Regulation of cellular quiescence by YAP/TAZ and Cyclin E1 in colon cancer cells: Implication in chemoresistance and cancer relapse.

Our aim was to decipher the role and clinical relevance of the YAP/TAZ transcriptional coactivators in the regulation of the proliferation/quiescence balance in human colon cancer cells (CCC) and survival after 5FU-based chemotherapy. The prognostic value of YAP/TAZ on tumor relapse and overall survival was assessed in a five-year follow-up study using specimens of liver metastases (n = 70) from colon cancer patients. In 5FU-chemoresistant HT29-5F31 and -chemosensitive HCT116 and RKO CCC, a reversible G0 quiescent state mediated by Cyclin E1 down-regulation was induced by 5FU in 5F31 cells and recapitulated in CCC by either YAP/TAZ or Cyclin E1 siRNAs or the YAP inhibitor Verteporfin. Conversely, the constitutive active YAPdc-S127A mutant restricted cellular quiescence in 5FU-treated 5F31 cells and sustained high Cyclin E1 levels through CREB Ser-133 phosphorylation and activation. In colon cancer patients, high YAP/TAZ level in residual liver metastases correlated with the proliferation marker Ki-67 (p < 0.0001), high level of the YAP target CTGF (p = 0.01), shorter disease-free and overall survival (p = 0.008 and 0.04, respectively). By multivariate analysis and Cox regression model, the YAP/TAZ level was an independent factor of overall (Hazard ratio [CI 95%] 2.06 (1.02-4.16) p = 0.045) and disease-free survival (Hazard ratio [CI 95%] 1.98 (1.01-3.86) p = 0.045). Thus, YAP/ TAZ pathways contribute to the proliferation/quiescence switch during 5FU treatment according to the concerted regulation of Cyclin E1 and CREB. These findings provide a rationale for therapeutic interventions targeting these transcriptional regulators in patients with residual chemoresistant liver metastases expressing high YAP/TAZ levels.

Melanoma dormancy in a mouse model is linked to GILZ/FOXO3A-dependent quiescence of disseminated stem-like cells.

Metastatic cancer relapses following the reactivation of dormant, disseminated tumour cells; however, the cells and factors involved in this reactivation are just beginning to be identified. Using an immunotherapy-based syngeneic model of melanoma dormancy and GFP-labelled dormant cell-derived cell lines, we determined that vaccination against melanoma prevented tumour growth but did not prevent tumour cell dissemination or eliminate all tumour cells. The persistent disseminated melanoma tumour cells were quiescent and asymptomatic for one year. The quiescence/activation of these cells in vitro and the dormancy of melanoma in vivo appeared to be regulated by glucocorticoid-induced leucine zipper (GILZ)-mediated immunosuppression. GILZ expression was low in dormant cell-derived cultures, and re-expression of GILZ inactivated FOXO3A and its downstream target, p21CIP1. The ability of dormancy-competent cells to re-enter the cell cycle increased after a second round of cellular dormancy in vivo in association with shortened tumour dormancy period and faster and more aggressive melanoma relapse. Our data indicate that future cancer treatments should be adjusted according to the stage of disease progression.

[Reply to « Why is it difficult to implement biomedical policy? The ase of the Cancéropôles, the French cancer organizations ¼, by Audrey Vézian, Med Sci (Paris) 2014 Aug-Sep; 30 (8-9) : 803-7]. / Contribution des responsables des Cancéropôles au Forum/Débat sur le bilan des actions des Cancéropôles - Répartie à l'analyse proposée par Audrey Vézian.

The authors, all in charge of the administration of one of the 7 French Cancéropôles, reply to the article authored by Audrey Vézian, and -provide an alternative and more supportive view of the initiatives -sponsored by these regional cancer research networks.

Transient TNF regulates the self-renewing capacity of stem-like label-retaining cells in sphere and skin equivalent models of melanoma.

