Bcl-2 dependent modulation of Hippo pathway in cancer cells.

INTRODUCTION: Bcl-2 and Bcl-xL are the most studied anti-apoptotic members of Bcl-2 family proteins. We previously characterized both of them, not only for their role in regulating apoptosis and resistance to therapy in cancer cells, but also for their non-canonical functions, mainly including promotion of cancer progression, metastatization, angiogenesis, and involvement in the crosstalk among cancer cells and components of the tumor microenvironment. Our goal was to identify transcriptional signature and novel cellular pathways specifically modulated by Bcl-2. METHODS: We performed RNAseq analysis of siRNA-mediated transient knockdown of Bcl-2 or Bcl-xL in human melanoma cells and gene ontology analysis to identify a specific Bcl-2 transcriptional signature. Expression of genes modulated by Bcl-2 and associated to Hippo pathway were validated in human melanoma, breast adenocarcinoma and non-small cell lung cancer cell lines by qRT-PCR. Western blotting analysis were performed to analyse protein expression of upstream regulators of YAP and in relation to different level of Bcl-2 protein. The effects of YAP silencing in Bcl-2 overexpressing cancer cells were evaluated in migration and cell viability assays in relation to different stiffness conditions. In vitro wound healing assays and co-cultures were used to evaluate cancer-specific Bcl-2 ability to activate fibroblasts. RESULTS: We demonstrated the Bcl-2-dependent modulation of Hippo Pathway in cancer cell lines from different tumor types by acting on upstream YAP regulators. YAP inhibition abolished the ability of Bcl-2 to increase tumor cell migration and proliferation on high stiffness condition of culture, to stimulate in vitro fibroblasts migration and to induce fibroblasts activation. CONCLUSIONS: We discovered that Bcl-2 regulates the Hippo pathway in different tumor types, promoting cell migration, adaptation to higher stiffness culture condition and fibroblast activation. Our data indicate that Bcl-2 inhibitors should be further investigated to counteract cancer-promoting mechanisms.

Phytochemicals as Immunomodulatory Agents in Melanoma.

Cutaneous melanoma is an immunogenic highly heterogenic tumor characterized by poor outcomes when it is diagnosed late. Therefore, immunotherapy in combination with other anti-proliferative approaches is among the most effective weapons to control its growth and metastatic dissemination. Recently, a large amount of published reports indicate the interest of researchers and clinicians about plant secondary metabolites as potentially useful therapeutic tools due to their lower presence of side effects coupled with their high potency and efficacy. Published evidence was reported in most cases through in vitro studies but also, with a growing body of evidence, through in vivo investigations. Our aim was, therefore, to review the published studies focused on the most interesting phytochemicals whose immunomodulatory activities and/or mechanisms of actions were demonstrated and applied to melanoma models.

Negative Modulation of the Angiogenic Cascade Induced by Allosteric Kinesin Eg5 Inhibitors in a Gastric Adenocarcinoma In Vitro Model.

Eg5 is a kinesin essential in bipolar spindle formation, overexpressed in tumours, thus representing a new target in cancer therapy. We aimed at evaluating the anti-cancer activity of Eg5 thiadiazoline inhibitors 2 and 41 on gastric adenocarcinoma cells (AGS), focusing on the modulation of angiogenic signalling. Docking studies confirmed a similar interaction with Eg5 to that of the parent compound K858. Thiadiazolines were also tested in combination with Hesperidin (HSD). Cell cycle analysis reveals a reduction of G1 and S phase percentages when 41 is administered as well as HSD in combination with K858. Western blot reveals Eg5 inhibitors capability to reduce PI3K, p-AKT/Akt and p-Erk/Erk expressions; p-Akt/Akt ratio is even more decreased in HSD+2 sample than the p-Erk/Erk ratio in HSD+41 or K858. VEGF expression is reduced when HSD+2 and HSD+41 are administered with respect to compounds alone, after 72 h. ANGPT2 gene expression increases in cells treated with 41 and HSD+2 compared to K858. The wound-healing assay highlights a reduction in the cut in HSD+2 sample compared to 2 and HSD. Thus, Eg5 inhibitors appear to modulate angiogenic signalling by controlling VEGF activity even better if combined with HSD. Overall, Eg5 inhibitors can represent a promising starting point to develop innovative anti-cancer strategies.

