Targeting autophagy inhibits melanoma growth by enhancing NK cells infiltration in a CCL5-dependent manner.

While blocking tumor growth by targeting autophagy is well established, its role on the infiltration of natural killer (NK) cells into tumors remains unknown. Here, we investigate the impact of targeting autophagy gene Beclin1 (BECN1) on the infiltration of NK cells into melanomas. We show that, in addition to inhibiting tumor growth, targeting BECN1 increased the infiltration of functional NK cells into melanoma tumors. We provide evidence that driving NK cells to the tumor bed relied on the ability of autophagy-defective tumors to transcriptionally overexpress the chemokine gene CCL5 Such infiltration and tumor regression were abrogated by silencing CCL5 in BECN1-defective tumors. Mechanistically, we show that the up-regulated expression of CCL5 occurred through the activation of its transcription factor c-Jun by a mechanism involving the impairment of phosphatase PP2A catalytic activity and the subsequent activation of JNK. Similar to BECN1, targeting other autophagy genes, such as ATG5, p62/SQSTM1, or inhibiting autophagy pharmacologically by chloroquine, also induced the expression of CCL5 in melanoma cells. Clinically, a positive correlation between CCL5 and NK cell marker NKp46 expression was found in melanoma patients, and a high expression level of CCL5 was correlated with a significant improvement of melanoma patients' survival. We believe that this study highlights the impact of targeting autophagy on the tumor infiltration by NK cells and its benefit as a novel therapeutic approach to improve NK-based immunotherapy.

The Selective Degradation of Synaptic Connexin 43 Protein by Hypoxia-induced Autophagy Impairs Natural Killer Cell-mediated Tumor Cell Killing.

Although natural killer (NK) cells play an important role in the control of melanoma, hypoxic stress in the tumor microenvironment may impair NK-mediated tumor cell killing by mechanisms that are not fully understood. In this study, we investigated the effect of hypoxia on the expression and channel activity of connexin 43 (Cx43) in melanoma cells and its impact on their susceptibility to NK cell-mediated lysis. Our results demonstrated that hypoxic stress increases Cx43 expression in melanoma cells via hypoxia-inducible factor-1α (HIF-1α) transcriptional activity. Hypoxic cells displaying increased Cx43 expression were less susceptible to NK cell-mediated lysis compared with normoxic cells expressing a moderate level of Cx43. Conversely, when overexpressed in normoxic tumor cells, Cx43 improves their susceptibility to N cell-mediated killing. We show that the NK cell immune synapse formed with normoxic melanoma cells is more stable and contains a high level of gap-junctional Cx43 whereas that formed with hypoxic cells is less stable and contains a significant lower level of gap-junctional Cx43. We provide evidence that the activation of autophagy in hypoxic melanoma cells selectively degrades gap-junctional Cx43, leading to the destabilization of the immune synapse and the impairment of NK cell-mediated killing. Inhibition of autophagy by genetic or pharmacological approaches as well as expression of the non-degradable form of Cx43 significantly restore its accumulation at the immune synapse and improves N cell-mediated lysis of hypoxic melanoma cells. This study provides the first evidence that the hypoxic microenvironment negatively affects the immune surveillance of tumors by NK cells through the modulation of Cx43-mediated intercellular communications.

Granzyme B degradation by autophagy decreases tumor cell susceptibility to natural killer-mediated lysis under hypoxia.

Recent studies demonstrated that autophagy is an important regulator of innate immune response. However, the mechanism by which autophagy regulates natural killer (NK) cell-mediated antitumor immune responses remains elusive. Here, we demonstrate that hypoxia impairs breast cancer cell susceptibility to NK-mediated lysis in vitro via the activation of autophagy. This impairment was not related to a defect in target cell recognition by NK cells but to the degradation of NK-derived granzyme B in autophagosomes of hypoxic cells. Inhibition of autophagy by targeting beclin1 (BECN1) restored granzyme B levels in hypoxic cells in vitro and induced tumor regression in vivo by facilitating NK-mediated tumor cell killing. Together, our data highlight autophagy as a mechanism underlying the resistance of hypoxic tumor cells to NK-mediated lysis. The work presented here provides a cutting-edge advance in our understanding of the mechanism by which hypoxia-induced autophagy impairs NK-mediated lysis in vitro and paves the way for the formulation of more effective NK cell-based antitumor therapies.

