Philippe Juin

Bax activation and mitochondrial insertion during apoptosis.

The mitochondrial apoptotic pathway is a highly regulated biological mechanism which determines cell fate. It is defined as a cascade of events, going from an apoptotic stimulus to the MOM permeabilization, resulting in the activation of the so-called executive phase. This pathway is very often altered in cancer cells.The mitochondrial permeabilization is under the control of the Bcl-2 family of proteins (pBcls). These proteins share one to four homology domains (designed BH1-4) with Bcl-2, and are susceptible of homo- and/or hetero-dimerization. In spite of a poor amino-acid sequence homology, these proteins exhibit very similar tertiary structures. Strikingly, while some of these proteins are anti-apoptotic, the others are pro-apoptotic. Pro-apoptotic proteins are further divided in two sub-classes: multi-domains proteins, among which Bax and Bak, which exhibit BH1-3 domains, and BH3-only proteins (or BOPs). Schematically, BOPs and anti-apoptotic proteins antagonistically regulate the activation of the multi-domain proteins Bax and Bak and their oligomerization in the MOM, the latter process being responsible for the apoptotic mitochondrial permeabilization.Considering the critical role of Bax in cancer cells apoptosis, we focus in this review on the molecular events of Bax activation through its interaction with the other proteins from the Bcl-2 family. The mechanism by which Bax triggers the MOM permeabilization once activated will be discussed in some other reviews in this special issue.

Induction of a Caspase-3-like Activity by Calcium in Normal Cytosolic Extracts Triggers Nuclear Apoptosis in a Cell-free System

Calcium is involved in several steps of the apoptotic process. In nuclei, endonucleases are presumed to be the main targets of calcium; however, little is known about its role during the cytosolic phase of apoptosis. We used a cell-free system to address this question. Our results show that CaCl2 triggered nuclear apoptosis (i.e. typical morphological change and DNA fragmentation) at concentrations of 5 mm. This concentration was lowered 10-fold by the co-incubation with cytosolic extracts from nonapoptotic cells. Apoptotic changes induced by the incubation of nuclei with CaCl2 in the presence of these cytosols were strongly reduced in the presence of an inhibitor of caspase-3 and to a lesser extent by an inhibitor of caspase-1. We also show that calcium-induced apoptosis is affected by protease inhibitors such as N-tosyl-l-phenylalanine chloromethyl ketone, but not by calpain or several lysosomal protease inhibitors. The addition of CaCl2 to the cell-free system increased a caspase-3 activity in nonapoptotic cytosols as shown by specific antibodies and an enzymatic assay. No activation of a caspase-3-like activity by the addition of cytochrome c was observed in these extracts under similar conditions. The enhanced caspase-3 activity induced by calcium was inhibited by protease inhibitors affecting morphological nuclear apoptosis except for those responsible for the degradation of lamin A. These results suggest that CaCl2 could trigger, in normal cells, an apoptotic cascade through the activation of cytosolic caspase-3 activity.

Serum-Nutrient Starvation Induces Cell Death Mediated by Bax and Puma That Is Counteracted by p21 and Unmasked by Bcl-xL Inhibition

The cyclin-dependent kinase inhibitor p21 (p21WAF1/Cip1) is a multifunctional protein known to promote cell cycle arrest and survival in response to p53-dependent and p53 independent stimuli. We herein investigated whether and how it might contribute to the survival of cancer cells that are in low-nutrient conditions during tumour growth, by culturing isogenic human colorectal cancer cell lines (HCT116) and breast cancer cell lines in a medium deprived in amino acids and serum. We show that such starvation enhances, independently from p53, the expression of p21 and that of the pro-apoptotic BH3-only protein Puma. Under these conditions, p21 prevents Puma and its downstream effector Bax from triggering the mitochondrial apoptotic pathway. This anti-apoptotic effect is exerted from the cytosol but it is unrelated to the ability of p21 to interfere with the effector caspase 3. The survival function of p21 is, however, overcome by RNA interference mediated Bcl-xL depletion, or by the pharmacological inhibitor ABT-737. Thus, an insufficient supply in nutrients may not have an overt effect on cancer cell viability due to p21 induction, but it primes these cells to die, and sensitizes them to the deleterious effects of Bcl-xL inhibitors regardless of their p53 status.