BACKGROUND: It is well established that inflammation promotes cancer, including melanoma, although the exact mechanisms involved are less known. In this study, we tested the hypothesis that inflammatory factors affect the cancer stem cell (CSC) compartment responsible for tumor development and relapse. RESULTS: Using an inducible histone 2B-GFP fusion protein as a tracer of cell divisional history, we determined that tumor necrosis factor (TNF), which is a classical pro-inflammatory cytokine, enlarged the CSC pool of GFP-positive label-retaining cells (LRCs) in tumor-like melanospheres. Although these cells acquired melanoma stem cell markers, including ABCB5 and CD271, and self-renewal ability, they lost their capacity to differentiate, as evidenced by the diminished MelanA expression in melanosphere cells and the loss of pigmentation in a skin equivalent model of human melanoma. The undifferentiated cell phenotype could be reversed by LY294002, which is an inhibitor of the PI3K/AKT signaling pathway, and this reversal was accompanied by a significant reduction in CSC phenotypic markers and functional properties. Importantly, the changes induced by a transient exposure to TNF were long-lasting and observed for many generations after TNF withdrawal. CONCLUSIONS: We conclude that pro-inflammatory TNF targets the quiescent/slow-cycling melanoma SC compartment and promotes PI3K/AKT-driven expansion of melanoma SCs most likely by preventing their asymmetrical self-renewal. This TNF effect is maintained and transferred to descendants of LRC CSCs and is manifested in the absence of TNF, suggesting that a transient exposure to inflammatory factors imprints long-lasting molecular and/or cellular changes with functional consequences long after inflammatory signal suppression. Clinically, these results may translate into an inflammation-triggered accumulation of quiescent/slow-cycling CSCs and a post-inflammatory onset of an aggressive tumor.

Another facet to the anticancer response to lamellarin D: induction of cellular senescence through inhibition of topoisomerase I and intracellular Ros production.

Lamellarin D (LamD) is a marine alkaloid with broad spectrum antitumor activities. Multiple intracellular targets of LamD, which affect cancer cell growth and induce apoptosis, have been identified. These include nuclear topoisomerase I, relevant kinases (such as cyclin-dependent kinase 2) and the mitochondrial electron transport chain. While we have previously demonstrated that LamD at micromolar range deploys strong cytotoxicity by inducing mitochondrial apoptosis, mechanisms of its cytostatic effect have not yet been characterized. Here, we demonstrated that induction of cellular senescence (depicted by cell cycle arrest in G2 associated with ß-galactosidase activity) is a common response to subtoxic concentrations of LamD. Cellular senescence is observed in a large panel of cancer cells following in vitro or in vivo exposure to LamD. The onset of cellular senescence is dependent on the presence of intact topoisomerase I since topoisomerase I-mutated cells are resistant to senescence induced by LamD. LamD-induced senescence occurs without important loss of telomere integrity. Instead, incubation with LamD results in the production of intracellular reactive oxygen species (ROS), which are critical for senescence as demonstrated by the inhibitory effect of antioxidants. In addition, cancer cells lacking mitochondrial DNA also exhibit cellular senescence upon LamD exposure indicating that LamD can trigger senescence, unlike apoptosis, in the absence of functional mitochondria. Overall, our results identify senescence-associated growth arrest as a powerful effect of LamD and add compelling evidence for the pharmacological interest of lamellarins as potential anticancer agents.

Insulin-like growth factor 1 receptor and p38 mitogen-activated protein kinase signals inversely regulate signal transducer and activator of transcription 3 activity to control human dental pulp stem cell quiescence, propagation, and differentiation.

Dental pulp stem cells (DPSCs) remain quiescent until activated in response to severe dental pulp damage. Once activated, they exit quiescence and enter regenerative odontogenesis, producing reparative dentin. The factors and signaling molecules that control the quiescence/activation and commitment to differentiation of human DPSCs are not known. In this study, we determined that the inhibition of insulin-like growth factor 1 receptor (IGF-1R) and p38 mitogen-activated protein kinase (p38 MAPK) signaling commonly activates DPSCs and promotes their exit from the G0 phase of the cell cycle as well as from the pyronin Y(low) stem cell compartment. The inhibition of these two pathways, however, inversely determines DPSC fate. In contrast to p38 MAPK inhibitors, IGF-1R inhibitors enhance dental pulp cell sphere-forming capacity and reduce the cells' colony-forming capacity without inducing cell death. The inverse cellular changes initiated by IGF-1R and p38 MAPK inhibitors were accompanied by inverse changes in the levels of active signal transducer and activator of transcription 3 (STAT3) factor, inactive glycogen synthase kinase 3, and matrix extracellular phosphoglycoprotein, a marker of early odontoblast differentiation. Our data suggest that there is cross talk between the IGF-1R and p38 MAPK signaling pathways in DPSCs and that the signals provided by these pathways converge at STAT3 and inversely regulate its activity to maintain quiescence or to promote self-renewal and differentiation of the cells. We propose a working model that explains the possible interactions between IGF-1R and p38 MAPK at the molecular level and describes the cellular consequences of these interactions. This model may inspire further fundamental study and stimulate research on the clinical applications of DPSC in cellular therapy and tissue regeneration.