MicroRNA as biomarkers and regulators in B-cell chronic lymphocytic leukemia.

Early cancer detection and disease stratification or classification are critical to successful treatment. Accessible, reliable, and informative cancer biomarkers can be medically valuable and can provide some relevant insights into cancer biology. Recent studies have suggested improvements in detecting malignancies by the use of specific extracellular microRNAs (miRNAs) in plasma. In chronic lymphocytic leukemia (CLL), an incurable hematologic disorder, sensitive, early, and noninvasive diagnosis and better disease classification would be very useful for more effective therapies. We show here that circulating miRNAs can be sensitive biomarkers for CLL, because certain extracellular miRNAs are present in CLL patient plasma at levels significantly different from healthy controls and from patients affected by other hematologic malignancies. The levels of several of these circulating miRNAs also displayed significant differences between zeta-associated protein 70 (ZAP-70)(+) and ZAP-70(-) CLL. We also determined that the level of circulating miR-20a correlates reliably with diagnosis-to-treatment time. Network analysis of our data, suggests a regulatory network associated with BCL2 and ZAP-70 expression in CLL. This hypothesis suggests the possibility of using the levels of specific miRNAs in plasma to detect CLL and to determine the ZAP-70 status.

Improving STING agonist-based cancer therapy by inhibiting the autophagy-related protein VPS34.

We have recently demonstrated that inhibiting VPS34 enhances T-cell-recruiting chemokines through the activation of the cGAS/STING pathway using the STING agonist ADU-S100. Combining VPS34 inhibitors with ADU-S100 increased cytokine release and improved tumor control in mouse models, suggesting a potential synergy between VPS34 inhibition and therapies based on STING agonists.

Combining VPS34 inhibitors with STING agonists enhances type I interferon signaling and anti-tumor efficacy.

An immunosuppressive tumor microenvironment promotes tumor growth and is one of the main factors limiting the response to cancer immunotherapy. We have previously reported that inhibition of vacuolar protein sorting 34 (VPS34), a crucial lipid kinase in the autophagy/endosomal trafficking pathway, decreases tumor growth in several cancer models, increases infiltration of immune cells and sensitizes tumors to anti-programmed cell death protein 1/programmed cell death 1 ligand 1 therapy by upregulation of C-C motif chemokine 5 (CCL5) and C-X-C motif chemokine 10 (CXCL10) chemokines. The purpose of this study was to investigate the signaling mechanism leading to the VPS34-dependent chemokine increase. NanoString gene expression analysis was applied to tumors from mice treated with the VPS34 inhibitor SB02024 to identify key pathways involved in the anti-tumor response. We showed that VPS34 inhibitors increased the secretion of T-cell-recruitment chemokines in a cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes protein (STING)-dependent manner in cancer cells. Both pharmacological and small interfering RNA (siRNA)-mediated VPS34 inhibition increased cGAS/STING-mediated expression and secretion of CCL5 and CXCL10. The combination of VPS34 inhibitor and STING agonist further induced cytokine release in both human and murine cancer cells as well as monocytic or dendritic innate immune cells. Finally, the VPS34 inhibitor SB02024 sensitized B16-F10 tumor-bearing mice to STING agonist treatment and significantly improved mice survival. These results show that VPS34 inhibition augments the cGAS/STING pathway, leading to greater tumor control through immune-mediated mechanisms. We propose that pharmacological VPS34 inhibition may synergize with emerging therapies targeting the cGAS/STING pathway.

SNAI1-dependent upregulation of CD73 increases extracellular adenosine release to mediate immune suppression in TNBC.