Resistance to HER2 inhibitors: Is addition better than substitution? Rationale for the hypothetical concept of drug sedimentation

Twenty years were passed between the discovery of oncogene HER2, the description of its implication in mammary carcinogenesis, and the development of specific targeted therapies. To date, trastuzumab and lapatinib are the two anti-HER2 targeted therapies commonly used, demonstrating therapeutic effects. Although their clinical efficacy seems to be exclusively related to the amplification of the HER2 gene or to the overexpression of the protein, these factors are not sufficient since tumors can develop resistance. Because of a better knowledge in those mechanisms of resistance, novel therapeutic agents could help to bypass them. How should these be used with respect to current anti-HER2 targeted therapies? Recent notions such as oncogene addiction, tumor cell dormancy and residual disease led us to propose a new entity that we named the sedimentation strategy, in which distinct targeted approaches are summed during the treatment of metastatic breast cancer patients.

pRb/E2F-1-mediated caspase-dependent induction of Noxa amplifies the apoptotic effects of the Bcl-2/Bcl-xL inhibitor ABT-737

Although Bcl-2 family members control caspase activity by regulating mitochondrial permeability, caspases can, in turn, amplify the apoptotic process upstream of mitochondria by ill-characterized mechanisms. We herein show that treatment with a potent inhibitor of Bcl-2 and Bcl-xL, ABT-737, triggers caspase-dependent induction of the BH3-only protein, Mcl-1 inhibitor, Noxa. RNA interference experiments reveal that induction of Noxa, and subsequent cell death, rely not only on the transcription factor E2F-1 but also on its regulator pRb. In response to ABT-737, pRb is cleaved by caspases into a p68Rb form that still interacts with E2F-1. Moreover, pRb occupies the noxa promoter together with E2F-1, in a caspase-dependent manner upon ABT-737 treatment. Thus, caspases contribute to trigger the mitochondrial apoptotic pathway by coupling Bcl-2/Bcl-xL inhibition to that of Mcl-1, via the pRb/E2F-1-dependent induction of Noxa.

Impact of proapoptotic proteins Bax and Bak in tumor progression and response to treatment

The cell death program, apoptosis, is currently viewed as the ultimate obstacle in cancer therapy. Inhibition of apoptosis is thought to be involved in both tumorigenesis and resistance to chemo- and radiotherapy. Considerable efforts are underway to design new tools capable of overcoming this inhibition. In this review, the current understanding of mechanisms of apoptosis in normal and tumor cells as well as possible or existing strategies designed to induce specific and efficient cell death in cancers are summarized.

ABT-737 and/or folate reverse the PDGF-induced alterations in the mitochondrial apoptotic pathway in low-grade glioma patients

Elevated activation of the platelet-derived growth factor (PDGF) pathway, apoptosis evasion phenotype, and global DNA hypomethylation are hallmarks frequently observed in cancers, such as in low-grade glioma (LGG). However, the orchestration of these malignant functions is not fully elucidated in LGG. Our study reveals that the co-presence of these hallmarks in the same LGG is frequent and confers poor prognosis in patients with LGG. Our data also indicate that the apoptosis evasion phenotype of these cells harboring a hypomethylation-induced activation of the PDGF pathway is associated with a hypomethylation of the bcl-xl and bcl-w genes and the phosphorylation and/or downregulation of three major pro-apoptotic BH3-only proteins: PUMA, Bad, and Bim. Consistent with this, we demonstrate that the use of folate, a DNA-methylating agent, promotes the reprogramming of the sensitivity of glioma cells to ABT-737/etoposide-induced apoptosis and reduces the dose of ABT-737 required to promote etoposide-induced apoptosis. This work supports the idea that the inclusion of folate and/or ABT-737 could be a promising adjuvant in the design of anti-glioma therapeutic protocols in clinical studies.

Échappement tumoral aux inhibiteurs de HER2 : théorie de la sédimentation

Twenty years have passed between the discovery of oncogene HER2, the description of its implication in mammary carcinogenesis, and the development of specific targeted therapies. To date, trastuzumab and lapatinib are the two anti-HER2 targeted therapies commonly used, demonstrating therapeutic effects. Although their clinical efficacy seems to be exclusively related to the amplification of the HER2 gene or to the overexpression of the protein, these factors are not sufficient since tumors can develop resistance. Because of a better knowledge in those mechanisms of resistance, novel therapeutic agents could help to bypass them. How should these be used with respect to current anti-HER2 targeted therapies? Recent notions such as oncogene addiction, tumor cell dormancy and residual disease led us to propose a new entity that we named the sedimentation strategy, in which distinct targeted approaches are summed during the treatment of metastatic breast cancer patients.

mTORC1 Activity Contributes to the Mcl-1 Dependence of HER2 Amplified Breast Cancer Cells.

