melanoma cell lines with cycloheximide, a general inhibitor of de novo protein synthesis, sensitizes them to anti-Fas monoclonal antibody (mAb)–induced apoptosis. The cycloheximide-induced sensitization to Fas-induced apoptosis is associated with a rapid down-regulation of Mcl-1 protein levels, but not that of Bcl-2 or Bcl-xL. Targeting Mcl-1 in these melanoma cell lines with specific small interfering RNA was sufficient to sensitize them to both anti-Fas mAb-induced apoptosis and activation of caspase-9. Furthermore, ectopic expression of Mcl-1 in a Fas-sensitive melanoma cell line rescues the cells from Fas-mediated apoptosis. Our results further show that the expression of Mcl-1 in melanoma cells is regulated by the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and not by phosphatidylinositol 3-kinase/AKT signaling pathway. Inhibition of ERK signaling with the mitogen-activated protein/ERK kinase-1 inhibitor or by expressing a dominant negative form of mitogen-activated protein/ERK kinase-1 also sensitizes resistant melanoma cells to anti-Fas mAb-induced apoptosis. Thus, our study identifies mitogen-activated protein kinase/ERK/Mcl-1 as an important survival signaling pathway in the resistance of melanoma cells to Fas-mediated apoptosis and suggests that its targeting may contribute to the elimination of melanoma tumors by the immune system. (Mol Cancer Res 2008;6(1):42–52)

Melanoma Spheroids Grown Under Neural Crest Cell Conditions Are Highly Plastic Migratory/Invasive Tumor Cells Endowed with Immunomodulator Function

Background The aggressiveness of melanoma tumors is likely to rely on their well-recognized heterogeneity and plasticity. Melanoma comprises multi-subpopulations of cancer cells some of which may possess stem cell-like properties. Although useful, the sphere-formation assay to identify stem cell-like or tumor initiating cell subpopulations in melanoma has been challenged, and it is unclear if this model can predict a functional phenotype associated with aggressive tumor cells. Methodology/Principal Findings We analyzed the molecular and functional phenotypes of melanoma spheroids formed in neural crest cell medium. Whether from metastatic or advanced primary tumors, spheroid cells expressed melanoma-associated markers. They displayed higher capacity to differentiate along mesenchymal lineages and enhanced expression of SOX2, NANOG, KLF4, and/or OCT4 transcription factors, but not enhanced self-renewal or tumorigenicity when compared to their adherent counterparts. Gene expression profiling attributed a neural crest cell signature to these spheroids and indicated that a migratory/invasive and immune-function modulating program could be associated with these cells. In vitro assays confirmed that spheroids display enhanced migratory/invasive capacities. In immune activation assays, spheroid cells elicited a poorer allogenic response from immune cells and inhibited mitogen-dependent T cells activation and proliferation more efficiently than their adherent counterparts. Our findings reveal a novel immune-modulator function of melanoma spheroids and suggest specific roles for spheroids in invasion and in evasion of antitumor immunity. Conclusion/Significance The association of a more plastic, invasive and evasive, thus a more aggressive tumor phenotype with melanoma spheroids reveals a previously unrecognized aspect of tumor cells expanded as spheroid cultures. While of limited efficiency for melanoma initiating cell identification, our melanoma spheroid model predicted aggressive phenotype and suggested that aggressiveness and heterogeneity of melanoma tumors can be supported by subpopulations other than cancer stem cells. Therefore, it could be constructive to investigate melanoma aggressiveness, relevant to patients and clinical transferability.

MHC Class II Engagement by Its Ligand LAG-3 (CD223) Contributes to Melanoma Resistance to Apoptosis

Melanoma is the most aggressive skin cancer in humans that often expresses MHC class II (MHC II) molecules, which could make these tumors eliminable by the immune system. However, this MHC II expression has been associated with poor prognosis, and there is a lack of immune-mediated eradication. The lymphocyte activation gene-3 (LAG-3) is a natural ligand for MHC II that is substantially expressed on melanoma-infiltrating T cells including those endowed with potent immune-suppressive activity. Based on our previous data showing the signaling capacity of MHC II in melanoma cells, we hypothesized that LAG-3 could contribute to melanoma survival through its MHC II signaling capacity in melanoma cells. In this study, we demonstrate that both soluble LAG-3 and LAG-3–transfected cells can protect MHC II-positive melanoma cells, but not MHC II-negative cells, from FAS-mediated and drug-induced apoptosis. Interaction of LAG-3 with MHC II expressed on melanoma cells upregulates both MAPK/Erk and PI3K/Akt pathways, albeit with different kinetics. Inhibition studies using specific inhibitors of both pathways provided evidence of their involvement in the LAG-3–induced protection from apoptosis. Altogether, our data suggest that the LAG-3–MHC II interaction could be viewed as a bidirectional immune escape pathway in melanoma, with direct consequences shared by both melanoma and immune cells. In the future, compounds that efficiently hinder LAG-3–MHC II interaction might be used as an adjuvant to current therapy for MHC II-positive melanoma.

Interleukin-7 promotes the survival of human CD4+ effector/memory T cells by up-regulating Bcl-2 proteins and activating the JAK/STAT signalling pathway.