Triple-negative subtype of breast cancer (TNBC) is hallmarked by frequent disease relapse and shows highest mortality rate. Although PD-1/PD-L1 immune checkpoint blockades have recently shown promising clinical benefits, the overall response rate remains largely insufficient. Hence, alternative therapeutic approaches are warranted. Given the immunosuppressive properties of CD73-mediated adenosine release, CD73 blocking approaches are emerging as attractive strategies in cancer immunotherapy. Understanding the precise mechanism regulating the expression of CD73 is required to develop effective anti-CD73-based therapy. Our previous observations demonstrate that the transcription factors driving epithelial-to-mesenchymal transition (EMT-TF) can regulate the expression of several inhibitory immune checkpoints. Here we analyzed the role of the EMT-TF SNAI1 in the regulation of CD73 in TNBC cells. We found that doxycycline-driven SNAI1 expression in the epithelial -like TNBC cell line MDA-MB-468 results in CD73 upregulation by direct binding to the CD73 proximal promoter. SNAI1-dependent upregulation of CD73 leads to increased production and release of extracellular adenosine by TNBC cells and contributes to the enhancement of TNBC immunosuppressive properties. Our data are validated in TNBC samples by showing a positive correlation between the mRNA expression of CD73 and SNAI1. Overall, our results reveal a new CD73 regulation mechanism in TNBC that participates in TNBC-mediated immunosuppression and paves the way for developing new treatment opportunities for CD73-positive TNBC.

Hyal2 is a glycosylphosphatidylinositol-anchored, lipid raft-associated hyaluronidase.

The rapid turnover rate of hyaluronan (HA), the major unbranched glycosaminoglycan of the extracellular matrix, is dependent on hyaluronidases. One of them, hyaluronidase-2 (Hyal2), degrades HA into smaller fragments endowed with specific biological activities such as inflammation and angiogenesis. Yet the cellular environment of Hyal2, a purported glycosylphosphatidylinositol (GPI)-anchored protein, remains uncertain. We have examined the membrane association of Hyal2 in MDA-MB231 cancer cells where it is highly expressed and in COS-7 cells transfected with native or fluorescent Hyal2 constructs. In both cell types, Hyal2 was strongly associated with cell membrane fractions from which it could be extracted using a Triton X-114 treatment (hydrophobic phase) but not an osmotic shock or an alkaline carbonate solution. Treatment of membrane preparations with phosphatidylinositol-specific phospholipase C released immunoreactive Hyal2 into the aqueous phase, confirming the protein is attached to the membrane through a functional GPI anchor. Hyal2 transfected in COS-7 cells was associated with detergent-resistant, cholesterol-rich membranes known as lipid rafts. The cellular immunofluorescent pattern of Hyal2 was conditioned by the presence of a GPI anchor. In summary, the strong membrane association of Hyal2 through its GPI anchor demonstrated in this study using biochemical methods suggests that the main activity of this enzyme is located at the level of the plasma membrane in close contact with the pericellular HA-rich glycocalyx, the extracellular matrix, or possibly endocytic vesicles.

Targeting HIF-1 alpha transcriptional activity drives cytotoxic immune effector cells into melanoma and improves combination immunotherapy.

Hypoxia is a key factor responsible for the failure of therapeutic response in most solid tumors and promotes the acquisition of tumor resistance to various antitumor immune effectors. Reshaping the hypoxic immune suppressive tumor microenvironment to improve cancer immunotherapy is still a relevant challenge. We investigated the impact of inhibiting HIF-1α transcriptional activity on cytotoxic immune cell infiltration into B16-F10 melanoma. We showed that tumors expressing a deleted form of HIF-1α displayed increased levels of NK and CD8+ effector T cells in the tumor microenvironment, which was associated with high levels of CCL2 and CCL5 chemokines. We showed that combining acriflavine, reported as a pharmacological agent preventing HIF-1α/HIF-1ß dimerization, dramatically improved the benefit of cancer immunotherapy based on TRP-2 peptide vaccination and anti-PD-1 blocking antibody. In melanoma patients, we revealed that tumors exhibiting high CCL5 are less hypoxic, and displayed high NK, CD3+, CD4+ and CD8+ T cell markers than those having low CCL5. In addition, melanoma patients with high CCL5 in their tumors survive better than those having low CCL5. This study provides the pre-clinical proof of concept for a novel triple combination strategy including blocking HIF-1α transcription activity along vaccination and PD-1 blocking immunotherapy.