Bcl-xL: A Focus on Melanoma Pathobiology.

Apoptosis is the main mechanism by which multicellular organisms eliminate damaged or unwanted cells. To regulate this process, a balance between pro-survival and pro-apoptotic proteins is necessary in order to avoid impaired apoptosis, which is the cause of several pathologies, including cancer. Among the anti-apoptotic proteins, Bcl-xL exhibits a high conformational flexibility, whose regulation is strictly controlled by alternative splicing and post-transcriptional regulation mediated by transcription factors or microRNAs. It shows relevant functions in different forms of cancer, including melanoma. In melanoma, Bcl-xL contributes to both canonical roles, such as pro-survival, protection from apoptosis and induction of drug resistance, and non-canonical functions, including promotion of cell migration and invasion, and angiogenesis. Growing evidence indicates that Bcl-xL inhibition can be helpful for cancer patients, but at present, effective and safe therapies targeting Bcl-xL are lacking due to toxicity to platelets. In this review, we summarized findings describing the mechanisms of Bcl-xL regulation, and the role that Bcl-xL plays in melanoma pathobiology and response to therapy. From these findings, it emerged that even if Bcl-xL plays a crucial role in melanoma pathobiology, we need further studies aimed at evaluating the involvement of Bcl-xL and other members of the Bcl-2 family in the progression of melanoma and at identifying new non-toxic Bcl-xL inhibitors.

The Combination of the M2 Muscarinic Receptor Agonist and Chemotherapy Affects Drug Resistance in Neuroblastoma Cells.

One of the major limits of chemotherapy is depending on the ability of the cancer cells to elude and adapt to different drugs. Recently, we demonstrated how the activation of the M2 muscarinic receptor could impair neuroblastoma cell proliferation. In the present paper, we investigate the possible effects mediated by the preferential M2 receptor agonist arecaidine propargyl ester (APE) on drug resistance in two neuroblastoma cell lines, SK-N-BE and SK-N-BE(2C), a sub-clone presenting drug resistance. In both cell lines, we compare the expression of the M2 receptor and the effects mediated by the M2 agonist APE on cell cycle, demonstrating a decreased percentage of cells in S phase and an accumulation of SK-N-BE cells in G1 phase, while the APE treatment of SK-N-BE(2C) cells induced a block in G2/M phase. The withdrawal of the M2 agonist from the medium shows that only the SK-N-BE(2C) cells are able to rescue cell proliferation. Further, we demonstrate that the co-treatment of low doses of APE with doxorubicin or cisplatin significantly counteracts cell proliferation when compared with the single treatment. Analysis of the expression of ATP-binding cassette (ABC) efflux pumps demonstrates the ability of the M2 agonist to downregulate their expression and that this negative modulation may be dependent on N-MYC decreased expression induced by the M2 agonist. Our data demonstrate that the combined effect of low doses of conventional drugs and the M2 agonist may represent a new promising therapeutic approach in neuroblastoma treatment, in light of its significant impact on drug resistance and the possible reduction in the side effects caused by high doses of chemotherapy drugs.

Interleukin 8 mediates bcl-xL-induced enhancement of human melanoma cell dissemination and angiogenesis in a zebrafish xenograft model.

The protein bcl-xL is able to enhance the secretion of the proinflammatory chemokine interleukin 8 (CXCL8) in human melanoma lines. In this study, we investigate whether the bcl-xL/CXCL8 axis is important for promoting melanoma angiogenesis and aggressiveness in vivo, using angiogenesis and xenotransplantation assays in zebrafish embryos. When injected into wild-type embryos, bcl-xL-overexpressing melanoma cells showed enhanced dissemination and angiogenic activity compared with control cells. Human CXCL8 protein elicited a strong proangiogenic activity in zebrafish embryos and zebrafish Cxcr2 receptor was identified as the mediator of CXCL8 proangiogenic activity using a morpholino-mediated gene knockdown. However, human CXCL8 failed to induce neutrophil recruitment in contrast to its zebrafish homolog. Interestingly, the greater aggressiveness of bcl-xL-overexpressing melanoma cells was mediated by an autocrine effect of CXCL8 on its CXCR2 receptor, as confirmed by an shRNA approach. Finally, correlation studies of gene expression and survival analyses using microarray and RNA-seq public databases of human melanoma biopsies revealed that bcl-xL expression significantly correlated with the expression of CXCL8 and other markers of melanoma progression. More importantly, a high level of co-expression of bcl-xL and CXCL8 was associated with poor prognosis in melanoma patients. In conclusion, these data demonstrate the existence of an autocrine CXCL8/CXCR2 signaling pathway in the bcl-xL-induced melanoma aggressiveness, encouraging the development of novel therapeutic approaches for high bcl-xL-expressing melanoma.