A structural explanation of the effects of dissociated glucocorticoids on glucocorticoid receptor transactivation.

There is a therapeutic need for glucocorticoid receptor (GR) ligands that distinguish between the transrepression and transactivation activity of the GR, the later thought to be responsible for side effects. These ligands are known as "dissociated glucocorticoids" (dGCs). The first published dGCs, RU24782 (9α-fluoro-11ß-hydroxy-16α-methylpregna-21-thiomethyl-1,4-diene-3,20-dione) and RU24858 (9α-fluoro-11ß-hydroxy-16α-methylpregna-21-cyanide-1,4-diene-3,20-dione), do not have the 17α-hydroxyl group that characterizes dexamethasone (Dex; 9α-fluoro-11ß,17α,21-trihydroxy-16α-methylpregna-1,4-diene-3,20-dione), and they differ from one another by having C21-thiomethyl and C21-cyanide moieties, respectively. Our aim was therefore to establish the structural basis of their activity. Both RU24782 and RU24858 induced a transactivation activity highly dependent on the GR expression level but always lower than dexamethasone. They also display less ability than dexamethasone to trigger steroid receptor coactivator 1 (SRC-1) recruitment and histone H3 acetylation. Docking studies, validated by mutagenesis experiments, revealed that dGCs are not anchored by Gln642, in contrast to Dex, which is hydrogen bonded to this residue via its 17α-hydroxyl group. This contact is essential for SRC-1 recruitment and subsequent dexamethasone-induced GR transactivation, but not transrepression. The ability of dGCs to make contacts with Ile747, for both RU24858 and RU24782 and with Asn564 for RU24858 are not strong enough to maintain GR in a conformation able to efficiently recruit SRC-1, unless SRC-1 is overexpressed. Overall, our findings provide some structural guidelines for the synthesis of potential new dissociated glucocorticoids with a better therapeutic ratio.

Colon cancer cells escape 5FU chemotherapy-induced cell death by entering stemness and quiescence associated with the c-Yes/YAP axis.

PURPOSE: Metastasis and drug resistance are the major limitations in the survival and management of patients with cancer. This study aimed to identify the mechanisms underlying HT29 colon cancer cell chemoresistance acquired after sequential exposure to 5-fluorouracil (5FU), a classical anticancer drug for treatment of epithelial solid tumors. We examined its clinical relevance in a cohort of patients with colon cancer with liver metastases after 5FU-based neoadjuvant chemotherapy and surgery. RESULTS: We show that a clonal 5F31 cell population, resistant to 1 µmol/L 5FU, express a typical cancer stem cell-like phenotype and enter into a reversible quiescent G0 state upon reexposure to higher 5FU concentrations. These quiescent cells overexpressed the tyrosine kinase c-Yes that became activated and membrane-associated upon 5FU exposure. This enhanced signaling pathway induced the dissociation of the Yes/YAP (Yes-associated protein) molecular complex and depleted nuclear YAP levels. Consistently, YES1 silencing decreased nuclear YAP accumulation and induced cellular quiescence in 5F31 cells cultured in 5FU-free medium. Importantly, YES1 and YAP transcript levels were higher in liver metastases of patients with colon cancer after 5FU-based neoadjuvant chemotherapy. Moreover, the YES1 and YAP transcript levels positively correlated with colon cancer relapse and shorter patient survival (P < 0.05 and P < 0.025, respectively). CONCLUSIONS: We identified c-Yes and YAP as potential molecular targets to eradicate quiescent cancer cells and dormant micrometastases during 5FU chemotherapy and resistance and as predictive survival markers for colon cancer.

Mitochondrial oxidative stress is the Achille's heel of melanoma cells resistant to Braf-mutant inhibitor.