Determination of genes and microRNAs involved in the resistance to fludarabine in vivo in chronic lymphocytic leukemia.

BACKGROUND: Chronic lymphocytic leukemia (CLL) cells are often affected by genomic aberrations targeting key regulatory genes. Although fludarabine is the standard first line therapy to treat CLL, only few data are available about the resistance of B cells to this purine nucleoside analog in vivo. Here we sought to increase our understanding of fludarabine action and describe the mechanisms leading to resistance in vivo. We performed an analysis of genomic aberrations, gene expression profiles, and microRNAs expression in CLL blood B lymphocytes isolated during the course of patients' treatment with fludarabine. RESULTS: In sensitive patients, the differentially expressed genes we identified were mainly involved in p53 signaling, DNA damage response, cell cycle and cell death. In resistant patients, uncommon genomic abnormalities were observed and the resistance toward fludarabine could be characterized based on the expression profiles of genes implicated in lymphocyte proliferation, DNA repair, and cell growth and survival. Of particular interest in some patients was the amplification of MYC (8q) observed both at the gene and transcript levels, together with alterations of myc-transcriptional targets, including genes and miRNAs involved in the regulation of cell cycle and proliferation. Differential expression of the sulfatase SULF2 and of miR-29a, -181a, and -221 was also observed between resistant and sensitive patients before treatment. These observations were further confirmed on a validation cohort of CLL patients treated with fludarabine in vitro. CONCLUSION: In the present study we identified genes and miRNAs that may predict clinical resistance of CLL to fludarabine, and describe an interesting oncogenic mechanism in CLL patients resistant to fludarabine by which the complete MYC-specific regulatory network was altered (DNA and RNA levels, and transcriptional targets). These results should prove useful for understanding and overcoming refractoriness to fludarabine and also for predicting the clinical outcome of CLL patients before or early during their treatment.

Inhibition of Vps34 reprograms cold into hot inflamed tumors and improves anti-PD-1/PD-L1 immunotherapy.

One of the major challenges limiting the efficacy of anti-PD-1/PD-L1 therapy in nonresponding patients is the failure of T cells to penetrate the tumor microenvironment. We showed that genetic or pharmacological inhibition of Vps34 kinase activity using SB02024 or SAR405 (Vps34i) decreased the tumor growth and improved mice survival in multiple tumor models by inducing an infiltration of NK, CD8+, and CD4+ T effector cells in melanoma and CRC tumors. Such infiltration resulted in the establishment of a T cell-inflamed tumor microenvironment, characterized by the up-regulation of pro-inflammatory chemokines and cytokines, CCL5, CXCL10, and IFNγ. Vps34i treatment induced STAT1 and IRF7, involved in the up-regulation of CCL5 and CXCL10. Combining Vps34i improved the therapeutic benefit of anti-PD-L1/PD-1 in melanoma and CRC and prolonged mice survival. Our study revealed that targeting Vps34 turns cold into hot inflamed tumors, thus enhancing the efficacy of anti-PD-L1/PD-1 blockade.

Peroxisome proliferator-activated receptor gamma agonists potentiate the cytotoxic effect of valproic acid in multiple myeloma cells.