Non-canonical roles of Bcl-2 and Bcl-xL proteins: relevance of BH4 domain.

Bcl-2 protein family is constituted by multidomain members originally identified as modulators of programmed cell death and whose expression is frequently misbalanced in cancer cells. The lead member Bcl-2 and its homologue Bcl-xL proteins are characterized by the presence of all four conserved BH domain and exert their antiapoptotic role mainly through the involvement of BH1, BH2 and BH3 homology domains, that mediate the interaction with the proapoptotic members of the same Bcl-2 family. The N-terminal BH4 domain of Bcl-2 and Bcl-xL is responsible for the interaction with other proteins that do not belong to Bcl-2 protein family. Beyond a classical role in inhibiting apoptosis, BH4 domain has been characterized as a crucial regulator of other important cellular functions attributed to Bcl-2 and Bcl-xL, including proliferation, autophagy, differentiation, DNA repair, cell migration, tumor progression and angiogenesis. During the last two decades a strong effort has been made to dissect the molecular pathways involved the capability of BH4 domain to regulate the canonical antiapoptotic and the non-canonical activities of Bcl-2 and Bcl-xL, creating the basis for the development of novel anticancer agents targeting this domain. Indeed, recent evidences obtained on in vitro and in vivo model of different cancer histotypes are confirming the promising therapeutic potential of BH4 domain inhibitors supporting their future employment as a novel anticancer strategy.

Metabolite profiling of ascidian Styela plicata using LC-MS with multivariate statistical analysis and their antitumor activity.

To identify the metabolite distribution in ascidian, we have applied an integrated liquid chromatography- tandem mass spectrometry (LC-MS) metabolomics approach to explore and identify patterns in chemical diversity of invasive ascidian Styela plicata. A total of 71 metabolites were reported among these alkaloids, fatty acids and lipids are the most dominant chemical group. Multivariate statistical analysis, principal component analysis (PCA) showed a clear separation according to chemical diversity and taxonomic groups. PCA and partial least square discriminant analysis were applied to discriminate the chemical group of S. plicata crude compounds and classify the compounds with unknown biological activities. In this study, we reported for the first time that a partially purified methanol extract prepared from the ascidian S. plicata and Ascidia mentula possess antitumor activity against four tumor cell lines with different tumor histotype, such as HeLa (cervical carcinoma), HT29 (colon carcinoma), MCF-7 (breast carcinoma) and M14 (melanoma). S. plicata fraction SP-50 showed strong inhibition of cell proliferation and induced apoptosis in HeLa and HT29 cells, thus indicating S. plicata fraction SP-50 a potential lead compound for anticancer therapy. The molecular mechanism of action and chemotherapeutic potential of these ascidian unknown biomolecules need further research.

VEGF-induced neoangiogenesis is mediated by NAADP and two-pore channel-2-dependent Ca2+ signaling.

Vascular endothelial growth factor (VEGF) and its receptors VEGFR1/VEGFR2 play major roles in controlling angiogenesis, including vascularization of solid tumors. Here we describe a specific Ca(2+) signaling pathway linked to the VEGFR2 receptor subtype, controlling the critical angiogenic responses of endothelial cells (ECs) to VEGF. Key steps of this pathway are the involvement of the potent Ca(2+) mobilizing messenger, nicotinic acid adenine-dinucleotide phosphate (NAADP), and the specific engagement of the two-pore channel TPC2 subtype on acidic intracellular Ca(2+) stores, resulting in Ca(2+) release and angiogenic responses. Targeting this intracellular pathway pharmacologically using the NAADP antagonist Ned-19 or genetically using Tpcn2(-/-) mice was found to inhibit angiogenic responses to VEGF in vitro and in vivo. In human umbilical vein endothelial cells (HUVECs) Ned-19 abolished VEGF-induced Ca(2+) release, impairing phosphorylation of ERK1/2, Akt, eNOS, JNK, cell proliferation, cell migration, and capillary-like tube formation. Interestingly, Tpcn2 shRNA treatment abolished VEGF-induced Ca(2+) release and capillary-like tube formation. Importantly, in vivo VEGF-induced vessel formation in matrigel plugs in mice was abolished by Ned-19 and, most notably, failed to occur in Tpcn2(-/-) mice, but was unaffected in Tpcn1(-/-) animals. These results demonstrate that a VEGFR2/NAADP/TPC2/Ca(2+) signaling pathway is critical for VEGF-induced angiogenesis in vitro and in vivo. Given that VEGF can elicit both pro- and antiangiogenic responses depending upon the balance of signal transduction pathways activated, targeting specific VEGFR2 downstream signaling pathways could modify this balance, potentially leading to more finely tailored therapeutic strategies.

