Synthesis of peptidomimetics and chemo-biological tools for CD95/PLCγ1 interaction analysis.

The death receptor CD95 (also known as Fas) induces apoptosis through protein/protein association and the formation of the death-inducing signaling complex. On the other hand, in certain biological conditions, this receptor recruits different proteins and triggers the formation of another complex designated motility-inducing signaling complex, which promotes cell migration and inflammation. This pathway relies on a short sequence of CD95, called calcium-inducing domain (CID), which interacts with the phospholipase PLCγ1. To better understand how CID/PLCγ1 interaction occurs, we synthesized different α-AA peptides mimicking CID. Some of these peptidomimetics are as potent as the natural peptide to disrupt the CID/PLCγ1 interaction and cell migration, and showed improved pharmacokinetic properties. We also generated biotinyl- and palmitoyl-labelled peptidomimetics, useful chemico-biological tools to further explore the pro-inflammatory signal of CD95, which plays an important role in the pathogenesis of lupus and other autoimmune diseases.

Chemotherapy elicits pro-metastatic extracellular vesicles in breast cancer models.

Cytotoxic chemotherapy is an effective treatment for invasive breast cancer. However, experimental studies in mice also suggest that chemotherapy has pro-metastatic effects. Primary tumours release extracellular vesicles (EVs), including exosomes, that can facilitate the seeding and growth of metastatic cancer cells in distant organs, but the effects of chemotherapy on tumour-derived EVs remain unclear. Here we show that two classes of cytotoxic drugs broadly employed in pre-operative (neoadjuvant) breast cancer therapy, taxanes and anthracyclines, elicit tumour-derived EVs with enhanced pro-metastatic capacity. Chemotherapy-elicited EVs are enriched in annexin A6 (ANXA6), a Ca2+-dependent protein that promotes NF-κB-dependent endothelial cell activation, Ccl2 induction and Ly6C+CCR2+ monocyte expansion in the pulmonary pre-metastatic niche to facilitate the establishment of lung metastasis. Genetic inactivation of Anxa6 in cancer cells or Ccr2 in host cells blunts the pro-metastatic effects of chemotherapy-elicited EVs. ANXA6 is detected, and potentially enriched, in the circulating EVs of breast cancer patients undergoing neoadjuvant chemotherapy.

Disrupting the CD95-PLCγ1 interaction prevents Th17-driven inflammation.

CD95L is a transmembrane ligand (m-CD95L) that is cleaved by metalloproteases to release a soluble ligand (s-CD95L). Unlike m-CD95L, interaction between s-CD95L and CD95 fails to recruit caspase-8 and FADD to trigger apoptosis and instead induces a Ca2+ response via docking of PLCγ1 to the calcium-inducing domain (CID) within CD95. This signaling pathway induces accumulation of inflammatory Th17 cells in damaged organs of lupus patients, thereby aggravating disease pathology. A large-scale screen revealed that the HIV protease inhibitor ritonavir is a potent disruptor of the CD95-PLCγ1 interaction. A structure-activity relationship approach highlighted that ritonavir is a peptidomimetic that shares structural characteristics with CID with respect to docking to PLCγ1. Thus, we synthesized CID peptidomimetics abrogating both the CD95-driven Ca2+ response and transmigration of Th17 cells. Injection of ritonavir and the CID peptidomimetic into lupus mice alleviated clinical symptoms, opening a new avenue for the generation of drugs for lupus patients.

Tumor analysis: freeze-thawing cycle of triple-negative breast cancer cells alters tumor CD24/CD44 profiles and the percentage of tumor-infiltrating immune cells.

OBJECTIVE: The use of novel methods to characterize living tumor cells relies on well-conceived biobanks. Herein, we raised the question of whether the composition of fresh and freeze/thawed dissociated tumor samples is comparable in terms of quantitative and qualitative profiling. RESULTS: Breast cancer is a heterogeneous disease, encompassing luminal A and B, basal/triple-negative breast cancer (TNBC), and ERBB2-like tumors. We examined living cells dissociated from TNBC and found that a classical freeze/thaw protocol leads to a marked reduction in the number of CD45-CD44LowCD24Low tumor cells. This, in turn, changed the percentage of tumor cells with certain CD44/CD24 expression patterns and changed the percentage of tumor-infiltrating immune cells. These cryopreservation-driven alterations in cellular phenotype make it impossible to compare fresh and frozen samples from the same patient directly. Moreover, the freeze/thaw process changed the transcriptomic signatures of triple-negative cancer stem cells in such a manner that hierarchical clustering no longer ranked them according to expected inter-individual differences. Overall, this study suggests that all analyses of living tumor cells should be conducted only using freshly dissociated tumors if we are to generate a robust scoring system for prognostic/predictive markers.