Vemurafenib/PLX4032, a selective inhibitor of mutant BRAFV600E, constitutes a paradigm shift in melanoma therapy. Unfortunately, acquired resistance, which unavoidably occurs, represents one major limitation to clinical responses. Recent studies have highlighted that vemurafenib activated oxidative metabolism in BRAFV600E melanomas expressing PGC1α. However, the oxidative state of melanoma resistant to BRAF inhibitors is unknown. We established representative in vitro and in vivo models of human melanoma resistant to vemurafenib including primary specimens derived from melanoma patients. Firstly, our study reveals that vemurafenib increased mitochondrial respiration and ROS production in BRAFV600E melanoma cell lines regardless the expression of PGC1α. Secondly, melanoma cells that have acquired resistance to vemurafenib displayed intrinsically high rates of mitochondrial respiration associated with elevated mitochondrial oxidative stress irrespective of the presence of vemurafenib. Thirdly, the elevated ROS level rendered vemurafenib-resistant melanoma cells prone to cell death induced by pro-oxidants including the clinical trial drug, elesclomol. Based on these observations, we propose that the mitochondrial oxidative signature of resistant melanoma constitutes a novel opportunity to overcome resistance to BRAF inhibition.

Patient-derived tumor xenograft model to guide the use of BRAF inhibitors in metastatic melanoma.

Recently, the BRAF V600 inhibitor, vemurafenib, has revolutionized the therapeutic management of metastatic melanoma. However, adverse effects and the onset of resistance are frequently observed, limiting the efficacy of this treatment. Patient-derived tumor xenografts (PDTX) engrafted in immunocompromised mice have been proposed as valuable preclinical models that can predict clinical response to treatments. Here, we established a PDTX model of BRAF V600E melanoma useful for testing the efficacy of vemurafenib. First, we validated the stability of the model that was similar to the original tumor with respect to histology, immunohistochemistry, mutational status, and fluorine-18 fluorodeoxyglucose ([(18)F]FDG)-PET/computed tomography (CT). Next, the sensitivity of the xenografts to vemurafenib was determined by tumor growth inhibition and decreased in standardized uptake value on [(18)F]FDG-PET/CT. Finally, this result, using personalized PDTX, allowed successful rechallenge with vemurafenib in a patient who was administered a lower dose of vemurafenib because of the onset of adverse events. Overall, we found that PDTX provides 'real-time' results in an animal that phenocopies the biology and expected vemurafenib responses of the tumor in a patient with BRAF V600E melanoma. Thus, this 'coclinical' trial using PDTX can help guide vemurafenib treatment for metastatic melanoma.

The PI3K/AKT signaling pathway controls the quiescence of the low-Rhodamine123-retention cell compartment enriched for melanoma stem cell activity.

Melanoma is one of the most aggressive and extremely resistant to conventional therapies neoplasms. Recently, cellular resistance was linked to the cancer stem cell phenotype, still controversial and not well-defined. In this study, we used a Rhodamine 123 (Rh123) exclusion assay to functionally identify stem-like cells in metastatic human melanomas and melanoma cell lines. We demonstrate that a small subset of Rh123-low-retention (Rh123(low)) cells is enriched for stem cell-like activities, including the ability to self-renew and produce nonstem Rh123(high) progeny and to form melanospheres, recapitulating the phenotypic profile of the parental tumor. Rh123(low) cells are relatively quiescent and chemoresistant. At the molecular level, we show that melanoma Rh123(low) cells overexpress HIF1α, pluripotency factor OCT4, and the ABCB5 marker of melanoma stem cells and downregulate the expression of Cyclin D1 and CDK4. Interestingly, a short treatment with LY294002, an inhibitor of the PI3K/AKT pathway, specifically reverts a subset of Rh123(high) cells to the Rh123(low) phenotype, whereas treatment with inhibitors of mammalian target of rapamycin, phosphatase and tensin homolog or mitogen-activated protein kinase signaling does not. This phenotypic switching was associated with reduced levels of the HIF1α transcript and an increase in the level of phosphorylated nuclear FOXO3a preferentially in Rh123(low) cells. Moreover, the Rh123(low) cells became less quiescent and displayed a significant increase in their melanosphere-forming ability. All the above indicates that the Rh123(low) melanoma stem cell pool is composed of cycling and quiescent cells and that the PI3K/AKT signaling while maintaining the quiescence of Rh123(low) G0 cells promotes the exit of cycling cells from the stem cell compartment.

Inactivation of the HIF-1α/PDK3 signaling axis drives melanoma toward mitochondrial oxidative metabolism and potentiates the therapeutic activity of pro-oxidants.