miR-211 and MITF modulation by Bcl-2 protein in melanoma cells.

Melanoma, the most lethal form of skin cancer, is frequently associated with alterations in several genes, among which the Bcl-2 oncogene plays an important role in progression, chemosensitivity and angiogenesis. Also microRNA (miRNA) are emerging as modulators of melanoma development and progression, and among them, miR-211, located within the melastatin-1/TRPM1 (transient receptor potential cation channel, subfamily M, member 1 protein) gene, is prevalently expressed in the melanocyte lineage and acts as oncosuppressor. Using several human melanoma cell lines and their Bcl-2 stably overexpressing derivatives, we evaluated whether there was a correlation between expression of Bcl-2 and miR-211. Western blot analysis and quantitative real-time polymerase chain reaction demonstrated reduced expression of pri-miR-211, miR-211, TRPM1, and MLANA levels, after Bcl-2 overexpression, associated with increased expression of well-known miR-211 target genes. Overexpression of mature miR-211 in Bcl-2 overexpressing cells rescued Bcl-2 ability to increase cell migration. A decreased nuclear localization of microphthalmia-associated transcription factor (MITF), a co-regulator of both miR-211 and TRPM1, and a reduced MITF recruitment at the TRPM1 and MLANA promoters were also evidenced in Bcl-2 overexpressing cells by immunofluorescence and chromatin immunoprecipitation experiments, respectively. Reduction of Bcl-2 expression by small interference RNA confirmed the ability of Bcl-2 to modulate miR-211 and TRPM1 expression. © 2015 Wiley Periodicals, Inc.

TLR3 engagement induces IRF-3-dependent apoptosis in androgen-sensitive prostate cancer cells and inhibits tumour growth in vivo.

Toll-like receptors (TLRs) are a family of highly conserved transmembrane proteins expressed in epithelial and immune cells that recognize pathogen associated molecular patterns. Besides their role in immune response against infections, numerous studies have shown an important role of different TLRs in cancer, indicating these receptors as potential targets for cancer therapy. We previously demonstrated that the activation of TLR3 by the synthetic double-stranded RNA analogue poly I:C induces apoptosis of androgen-sensitive prostate cancer (PCa) LNCaP cells and, much less efficiently, of the more aggressive PC3 cell line. Therefore, in this study we selected LNCaP cells to investigate the mechanism of TLR3-mediated apoptosis and the in vivo efficacy of poly I:C-based therapy. We show that interferon regulatory factor-3 (IRF-3) signalling plays an essential role in TLR3-mediated apoptosis in LNCaP cells through the activation of the intrinsic and extrinsic apoptotic pathways. Interestingly, hardly any apoptosis was induced by poly I:C in normal prostate epithelial cells RWPE-1. We also demonstrate for the first time the direct anticancer effect of poly I:C as a single therapeutic agent in a well-established human androgen-sensitive PCa xenograft model, by showing that tumour growth is highly impaired in poly I:C-treated immunodeficient mice. Immunohistochemical analysis of PCa xenografts highlights the antitumour role of poly I:C in vivo both on cancer cells and, indirectly, on endothelial cells. Notably, we show the presence of TLR3 and IRF-3 in both human normal and PCa clinical samples, potentially envisaging poly I:C-based therapy for PCa.