CD95/Fas, Non-Apoptotic Signaling Pathways, and Kinases.

Endothelial cells lining new blood vessels that develop during inflammatory disorders or cancers act as doors that either allow or block access to the tumor or inflamed organ. Recent data show that these endothelial cells in cancer tissues and inflamed tissues of lupus patients overexpress CD95L, the biological role of which is a subject of debate. The receptor CD95 (also named Fas or apoptosis antigen 1) belongs to the tumor necrosis factor (TNF) receptor superfamily. Its cognate ligand, CD95L, is implicated in immune homeostasis and immune surveillance. Because mutations of this receptor or its ligand lead to autoimmune disorders such as systemic lupus erythematosus (SLE) and cancers, CD95 and CD95L were initially thought to play a role in immune homeostasis and tumor elimination via apoptotic signaling pathways. However, recent data reveal that CD95 also evokes non-apoptotic signals, promotes inflammation, and contributes to carcinogenesis; therefore, it is difficult to dissect its apoptotic effects from its non-apoptotic effects during pathogenesis of disease. CD95L is cleaved by metalloproteases and so exists in two different forms: a transmembrane form and a soluble ligand (s-CD95L). We recently observed that the soluble ligand is overexpressed in serum from patients with triple-negative breast cancer or SLE, in whom it contributes to disease severity by activating non-apoptotic signaling pathways and promoting either metastatic dissemination or accumulation of certain T cell subsets in damaged organs. Here, we discuss the roles of CD95 in modulating immune functions via induction of mainly non-apoptotic signaling pathways.

Boyden Chamber Assay to Study of Cell Migration Induced by Metalloprotease Cleaved-CD95L.

CD95 receptor, also called Fas or Apo-1, is a member of the tumor necrosis factor receptors (TNF-R) superfamily (Itoh and Nagata, J Biol Chem 268:10932-10937, 1993). Its cognate ligand, CD95L, is a transmembrane cytokine, which can be cleaved by metalloproteases (Matsuno et al., J Rheumatol 28:22-28, 2001; Vargo-Gogola et al., Arch Biochem Biophys 408:155-161, 2002; Kiaei et al., Exp Neurol 205:74-81, 2007; Schulte et al., Cell Death Differ 14:1040-1049, 2007) releasing a soluble ligand into the bloodstream. Recent work has shown that this metalloprotease-cleaved CD95L (cl-CD95L) is involved in carcinogenesis (Malleter et al., Cancer Res 73:6711-6721, 2013). Cl-CD95L also fuels the inflammatory process in patients affected by systemic lupus erythematosus by promoting the accumulation of activated T lymphocytes in enflamed organs (Tauzin et al., PLoS Biol 9:e1001090, 2011). This chapter aims at describing the methodology used to measure the chemoattractive effect of cl-CD95L on human cancer cells and lymphocytes.

CD95-Mediated Calcium Signaling Promotes T Helper 17 Trafficking to Inflamed Organs in Lupus-Prone Mice.

CD95 ligand (CD95L) is expressed by immune cells and triggers apoptotic death. Metalloprotease-cleaved CD95L (cl-CD95L) is released into the bloodstream but does not trigger apoptotic signaling. Hence, the pathophysiological role of cl-CD95L remains unclear. We observed that skin-derived endothelial cells from systemic lupus erythematosus (SLE) patients expressed CD95L and that after cleavage, cl-CD95L promoted T helper 17 (Th17) lymphocyte transmigration across the endothelial barrier at the expense of T regulatory cells. T cell migration relied on a direct interaction between the CD95 domain called calcium-inducing domain (CID) and the Src homology 3 domain of phospholipase Cγ1. Th17 cells stimulated with cl-CD95L produced sphingosine-1-phosphate (S1P), which promoted endothelial transmigration by activating the S1P receptor 3. We generated a cell-penetrating CID peptide that prevented Th17 cell transmigration and alleviated clinical symptoms in lupus mice. Therefore, neutralizing the CD95 non-apoptotic signaling pathway could be an attractive therapeutic approach for SLE treatment.