The main challenge in using chemotherapy to treat multiple myeloma (MM) is drug resistance. In order to evaluate the anti-neoplastic properties of a new drug combination in MM, two clinically available drugs, valproic acid (VPA) a histone deacetylase (HDAC) inhibitor and pioglitazone, a peroxisome proliferator-activated receptor gamma (PPARgamma) agonist, were tested in vitro on MM cell lines and MM patient cells. The sensitivity towards VPA alone was observed on several MM cell lines tested and also on primary myeloma cells and peripheral blood mononuclear cells from healthy donors. Importantly, the addition of a PPARgamma agonist to the VPA treatment increased the cytotoxic effect of VPA in a synergistic/additive manner on the different MM cell lines and MM patient cells. This effect was observed at the physiological range of VPA used to treat epileptic patients. The mechanisms underlying this increase induced a cell cycle arrest and caspase-dependent apoptosis. The potentiation of the effect of VPA by pioglitazone was mediated by higher acetylation levels of histones H3 and H4 compared to levels induced by HDAC inhibitors alone. This association reveals a new promising chemotherapeutic combination to be tested in MM.

Impact of hypoxic tumor microenvironment and tumor cell plasticity on the expression of immune checkpoints.

Compared to traditional therapies, such as surgery, radio-chemotherapy, or targeted approaches, immunotherapies based on immune checkpoint blockers (ICBs) have revolutionized the treatment of cancer. Although ICBs have yielded long-lasting results and have improved patient survival, this success has been seriously challenged by clinical observations showing that only a small fraction of patients benefit from this revolutionary therapy and no benefit has been found in patients with highly aggressive tumors. Efforts are currently ongoing to identify factors that predict the response to ICB. Among the different predictive markers established so far, the expression levels of immune checkpoint genes have proven to be important biomarkers for informing treatment choices. Therefore, understanding the mechanisms involved in the regulation of immune checkpoints is a key element that will facilitate novel combination approaches and optimize patient outcome. In this review, we discuss the impact of hypoxia and tumor cell plasticity on immune checkpoint gene expression and provide insight into the therapeutic value of the EMT signature and the rationale for novel combination approaches to improve ICB therapy and maximize the benefits for patients with cancer.

CD47 is a direct target of SNAI1 and ZEB1 and its blockade activates the phagocytosis of breast cancer cells undergoing EMT.

We report that CD47 was upregulated in different EMT-activated human breast cancer cells versus epithelial MCF7 cells. Overexpression of SNAI1 or ZEB1 in epithelial MCF7 cells activated EMT and upregulated CD47 while siRNA-mediated targeting of SNAI1 or ZEB1 in mesenchymal MDA-MB-231 cells reversed EMT and strongly decreased CD47. Mechanistically, SNAI1 and ZEB1 upregulated CD47 by binding directly to E-boxes in the human CD47 promoter. TCGA and METABRIC data sets from breast cancer patients revealed that CD47 correlated with SNAI1 and Vimentin. At functional level, different EMT-activated breast cancer cells were less efficiently phagocytosed by macrophages vs. MCF7 cells. The phagocytosis of EMT-activated cells was rescued by using CD47 blocking antibody or by genetic targeting of SNAI1, ZEB1 or CD47. These results provide a rationale for an innovative preclinical combination immunotherapy based on PD-1/PD-L1 and CD47 blockade along with EMT inhibitors in patients with highly aggressive, mesenchymal, and metastatic breast cancer.

The multifaceted role of autophagy in tumor evasion from immune surveillance.

While autophagy is constitutively executed at basal level in all cells, it is activated in cancer cells in response to various microenvironmental stresses including hypoxia. It is now well established that autophagy can act both as tumor suppressor or tumor promoter. In this regard, several reports indicate that the tumor suppressor function of autophagy is associated with its ability to scavenge damaged oxidative organelles, thereby preventing the accumulation of toxic oxygen radicals and limiting the genome instability. Paradoxically, in developed tumors, autophagy can promote the survival of cancer cells and therefore operates as a cell resistance mechanism. The consensus appears to be that autophagy has a dual role in suppressing tumor initiation and in promoting the survival of established tumors. This has inspired significant interest in applying anti-autophagy therapies as an entirely new approach to cancer treatment. While much remains to be learned about the regulation and context-dependent biological role of autophagy, it is now well established that modulation of this process could be an attractive approach for the development of novel anticancer therapeutic strategies. In this review, we will summarize recent reports describing how tumor cells, by activating autophagy, manage to resist the immune cell attack. Data described in this review strongly argue that targeting autophagy may represent a conceptual realm for new immunotherapeutic strategies aiming to block the immune escape and therefore providing rational approach to future tumor immunotherapy design.