Histone deacetylase inhibition synergistically enhances pemetrexed cytotoxicity through induction of apoptosis and autophagy in non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide. Pemetrexed, a multi-target folate antagonist, has demonstrated efficacy in NSCLC histological subtypes characterized by low thymidylate synthase (TS) expression. Among many other potential targets, histone deacetylase inhibitors (HDACi) modulate TS expression, potentially sensitizing to the cytotoxic action of anti-cancer drugs that target the folate pathway, such as pemetrexed. Since high levels of TS have been linked to clinical resistance to pemetrexed in NSCLC, herein we investigated the molecular and functional effects of combined pemetrexed and ITF2357, a pan-HDACi currently in clinical trials as an anti-cancer agent. RESULTS: In NSCLC cell lines, HDAC inhibition by ITF2357 induced histone and tubulin acetylation and downregulated TS expression at the mRNA and protein level. In combination experiments in vitro ITF2357 and pemetrexed demonstrated sequence-dependent synergistic growth-inhibitory effects, with the sequence pemetrexed followed by ITF2357 inducing a strikingly synergistic reduction in cell viability and induction of both apoptosis and autophagy in all cell line models tested, encompassing both adenocarcinoma and squamous cell carcinoma. Conversely, simultaneous administration of both drugs achieved frankly antagonistic effects, while the sequence of ITF2357 followed by pemetrexed had additive to slightly synergistic growth-inhibitory effects only in certain cell lines. Similarly, highly synergistic growth inhibition was also observed in patient-derived lung cancer stem cells (LCSC) exposed to pemetrexed followed by ITF2357. In terms of molecular mechanisms of interaction, the synergistic growth-inhibitory effects observed were only partially related to TS modulation by ITF2357, as genetic silencing of TS expression potentiated growth inhibition by either pemetrexed or ITF2357 and, to a lesser extent, by their sequential combination. Genetic and pharmacological approaches provided an interesting link between the autophagic and apoptotic pathways, and showed that sequential pemetrexed/ITF2357 causes a toxic form of autophagy with consequent activation of a caspase-dependent apoptotic program. In vivo experiments in NSCLC xenografts confirmed that sequential pemetrexed/ITF2357 is feasible and results in increased inhibition of tumor growth and increased mice survival. CONCLUSIONS: Overall, these data provide a strong rationale for the clinical development of sequential schedules employing pemetrexed followed by HDACi in NSCLC.

Overview of BH3 mimetics in ovarian cancer.

Ovarian carcinoma is the leading cause of gynecological cancer-related death, still with a dismal five-year prognosis, mainly due to late diagnosis and the emergence of resistance to cytotoxic and targeted agents. Bcl-2 family proteins have a key role in apoptosis and are associated with tumor development/progression and response to therapy in different cancer types, including ovarian carcinoma. In tumors, evasion of apoptosis is a possible mechanism of resistance to therapy. BH3 mimetics are small molecules that occupy the hydrophobic pocket on pro-survival proteins, allowing the induction of apoptosis, and are currently under study as single agents and/or in combination with cytotoxic and targeted agents in solid tumors. Here, we discuss recent advances in targeting anti-apoptotic proteins of the Bcl-2 family for the treatment of ovarian cancer, focusing on BH3 mimetics, and how these approaches could potentially offer an alternative/complementary way to treat patients and overcome or delay resistance to current treatments.

Correction: Zwergel et al. Novel Quinoline Compounds Active in Cancer Cells through Coupled DNA Methyltransferase Inhibition and Degradation. Cancers 2020, 12, 447.

In the original publication [...].

Notch3-regulated microRNAs impair CXCR4-dependent maturation of thymocytes allowing maintenance and progression of T-ALL.