Cancer cells can undergo a metabolic reprogramming from oxidative phosphorylation to glycolysis that allows them to adapt to nutrient-poor microenvironments, thereby imposing a selection for aggressive variants. However, the mechanisms underlying this reprogramming are not fully understood. Using complementary approaches in validated cell lines and freshly obtained human specimens, we report here that mitochondrial respiration and oxidative phosphorylation are slowed in metastatic melanomas, even under normoxic conditions due to the persistence of a high nuclear expression of hypoxia-inducible factor-1α (HIF-1α). Pharmacologic or genetic blockades of the HIF-1α pathway decreased glycolysis and promoted mitochondrial respiration via specific reduction in the expression of pyruvate dehydrogenase kinase-3 (PDK3). Inhibiting PDK3 activity by dichloroacetate (DCA) or siRNA-mediated attenuation was sufficient to increase pyruvate dehydrogenase activity, oxidative phosphorylation, and mitochondrial reactive oxygen species generation. Notably, DCA potentiated the antitumor effects of elesclomol, a pro-oxidative drug currently in clinical development, both by limiting cell proliferation and promoting cell death. Interestingly, this combination was also effective against BRAF V600E-mutant melanoma cells that were resistant to the BRAF inhibitor vemurafenib. Cotreatment of melanomas with DCA and elesclomol in vivo achieved a more durable response than single agent alone. Our findings offer a preclinical validation of the HIF-1/PDK3 bioenergetic pathway as a new target for therapeutic intervention in metastatic melanoma, opening the door to innovative combinations that might eradicate this disease.

Regulation by survivin of cancer cell death induced by F14512, a polyamine-containing inhibitor of DNA topoisomerase II.

F14512, an epipodophyllotoxin derivative equipped with a spermine moiety, is selectively taken up by the polyamine transport system over-active in tumor cells. F14512 was identified as a selective anticancer agent with a broad spectrum of antitumor activities and is currently undergoing phase I clinical trial in onco-hematology. However, the mechanism by which F14512 exerts its selective effects on neoplastic cells remains poorly understood. In this study, using mainly P388 leukemia cells, we showed that activation of the DNA damage response by F14512 did not induce immediate apoptosis but resulted in an early growth arrest. F14512-induced G2 arrest was accompanied by the appearance of a senescence-like phenotype (characterized by an increased ß-galactosidase staining) with up-regulation of the cyclin-dependent kinase inhibitor p16, and cyclin D1. The early senescence-based cell cycle block was characterized by a marked increase of the level of the IAP protein survivin, but not cIAP2, in P388 cells as well as in three other leukemia and melanoma cell types. The Thr(34)-phosphorylated form of survivin was observed within 4 h after F14512 exposure. Inhibition of survivin by siRNA resulted in a switch from senescence-like growth arrest to apoptosis. Compared with the parental drug etoposide, F14512-induced DNA damage signaling pathway resulted in greater senescence like-growth arrest and delayed apoptosis. Collectively, our data show that senescence arrest and subsequent apoptosis are powerful mechanisms mediating the chemotherapeutic effects of F14512 and identify survivin as the molecular determinant responsible for a qualitative shift in cell fate from senescence to apoptosis upon treatment with F14512.

Exploiting mitochondrial dysfunction for effective elimination of imatinib-resistant leukemic cells.

Challenges today concern chronic myeloid leukemia (CML) patients resistant to imatinib. There is growing evidence that imatinib-resistant leukemic cells present abnormal glucose metabolism but the impact on mitochondria has been neglected. Our work aimed to better understand and exploit the metabolic alterations of imatinib-resistant leukemic cells. Imatinib-resistant cells presented high glycolysis as compared to sensitive cells. Consistently, expression of key glycolytic enzymes, at least partly mediated by HIF-1α, was modified in imatinib-resistant cells suggesting that imatinib-resistant cells uncouple glycolytic flux from pyruvate oxidation. Interestingly, mitochondria of imatinib-resistant cells exhibited accumulation of TCA cycle intermediates, increased NADH and low oxygen consumption. These mitochondrial alterations due to the partial failure of ETC were further confirmed in leukemic cells isolated from some imatinib-resistant CML patients. As a consequence, mitochondria generated more ROS than those of imatinib-sensitive cells. This, in turn, resulted in increased death of imatinib-resistant leukemic cells following in vitro or in vivo treatment with the pro-oxidants, PEITC and Trisenox, in a syngeneic mouse tumor model. Conversely, inhibition of glycolysis caused derepression of respiration leading to lower cellular ROS. In conclusion, these findings indicate that imatinib-resistant leukemic cells have an unexpected mitochondrial dysfunction that could be exploited for selective therapeutic intervention.