Whether or not certain subtypes of mammary tumors specifically rely on a limited number of anti-apoptotic mechanisms for their maintenance, and how therapy might affect these mechanisms, is currently ill characterized.We have analyzed published gene-expression profiles of breast cancer patients for which HER2 status, evaluated by immunohistochemistry, was also available. A statistically significant enrichment in the expression of Mcl-1 in HER2 amplified breast tumors, compared to other breast tumors, was found.In vitro, knock down of Mcl-1 expression by RNA interference proved sufficient to induce apoptosis in the HER2 amplified cell line BT474. Combined RNA interference experiments showed that apoptosis induced by Mcl-1 knock down in these cells (ie, their “Mcl-1 dependence”) relied on the expression of the pro-apoptotic BH3-only protein Bim. This indicates that the Bim/Mcl-1 balance (possibly regulated by direct physical interactions between these two proteins) constitutes an integrating point towards which the numerous survival pathways that are known to lie downstream of HER2 should converge, to the very least in these cells.While seeking for signaling pathways that might significantly affect Mcl-1 dependence downstream of HER2, using diverse small molecule inhibitors, we found that mTORC1 inhibition in BT474 cells not only leaves Mcl-1 expression levels unaltered, but also prevents induction of apoptosis by Mcl-1 depletion. mTORC1 inhibition enhances the activity of the survival kinases Akt and ERK (p44 and p42). HER2 signaling is involved in this activation, since it is prevented by trastuzumab treatment.These data suggest that, due to the structure of the signaling network mTORC1 is embedded in (which includes the HER2 driven negative feedback loop described here), its activity locks HER2 amplified cells in a Mcl-1 dependent state. They also imply that combining inhibitors of mTORC1 with inhibitors of the HER2 pathway (such as trastuzumab) might restrain the effects on cell survival of the former, and enhance their clinical efficiency in HER2 amplified breast cancers. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2161.

Blockade of Notch Signaling by GSI Induces Apoptosis in Human Breast Cancer Cells through Various Molecular Mechanisms Involving Survivin and BH3-Only Members of the Bcl-2 Family.

The Notch pathway is multifunctional and controls key nodes in cell signaling. In most cases, its deregulation has oncogenic effects, and aberrant activation of Notch signaling has been evidenced in breast cancer (Stylianou S, CR 2006). In particular, a functional Notch pathway appears to be of critical importance in the maintenance of basal breast cancer (Lee CW, CR 2008, BCR 2008). Moreover, because Notch signaling is repressed by both ER (Rizzo P, CR 2008) and HER2 (Osipo C, Oncogene 2008) oncogenic pathways, its reactivation during tamoxifen or trastuzumab treatment may restrain the efficiency of these inhibitors. Thus, Notch signaling inhibitors, such as competitive inhibitors of the g-secretase, GSI, that prevent proteolytic processing of the Notch receptor and the activation of its intracellular domain, are being actively investigated for the treatment of breast cancer, even though the mechanisms that might underlie their effects are still poorly understood.We first observed that GSI XII (z-Ile-Leu-CHO) was more effective in inducing apoptosis, as a single agent, on ER-/PR-/Her2- breast cancer cell lines than GSI X (z-Leu-Leu-Nle-CHO). This effect was associated with decreased expression of the anti-apoptotic proteins Survivin and XIAP and increased expression of, depending on cell lines, either NOXA or PUMA, two pro-apoptotic BH3-only members of the Bcl2 family.In the opposite, ER+ breast cancer cell lines were more sensitive to induction of apoptosis by GSI X, but this was manifest only in combination with Tamoxifen. Interestingly, Tamoxifen treatment of these cells induced both GSI X-sensitive Notch transcriptional activity and Survivin expression. Moreover, Survivin knock-down by RNA interference increased Tamoxifen sensitivity. Thus, Survivin might be an important actor of survival signal triggered by Notch (re)activation during ER pathway inhibition. Our results corroborate previous data showing the interest to use GSI in breast cancer but underline also the need to unravel the sub-type and context dependent molecular events involved in their biological activity. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2160.