Bronchial airway gene expression in smokers with lung or head and neck cancer.

Cigarette smoking is the major cause of cancers of the respiratory tract, including non-small cell lung cancer (NSCLC) and head and neck cancer (HNC). In order to better understand carcinogenesis of the lung and upper airways, we have compared the gene expression profiles of tumor-distant, histologically normal bronchial biopsy specimens obtained from current smokers with NSCLC or HNC (SC, considered as a single group), as well as nonsmokers (NS) and smokers without cancer (SNC). RNA from a total of 97 biopsies was used for gene expression profiling (Affymetrix HG-U133 Plus 2.0 array). Differentially expressed genes were used to compare NS, SNC, and SC, and functional analysis was carried out using Ingenuity Pathway Analysis (IPA). Smoking-related cancer of the respiratory tract was found to affect the expression of genes encoding xenobiotic biotransformation proteins, as well as proteins associated with crucial inflammation/immunity pathways and other processes that protect the airway from the chemicals in cigarette smoke or contribute to carcinogenesis. Finally, we used the prediction analysis for microarray (PAM) method to identify gene signatures of cigarette smoking and cancer, and uncovered a 15-gene signature that distinguished between SNC and SC with an accuracy of 83%. Thus, gene profiling of histologically normal bronchial biopsy specimens provided insight into cigarette-induced carcinogenesis of the respiratory tract and gene signatures of cancer in smokers.

Blocking hypoxia-induced autophagy in tumors restores cytotoxic T-cell activity and promotes regression.

The relationship between hypoxic stress, autophagy, and specific cell-mediated cytotoxicity remains unknown. This study shows that hypoxia-induced resistance of lung tumor to cytolytic T lymphocyte (CTL)-mediated lysis is associated with autophagy induction in target cells. In turn, this correlates with STAT3 phosphorylation on tyrosine 705 residue (pSTAT3) and HIF-1α accumulation. Inhibition of autophagy by siRNA targeting of either beclin1 or Atg5 resulted in impairment of pSTAT3 and restoration of hypoxic tumor cell susceptibility to CTL-mediated lysis. Furthermore, inhibition of pSTAT3 in hypoxic Atg5 or beclin1-targeted tumor cells was found to be associated with the inhibition Src kinase (pSrc). Autophagy-induced pSTAT3 and pSrc regulation seemed to involve the ubiquitin proteasome system and p62/SQSTM1. In vivo experiments using B16-F10 melanoma tumor cells indicated that depletion of beclin1 resulted in an inhibition of B16-F10 tumor growth and increased tumor apoptosis. Moreover, in vivo inhibition of autophagy by hydroxychloroquine in B16-F10 tumor-bearing mice and mice vaccinated with tyrosinase-related protein-2 peptide dramatically increased tumor growth inhibition. Collectively, this study establishes a novel functional link between hypoxia-induced autophagy and the regulation of antigen-specific T-cell lysis and points to a major role of autophagy in the control of in vivo tumor growth.

The acquisition of resistance to TNFα in breast cancer cells is associated with constitutive activation of autophagy as revealed by a transcriptome analysis using a custom microarray.