Macromolecular synthesis inhibitors perturb glucocorticoid receptor trafficking.

The ability of inhibitors of transcription and translation to prevent glucocorticoid-induced apoptosis has been interpreted to indicate that the cell death machinery requires de novo protein synthesis. The transcriptional inhibitors actinomycin D (Act D) and DRB as well as the translational inhibitors CHX and puromycin inhibited early loss of mitochondrial membrane integrity in a dose-dependent manner. This effect was not observed with the transcriptional inhibitor α-amanitin suggesting they may have additional effects. Their role in the glucocorticoid receptor (GR) intracellular trafficking was therefore investigated. Here, we show that Act D and CHX reduced glucocorticoid binding, GR turnover and impaired GR nuclear translocation. We performed the same experiments in different thymocyte subpopulations of Balb/c mice. At the highest dose tested, actinomycin D and cycloheximide abolished glucocorticoid-induced cell death of CD4+CD8+ and CD4+CD8-. In all subsets, Act D, DRB, as well as CHX and puromycin prevented receptor nuclear translocation, indicating a general alteration of GR trafficking. Overall, our data support a direct effect of macromolecular inhibitors on GR activation and trafficking. Finally, direct alterations of the functional properties of the glucocorticoid receptor might be responsible for cell death prevention by actinomycin D, DRB, cycloheximide and puromycin.

Human umbilical cord blood cells form epidermis in the skin equivalent model.

Recently, human embryonic stem cells have been differentiated in vitro into functional epidermal keratinocytes. Here, we demonstrated that these cells can be generated also from non-embryonic, human umbilical cord blood (hUCB) cells that have the potential to differentiate into cells of non-hematopoietic lineage. Human UCB mono-nucleated cells were cultivated in monolayer and in three-dimensional skin equivalent cultures and assayed for the presence of phenotype-specific markers. Our results determined that after one month of culturing in serum containing medium, the hUCB cells produced morphologically homogeneous colonies of epithelial cells expressing keratinocyte-specific markers. They also formed stratified epidermis in organ cultures that contained sporadic CD1a-positive cells within the accurate strata. We concluded that hUCB cells have the capacity to differentiate into functional epidermal keratinocytes and may serve as a source of high-quality keratinocytes for clinical applications.

Inhibition of mitochondrial respiration mediates apoptosis induced by the anti-tumoral alkaloid lamellarin D.

Lamellarin D (Lam D), a marine alkaloid, exhibits a potent cytotoxicity against many different tumors. The pro-apoptotic function of Lam D has been attributed to its direct induction of mitochondrial permeability transition (MPT). This study was undertaken to explore the mechanisms through which Lam D promotes changes in mitochondrial function and as a result apoptosis. The use of eight Lam derivatives provides useful structure-apoptosis relationships. We demonstrate that Lam D and structural analogues induce apoptosis of cancer cells by acting directly on mitochondria inducing reduction of mitochondrial membrane potential, swelling and cytochrome c release. Cyclosporin A, a well-known inhibitor of MPT, completely prevents mitochondrial signs of apoptosis. The drug decreases calcium uptake by mitochondria but not by microsomes indicating that Lam D-dependent permeability is specific to mitochondrial membranes. In addition, upon Lam D exposure, a rapid decline of mitochondrial respiration and ATP synthesis occurs in isolated mitochondria as well as in intact cells. Evaluation of the site of action of Lam D on the electron-transport chain revealed that the activity of respiratory chain complex III is reduced by a half. To determine whether Lam D could induce MPT-dependent apoptosis by inhibiting mitochondrial respiration, we generated respiration-deficient cells (rho0) derived from human melanoma cells. In comparison to parental cells, rho0 cells are totally resistant to the induction of MPT-dependent apoptosis by Lam D. Our results indicate that functional mitochondria are required for Lam D-induced apoptosis. Inhibition of mitochondrial respiration is responsible for MPT-dependent apoptosis of cancer cells induced by Lam-D.

Asymmetric distribution of epidermal growth factor receptor directs the fate of normal and cancer keratinocytes in vitro.