Interleukin‐7 (IL‐7) is a crucial cytokine involved in T‐cell survival and development but its signalling in human T cells, particularly in effector/memory T cells, is poorly documented. In this study, we found that IL‐7 protects human CD4+ effector/memory T cells from apoptosis induced upon the absence of stimulation and cytokines. We show that IL‐7 up‐regulates not only Bcl‐2 but also Bcl‐xL and Mcl‐1 as well. Interleukin‐7‐induced activation of the janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway is sufficient for cell survival and up‐regulation of Bcl‐2 proteins. In contrast to previous studies with naive T cells, we found that IL‐7 is a weak activator of the phosphatidylinositol 3 kinase (PI3K)/AKT (also referred as protein kinase B) pathway and IL‐7‐mediated cell survival occurs independently from the PI3K/AKT pathway as well as from activation of the mitogen‐activated protein kinase/extracellular signal‐regulated kinase pathway. Considering the contribution of both IL‐7 and CD4+ effector/memory T cells to the pathogenesis of autoimmune diseases such as rheumatoid arthritis and colitis, our study suggests that IL‐7 can contribute to these diseases by promoting cell survival. A further understanding of the mechanisms of IL‐7 signalling in effector/memory T cells associated with autoimmune inflammatory diseases may lead to potential new therapeutic avenues.

α2β1 Integrin Promotes Chemoresistance against Doxorubicin in Cancer Cells through Extracellular Signal-regulated Kinase (ERK)

Background: Mechanisms by which β1 integrins regulate resistance of cancer cells to chemotherapy. Results: α2β1 integrin but not fibronectin-binding integrins inhibits doxorubicin-induced apoptosis of leukemic T cells via JNK inhibition and Mcl-1 up-regulation in an MAPK/ERK-dependent pathway. Conclusion: α2β1 integrin/ERK pathway promotes chemoresistance of cancer cells. Significance: Activation of this pathway can contribute to the appearance of the drug resistance phenotype. The role and the mechanisms by which β1 integrins regulate the survival and chemoresistance of T cell acute lymphoblastic leukemia (T-ALL) still are poorly addressed. In this study, we demonstrate in T-ALL cell lines and primary blasts, that engagement of α2β1 integrin with its ligand collagen I (ColI), reduces doxorubicin-induced apoptosis, whereas fibronectin (Fn) had no effect. ColI but not Fn inhibited doxorubicin-induced mitochondrial depolarization, cytochrome c release, and activation of caspase-9 and -3. ColI but not Fn also prevented doxorubicin from down-regulating the levels of the prosurvival Bcl-2 protein family member Mcl-1. The effect of ColI on Mcl-1 occurred through the inhibition of doxorubicin-induced activation of c-Jun N-terminal kinase (JNK). Mcl-1 knockdown experiments showed that the maintenance of Mcl-1 levels is essential for ColI-mediated T-ALL cell survival. Furthermore, activation of MAPK/ERK, but not PI3K/AKT, is required for ColI-mediated inhibition of doxorubicin-induced JNK activation and apoptosis and for ColI-mediated maintenance of Mcl-1 levels. Thus, our study identifies α2β1 integrin as an important survival pathway in drug-induced apoptosis of T-ALL cells and suggests that its activation can contribute to the generation of drug resistance.

Alpha2beta1 integrin is the major collagen‐binding integrin expressed on human Th17 cells

Growing evidence indicates that collagen‐binding integrins are important costimulatory molecules of effector T cells. In this study, we demonstrate that the major collagen‐binding integrin expressed by human Th17 cells is alpha2beta1 (α2β1) or VLA‐2, also known as the receptor for collagen I on T cells. Our results show that human naïve CD4+ T cells cultured under Th17 polarization conditions preferentially upregulate α2β1 integrin rather than α1β1 integrin, which is the receptor for collagen IV on T cells. Double staining analysis for integrin receptors and intracellular IL‐17 showed that α2 integrin but not α1 integrin is associated with Th17 cells. Cell adhesion experiments demonstrated that Th17 cells attach to collagen I and collagen II using α2β1 integrin but did not attach to collagen IV. Functional studies revealed that collagens I and II but not collagen IV costimulate the production of IL‐17A, IL‐17F and IFN‐γ by human Th17 cells activated with anti‐CD3. These results identify α2β1 integrin as the major collagen receptor expressed on human Th17 cells and suggest that it can be an important costimulatory molecule of Th17 cell responses.

Collagen/β1 integrin signaling up-regulates the ABCC1/MRP-1 transporter in an ERK/MAPK-dependent manner

Collagen/β1 integrin/extracellular signal-regulated kinase signaling up-regulates the expression and function of ABCC1 transporter. This suggests that its activation could represent an important pathway in cancer chemoresistance.

Implication of discoidin domain receptor 1 in T cell migration in three-dimensional collagen

T cell migration through extracellular matrix of the tissue is an important process in the development of inflammation. However, the mechanisms regulating this process are complex and still not well defined. In this study, we show that activation of human peripheral blood T cells with anti-CD3 mAb increases the mRNA and protein levels of the discoidin domain receptor 1 (DDR1), which is known to bind to collagens. Furthermore, our findings indicate that DDR1 is involved in the migration of activated T cells in three-dimensional (3D) collagen. Indeed, the use of a DDR1 blocking molecule (DDR1:Fc) reduced the capacity of anti-CD3-activated human T cells to migrate in 3D collagen, whereas a control immunoglobulin had no effect. As a control, the DDR1:Fc molecule did not interfere with the capacity of human T cells to migrate through fibronectin. Together these results suggest that DDR1 can represent an additional receptor regulating T cell movement in the tissues and therefore can contribute to the development of inflammatory diseases.