Malignant transformation of T-cell progenitors causes T-cell acute lymphoblastic leukemia (T-ALL), an aggressive childhood lymphoproliferative disorder. Activating mutations of Notch, Notch1 and Notch3, have been detected in T-ALL patients. In this study, we aimed to deeply characterize hyperactive Notch3-related pathways involved in T-cell dynamics within the thymus and bone marrow to propose these processes as an important step in facilitating the progression of T-ALL. We previously generated a transgenic T-ALL mouse model (N3-ICtg) demonstrating that aberrant Notch3 signaling affects early thymocyte maturation programs and leads to bone marrow infiltration by CD4+CD8+ (DP) T cells that are notably, Notch3highCXCR4high. Newly, our in vivo results suggest that an anomalous immature thymocyte subpopulation, such as CD4-CD8- (DN) over-expressing CD3ɛ, but with low CXCR4 expression, dominates N3-ICtg thymus-resident DN subset in T-ALL progression. MicroRNAs might be of significance in T-ALL pathobiology, however, whether required for leukemia maintenance is not fully understood. The selection of specific DN subsets demonstrates the inverse correlation between CXCR4 expression and a panel of Notch3-deregulated miRNAs. Interestingly, we found that within DN thymocyte subset hyperactive Notch3 inhibits CXCR4 expression through the cooperative effects of miR-139-5p and miR-150-5p, thus impinging on thymocyte differentiation with accumulation of DNCD3ɛ+CXCR4- cells. These data point out that deregulation of Notch3 in T-ALL, besides its role in sustaining dissemination of abnormal DP T cells, as we previously demonstrated, could play a role in selecting specific DN immature T cells within the thymus, thus impeding T cell development, to facilitate T-ALL progression inside the bone marrow.

BH4 domain of bcl-2 protein is required for its proangiogenic function under hypoxic condition.

Beyond its classical role as apoptosis inhibitor, bcl-2 protein promotes tumor angiogenesis and the removal of N-terminal bcl-2 homology (BH4) domain abrogates bcl-2-induced hypoxia-inducible factor 1 (HIF-1)-mediated vascular endothelial growth factor (VEGF) expression in hypoxic cancer cells. Using M14 human melanoma cell line and its derivative clones stably overexpressing bcl-2 wild-type or deleted of its BH4 domain, we found that conditioned media (CM) from cells expressing BH4-deleted bcl-2 protein showed a reduced capability to increase in vitro human endothelial cells proliferation and differentiation, and in vivo neovascularization compared with CM from cells overexpressing wild-type bcl-2. Moreover, xenografts derived from cells expressing bcl-2 lacking BH4 domain showed a reduction of metastatic potential compared with tumors derived from wild-type bcl-2 transfectants injection. Stably expressing the Flag-tagged N-terminal sequence of bcl-2 protein, encompassing BH4 domain, we found that this domain is sufficient to enhance the proangiogenic HIF-1/VEGF axis under hypoxic condition. Indeed, lacking of BH4 domain abolishes the interaction between bcl-2 and HIF-1α proteins and the capability of exogenous bcl-2 protein to localize in the nucleus. Moreover, when endoplasmic reticulum-targeted bcl-2 protein is overexpressed in cells, this protein lost the capability to synergize with hypoxia to induce the proangiogenic HIF-1/VEGF axis as shown by wild-type bcl-2 protein. These results demonstrate that BH4 domain of bcl-2 is required for the ability of this protein to increase tumor angiogenesis and progression and indicate that bcl-2 nuclear localization may be required for bcl-2-mediated induction of HIF-1/VEGF axis.

Exploring association of melanoma-specific Bcl-xL with tumor immune microenvironment.

BACKGROUND: Macrophages take center stage in the tumor microenvironment, a niche composed of extracellular matrix and a heterogeneous group of cells, including immune ones. They can evolve during tumor progression and acquire Tumor-Associated Macrophage (TAMs) phenotype. The release of cytokines by tumor and stromal cells, influence the secretion of cytokines by TAMs, which can guarantee tumor progression and influence the response to therapy. Among all factors able to recruit and polarize macrophages, we focused our attention on Bcl-xL, a multifaceted member of the Bcl-2 family, whose expression is deregulated in melanoma. It acts not only as a canonical pro-survival and anti-apoptotic protein, but also as a promoter of tumor progression. METHODS: Human melanoma cells silencing or overexpressing Bcl-xL protein, THP-1 monocytic cells and monocyte-derived macrophages were used in this study. Protein array and specific neutralizing antibodies were used to analyze cytokines and chemokines secreted by melanoma cells. qRT-PCR, ELISA and Western Blot analyses were used to evaluate macrophage polarization markers and protein expression levels. Transwell chambers were used to evaluate migration of THP-1 and monocyte-derived macrophages. Mouse and zebrafish models were used to evaluate the ability of melanoma cells to recruit and polarize macrophages in vivo. RESULTS: We demonstrated that melanoma cells overexpressing Bcl-xL recruit macrophages at the tumor site and induce a M2 phenotype. In addition, we identified that interleukin-8 and interleukin-1ß cytokines are involved in macrophage polarization, and the chemokine CCL5/RANTES in the macrophages recruitment at the tumor site. We also found that all these Bcl-xL-induced factors are regulated in a NF-kB dependent manner in human and zebrafish melanoma models. CONCLUSIONS: Our findings confirmed the pro-tumoral function of Bcl-xL in melanoma through its effects on macrophage phenotype.