While the autophagic process is mainly regulated at the post-translational level, a growing body of evidence suggests that autophagy might also be regulated at the transcriptional level. The identification of transcription factors involved in the regulation of autophagy genes has provided compelling evidence for such regulation. In this context, a powerful high throughput analysis tool to simultaneously monitor the expression level of autophagy genes is urgently needed. Here we describe setting up the first comprehensive human autophagy database (HADb, available at www.autophagy.lu) and the development of a companion Human Autophagy-dedicated cDNA Microarray which comprises 234 genes involved in or related to autophagy. The autophagy microarray tool used on breast adenocarcinoma MCF-7 cell line allowed the identification of 47 differentially expressed autophagy genes associated with the acquisition of resistance to the cytotoxic effect of TNFα. The autophagy-core machinery genes DRAM (Damage-Regulated Autophagy Modulator), BNIP3L (BCL2/adenovirus E1B 19 kDa interacting protein 3-like), BECN1 (Beclin 1), GABARAP (Gamma-AminoButyric Acid Receptor-Associated Protein) and UVRAG (UV radiation resistance associated gene) were found upregulated in TNF-resistant cells, suggesting a constitutive activation of the autophagy machinery in these cells. More interestingly, we identified NPC1 as the most upregulated genes in TNF-resistant compared to TNF-sensitive MCF-7 cells, suggesting a relation between the intracellular transport of cholesterol, the regulation of autophagy and NPC1 expression in TNF-resistant tumor cells. In conclusion, we describe here new tools that may help investigating autophagy gene regulation in various cellular models and diseases.

The histone deacetylase inhibitor MGCD0103 induces apoptosis in B-cell chronic lymphocytic leukemia cells through a mitochondria-mediated caspase activation cascade.

Clinical trials have shown activity of the isotype-selective histone deacetylase (HDAC) inhibitor MGCD0103 in different hematologic malignancies. There are data to support the use of HDAC inhibitors in association with other cancer therapies. To propose a rational combination therapy, it is necessary to depict the molecular basis behind the cytotoxic effect of MGCD0103. In this study, we found that MGCD0103 was substantially more toxic in neoplastic B cells relative to normal cells, and we described the death pathways activated by MGCD0103 in B-cell chronic lymphocytic leukemia (CLL) cells from 32 patients. MGCD0103 decreased the expression of Mcl-1 and induced translocation of Bax to the mitochondria, mitochondrial depolarization, and release of cytochrome c in the cytosol. Caspase processing in the presence of the caspase inhibitor Q-VD-OPh and time course experiments showed that caspase-9 was the apical caspase. Thus, MGCD0103 induced the intrinsic pathway of apoptosis in CLL cells. Moreover, MGCD0103 treatment resulted in the activation of a caspase cascade downstream of caspase-9, caspase-dependent amplification of mitochondrial depolarization, activation of calpain, and Bax cleavage. We propose a model whereby the intrinsic pathway of apoptosis triggered by MGCD0103 in CLL is associated with a mitochondrial death amplification loop.

Transforming growth factor beta1 signaling via interaction with cell surface Hyal-2 and recruitment of WWOX/WOX1.

Transforming growth factor beta (TGF-beta) initiates multiple signal pathways and activates many downstream kinases. Here, we determined that TGF-beta1 bound cell surface hyaluronidase Hyal-2 on microvilli in type II TGF-beta receptor-deficient HCT116 cells, as determined by immunoelectron microscopy. This binding resulted in recruitment of proapoptotic WOX1 (also named WWOX or FOR) and formation of Hyal-2.WOX1 complexes for relocation to the nuclei. TGF-beta1 strengthened the binding of the catalytic domain of Hyal-2 with the N-terminal Tyr-33-phosphorylated WW domain of WOX1, as determined by time lapse fluorescence resonance energy transfer analysis in live cells, co-immunoprecipitation, and yeast two-hybrid domain/domain mapping. In promoter activation assay, ectopic WOX1 or Hyal-2 alone increased the promoter activity driven by Smad. In combination, WOX1 and Hyal-2 dramatically enhanced the promoter activation (8-9-fold increases), which subsequently led to cell death (>95% of promoter-activated cells). TGF-beta1 supports L929 fibroblast growth. In contrast, transiently overexpressed WOX1 and Hyal-2 sensitized L929 to TGF-beta1-induced apoptosis. Together, TGF-beta1 invokes a novel signaling by engaging cell surface Hyal-2 and recruiting WOX1 for regulating the activation of Smad-driven promoter, thereby controlling cell growth and death.