Triple-negative breast cancer (TNBC) lacks targeted therapies and has a worse prognosis than other breast cancer subtypes, underscoring an urgent need for new therapeutic targets and strategies. IRE1 is an endoplasmic reticulum (ER) stress sensor, whose activation is predominantly linked to the resolution of ER stress and, in the case of severe stress, to cell death. Here we demonstrate that constitutive IRE1 RNase activity contributes to basal production of pro-tumorigenic factors IL-6, IL-8, CXCL1, GM-CSF, and TGFβ2 in TNBC cells. We further show that the chemotherapeutic drug, paclitaxel, enhances IRE1 RNase activity and this contributes to paclitaxel-mediated expansion of tumor-initiating cells. In a xenograft mouse model of TNBC, inhibition of IRE1 RNase activity increases paclitaxel-mediated tumor suppression and delays tumor relapse post therapy. We therefore conclude that inclusion of IRE1 RNase inhibition in therapeutic strategies can enhance the effectiveness of current chemotherapeutics.

Patients affected by chronic inflammatory disorders display high amounts of soluble CD95L. This homotrimeric ligand arises from the cleavage by metalloproteases of its membrane-bound counterpart, a strong apoptotic inducer. In contrast, the naturally processed CD95L is viewed as an apoptotic antagonist competing with its membrane counterpart for binding to CD95. Recent reports pinpointed that activation of CD95 may attract myeloid and tumoral cells, which display resistance to the CD95-mediated apoptotic signal. However, all these studies were performed using chimeric CD95Ls (oligomerized forms), which behave as the membrane-bound ligand and not as the naturally processed CD95L. Herein, we examine the biological effects of the metalloprotease-cleaved CD95L on CD95-sensitive activated T-lymphocytes. We demonstrate that cleaved CD95L (cl-CD95L), found increased in sera of systemic lupus erythematosus (SLE) patients as compared to that of healthy individuals, promotes the formation of migrating pseudopods at the leading edge of which the death receptor CD95 is capped (confocal microscopy). Using different migration assays (wound healing/Boyden Chamber/endothelial transmigration), we uncover that cl-CD95L promotes cell migration through a c-yes/Ca²⁺/PI3K-driven signaling pathway, which relies on the formation of a CD95-containing complex designated the MISC for Motility-Inducing Signaling Complex. These findings revisit the role of the metalloprotease-cleaved CD95L and emphasize that the increase in cl-CD95L observed in patients affected by chronic inflammatory disorders may fuel the local or systemic tissue damage by promoting tissue-filtration of immune cells. The “death receptor” CD95 (also known as Fas) plays an essential role in ensuring immune tolerance of self antigens as well as in the elimination of the body's cells that have been infected or transformed. This receptor is engaged by the membrane-bound ligand CD95L, which can be released into blood circulation after cleavage by metalloproteases. Hitherto, most of the studies on the CD95 signal have been performed with chimeric CD95Ls that mimic the membrane-bound ligand and exhibit a level of aggregation beyond that described for the metalloprotease-cleaved ligand. Multi-aggregated CD95L elicits a caspase-driven apoptotic signal. In this study, we observe that levels of soluble and naturally processed CD95L in sera of patients suffering from lupus correlate with disease severity. Strikingly, although this soluble CD95L fails to trigger cell death unlike its chimeric version, it induces a “non-canonical” Ca²⁺/c-yes/PI3K-dependent signaling pathway that promotes the transmigration of T-lymphocytes across the endothelial barrier. These findings shed light on an entirely new role for the soluble CD95L that may contribute to local or systemic tissue damage by enhancing the infiltration of activated T-lymphocytes. Overall, these findings underline the importance of revisiting the role of this “apoptotic cytokine” in the context of chronic inflammatory disorders.

Triple-negative breast cancers (TNBC) lacking estrogen and progesterone receptors and HER2 amplification have a relatively high risk of metastatic dissemination, but the mechanistic basis for this risk is not understood. Here, we report that serum levels of CD95 ligand (CD95L) are higher in patients with TNBC than in other patients with breast cancer. Metalloprotease-mediated cleavage of CD95L expressed by endothelial cells surrounding tumors generates a gradient that promotes cell motility due to the formation of an unconventional CD95-containing receptosome called the motility-inducing signaling complex. The formation of this complex was instrumental for Nox3-driven reactive oxygen species generation. Mechanistic investigations revealed a Yes–Orai1–EGFR–PI3K pathway that triggered migration of TNBC cells exposed to CD95L. Our findings establish a prometastatic function for metalloprotease-cleaved CD95L in TNBCs, revisiting its role in carcinogenesis. Cancer Res; 73(22); 6711–21. ©2013 AACR.

CD95 type I and II cells differ in their dependence on mitochondria to execute apoptosis, because antiapoptotic members of the Bcl-2 family render only type II cells resistant to death receptor-induced apoptosis. They can also be distinguished by a more efficient formation of the death-inducing signaling complex in type I cells. We have identified a soluble form of CD95 ligand (S2) that is cytotoxic to type II cells but does not kill type I cells. By testing 58 tumor cell lines of the National Cancer Institute's anticancer drug-screening panel for apoptosis sensitivity to S2 and performing death-inducing signaling complex analyses, we determined that half of the CD95-sensitive cells are type I and half are type II. Most of the type I cell lines fall into a distinct class of tumor cells expressing mesenchymal-like genes, whereas the type II cell lines preferentially express epithelium-like markers. This suggests that type I and II tumor cells represent different stages of carcinogenesis that resemble the epithelial–mesenchymal transition. We then screened the National Cancer Institute database of >42,000 compounds for reagents with patterns of growth inhibition that correlated with either type I or type II cell lines and found that actin-binding compounds selectively inhibited growth of type I cells, whereas tubulin-interacting compounds inhibited growth of type II cells. Our analysis reveals fundamental differences in programs of gene expression between type I and type II cells and could impact the way actin- and microtubule-disrupting antitumor agents are used in tumor therapy.