Pharmacological targeting of CBP/p300 drives a redox/autophagy axis leading to senescence-induced growth arrest in non-small cell lung cancer cells.

p300/CBP histone acetyltransferases (HAT) are critical transcription coactivators involved in multiple cellular activities. They act at multiple levels in non-small cell lung carcinoma (NSCLC) and appear, therefore, as promising druggable targets. Herein, we investigated the biological effects of A-485, the first selective (potent) drug-like HAT catalytic inhibitor of p300/CBP, in human NSCLC cell lines. A-485 treatment specifically reduced p300/CBP-mediated histone acetylation marks and caused growth arrest of lung cancer cells via activation of the autophagic pathway. Indeed, A-485 growth-arrested cells displayed phenotypic markers of cell senescence and failed to form colonies. Notably, disruption of autophagy by genetic and pharmacological approaches triggered apoptotic cell death. Mechanistically, A-485-induced senescence occurred through the accumulation of reactive oxygen species (ROS), which in turn resulted in DNA damage and activation of the autophagic pathway. Interestingly, ROS scavengers were able to revert senescence phenotype and restore cell viability, suggesting that ROS production had a key role in upstream events leading to growth arrest commitment. Altogether, our data provide new insights into the biological effects of the A-485 and uncover the importance of the autophagic/apoptotic response to design a new combinatorial anticancer strategy.

Caspase-8 as a novel mediator linking Src kinase signaling to enhanced glioblastoma malignancy.

Caspase-8 is a cysteine protease that plays an essential role in apoptosis. Consistently with its canonical proapoptotic function, cancer cells may genetically or epigenetically downregulate its expression. Unexpectedly, Caspase-8 is often retained in cancer, suggesting the presence of alternative mechanisms that may be exploited by cancer cells to their own benefit. In this regard, we reported that Src tyrosine kinase, which is aberrantly activated in many tumors, promotes Caspase-8 phosphorylation on Tyrosine 380 (Y380) preventing its full activation. Here, we investigated the significance of Caspase-8 expression and of its phosphorylation on Y380 in glioblastoma, a brain tumor where both Caspase-8 expression and Src activity are often aberrantly upregulated. Transcriptomic analyses identified inflammatory response as a major target of Caspase-8, and in particular, NFκB signaling as one of the most affected pathways. More importantly, we could show that Src-dependent phosphorylation of Caspase-8 on Y380 drives the assembly of a multiprotein complex that triggers NFκB activation, thereby inducing the expression of inflammatory and pro-angiogenic factors. Remarkably, phosphorylation on Y380 sustains neoangiogenesis and resistance to radiotherapy. In summary, our work identifies a novel interplay between Src kinase and Caspase-8 that allows cancer cells to hijack Caspase-8 to sustain tumor growth.

First-in-Class Selective Inhibitors of the Lysine Acetyltransferase KAT8.

KAT8 is a lysine acetyltransferase primarily catalyzing the acetylation of Lys16 of histone H4 (H4K16). KAT8 dysregulation is linked to the development and metastatization of many cancer types, including non-small cell lung cancer (NSCLC) and acute myeloid leukemia (AML). Few KAT8 inhibitors have been reported so far, none of which displaying selective activity. Based on the KAT3B/KDAC inhibitor C646, we developed a series of N-phenyl-5-pyrazolone derivatives and identified compounds 19 and 34 as low-micromolar KAT8 inhibitors selective over a panel of KATs and KDACs. Western blot, immunofluorescence, and CETSA experiments demonstrated that both inhibitors selectively target KAT8 in cells. Moreover, 19 and 34 exhibited mid-micromolar antiproliferative activity in different cancer cell lines, including NSCLC and AML, without impacting the viability of nontransformed cells. Overall, these compounds are valuable tools for elucidating KAT8 biology, and their simple structures make them promising candidates for future optimization studies.