Cancer cells are unequal in a tumor mass and in established cultures. This is attributable to cancer stem cells with the unique ability to self-renew and to generate differentiating progeny. This ability is controlled at the level of asymmetric division by mechanisms that are yet not well defined. We found that normal and cancer keratinocyte fate was linked to the asymmetric distribution of epidermal growth factor receptor (EGFR) during mitosis. Although essential for epithelial cell proliferation, differentiation, and survival, this receptor was not present on the surface of cells satisfying criteria for stem cells such as quiescence, competence to produce functionally distinct daughters, high proliferative and clonogenic potential, sphere formation ability, and expression of stem cell markers. In contrast, keratinocytes displaying EGFR acquired a more differentiated phenotype, suggesting that EGFR may be involved in a switch from stem to transient amplifying cell fate. This switch was associated with changes in the expression profile of cell cycle, survival, and mitochondria controlling proteins that varied between normal and cancer cells. In conclusion, it appears that an unequal distribution of EGFR at mitosis controls keratinocyte fate by balancing quiescence and cycling of EGFR(-) cells, clearly malfunctioning in cancer. We believe that our findings provide mechanistic insights into the development of resistance to anti-EGFR therapies.

Essential role of mitochondria in apoptosis of cancer cells induced by the marine alkaloid Lamellarin D.

Lamellarin D, a potent cytotoxic marine alkaloid, exerts its antitumor action through two complementary pathways: a nuclear route via topoisomerase I inhibition and a mitochondrial targeting. The present study was designed to investigate the contribution of these two pathways for apoptosis in cancer cells. Lamellarin D promoted nuclear apoptosis in leukemia cells without prominent cell cycle arrest. Signals transmitted by lamellarin D initiated apoptosis via the intrinsic apoptotic pathway. The drug induced conformational activation of Bax and decreased the expression levels of antiapoptotic proteins Bcl-2 and cIAP2 in association with activation of caspase-9 and caspase-3. Upon lamellarin D exposure, Fas and Fas-L expression was not modified in leukemia cells. Moreover, leukemia cells deficient in caspase-8 or Fas-associated protein with death domain underwent apoptosis through the typical mitochondrial apoptotic cascade, indicating that cell death induced by lamellarin D was independent of the extrinsic apoptotic pathway. Lamellarin D also exerted a topoisomerase I-mediated DNA damage response resulting in H2AX phosphorylation, and the upregulation of the DNA repair protein Rad51 and of p53, as well as the phosphorylation of p53 at serine 15. However, lamellarin D killed efficiently mutated p53 or p53 null cancer cells, and sensitivity to lamellarin D was abrogated neither by cycloheximide nor in enucleated cells. Lamellarin D-induced cytochrome c release occurs independently of nuclear factors in a cell-free system. These results suggest that lamellarin D exerts its cytotoxic effects primarily by inducing mitochondrial apoptosis independently of nuclear signaling. Thus, lamellarin D constitutes a new proapoptotic agent that may bypass certain forms of apoptosis resistance that occur in tumor cells.

Apoptosis-related mitochondrial dysfunction defines human monocyte-derived dendritic cells with impaired immuno-stimulatory capacities.

The death of dendritic cells (DCs) can potentially influence immune responses by affecting the duration of DC stimulation of lymphocytes. Here, we report that cultured mature monocyte-derived DCs manifest early mitochondrial damage (i.e. within 24 hrs), characterized by mitochondrial membrane potential (psi Delta m) disruption and mitochondrial release of pro-apoptotic factors, followed by reactive oxygen species (ROS) production and activation of caspases. Afterwards, DCs with mitochondrial alterations are condemned to undergo apoptosis and necrosis. Macroarray analysis results (validated by real time quantitative-PCR (QRT-PCR) and immunoblotting), showed up-regulation of the pro-apoptotic member of the Bcl-2 family, Bim, while expression of several anti-apoptotic molecules was down-regulated. Importantly, pre-apoptotic DCs (characterized by a low Delta psi m) showed a modified phenotype, with down-regulation of HLA-DR and of the co-stimulatory molecules CD80 and CD86. Moreover, sorted viable low psi Delta m DCs were unable to activate allogeneic T cells, indicating that pre-apoptotic DCs have already lost some of their immuno-stimulatory capabilities long before any detectable signs of death occur. Perturbations to mitochondrial respiration with rotenone identified the same modifications to DC immune functions. These data indicate a strong requirement for mitochondrial integrity for the immuno-stimulatory capacities of DC. Determining Delta psi m could be a useful parameter to select 'fully' functional DCs for anti-tumour vaccines.