Bromodomain Inhibitor JQ1 Provides Novel Insights and Perspectives in Rhabdomyosarcoma Treatment.

Rhabdomyosarcoma (RMS) is the most common type of pediatric soft tissue sarcoma. It is classified into two main subtypes: embryonal (eRMS) and alveolar (aRMS). MYC family proteins are frequently highly expressed in RMS tumors, with the highest levels correlated with poor prognosis. A pharmacological approach to inhibit MYC in cancer cells is represented by Bromodomain and Extra-Terminal motif (BET) protein inhibitors. In this paper, we evaluated the effects of BET inhibitor (+)-JQ1 (JQ1) on the viability of aRMS and eRMS cells. Interestingly, we found that the drug sensitivity of RMS cell lines to JQ1 was directly proportional to the expression of MYC. JQ1 induces G1 arrest in cells with the highest steady-state levels of MYC, whereas apoptosis is associated with MYC downregulation. These findings suggest BET inhibition as an effective strategy for the treatment of RMS alone or in combination with other drugs.

A Micro-Immunotherapy Sequential Medicine MIM-seq Displays Immunomodulatory Effects on Human Macrophages and Anti-Tumor Properties towards In Vitro 2D and 3D Models of Colon Carcinoma and in an In Vivo Subcutaneous Xenograft Colon Carcinoma Model.

In this study, the immunomodulatory effects of a sequential micro-immunotherapy medicine, referred as MIM-seq, were appraised in human primary M1 and M2 macrophages, in which the secretion of pro-inflammatory cytokines, such as interleukin (IL)-1ß, IL-6, IL-12, IL-23, and tumor necrosis factor (TNF)-alpha, was inhibited. In addition, the potential anti-proliferative effects of MIM-seq on tumor cells was assessed in three models of colorectal cancer (CRC): an in vitro two-dimensions (2D) model of HCT-116 cells, an in vitro tri-dimensional (3D) model of spheroids, and an in vivo model of subcutaneous xenografted mice. In these models, MIM-seq displayed anti-proliferative effects when compared with the vehicle. In vivo, the tumor growth was slightly reduced in MIM-seq-treated animals. Moreover, MIM-seq could slightly reduce the growth of our spheroid models, especially under serum-deprivation. When MIM-seq was combined with two well-known anti-cancerogenic agents, either resveratrol or etoposide, MIM-seq could even further reduce the spheroid's volume, pointing up the need to further assess whether MIM-seq could be beneficial for CRC patients as an adjuvant therapy. Altogether, these data suggest that MIM-seq could have anti-tumor properties against CRC and an immunomodulatory effect towards the mediators of inflammation, whose systemic dysregulation is considered to be a poor prognosis for patients.

Antiproliferative and proapoptotic effects of Inula viscosa extract on Burkitt lymphoma cell line.

Burkitt lymphoma is a very aggressive B-cell non-Hodgkin lymphoma. Although remarkable progress has been made in the therapeutic scenario for patients with Burkitt lymphoma, search and development of new effective anticancer agents to improve patient outcome and minimize toxicity has become an urgent issue. In this study, the antitumoral activity of Inula viscosa, a traditional herb obtained from plants collected on the Asinara Island, Italy, was evaluated in order to explore potential antineoplastic effects of its metabolites on Burkitt lymphoma. Raji human cell line was treated with increasing Inula viscosa extract concentration for cytotoxicity screening and subsequent establishment of cell cycle arrest and apoptosis. Moreover, gene expression profiles were performed to identify molecular mechanisms involved in the anticancer activities of this medical plant. The Inula viscosa extract exhibited powerful antiproliferative and cytotoxic activities on Raji cell line, showing a dose- and time-dependent decrease in cell viability, obtained by cell cycle arrest in the G2/M phase and an increase in cell apoptosis. The treatment with Inula viscosa caused downregulation of genes involved in cell cycle and proliferation (c-MYC, CCND1) and inhibition of cell apoptosis (BCL2, BCL2L1, BCL11A). The Inula viscosa extract causes strong anticancer effects on Burkitt lymphoma cell line. The molecular mechanisms underlying such antineoplastic activity are based on targeting and downregulation of genes involved in cell cycle and apoptosis. Our data suggest that Inula viscosa natural metabolites should be further exploited as potential antineoplastic agents against Burkitt lymphoma.

BET-Inhibitor I-BET762 and PARP-Inhibitor Talazoparib Synergy in Small Cell Lung Cancer Cells.

Small cell lung cancer (SCLC) is an aggressive type of lung cancer with high mortality that is caused by frequent relapses and acquired resistance. Despite that several target-based approaches with potential therapeutic impact on SCLC have been identified, numerous targeted drugs have not been successful in providing improvements in cancer patients when used as single agents. A combination of targeted therapies could be a strategy to induce maximum lethal effects on cancer cells. As a starting point in the development of new drug combination strategies for the treatment of SCLC, we performed a mid-throughput screening assay by treating a panel of SCLC cell lines with BETi or AKi in combination with PARPi or EZH2i. We observed drug synergy between I-BET762 and Talazoparib, BETi and PARPi, respectively, in SCLC cells. Combinatorial efficacy was observed in MYCs-amplified and MYCs-wt SCLC cells over SCLC cells with impaired MYC signaling pathway or non-tumor cells. We indicate that drug synergy between I-BET762 and Talazoparib is associated with the attenuation HR-DSBR process and the downregulation of various players of DNA damage response by BET inhibition, such as CHEK2, PTEN, NBN, and FANCC. Our results provide a rationale for the development of new combinatorial strategies for the treatment of SCLC.

A new parameter of growth inhibition for cell proliferation assays.

Cell proliferation assays are performed by four decades to test the anti-proliferative activity of natural products and synthetic compounds in cell cultures. In cancer research, they are widely employed to evaluate drug efficacy in in vitro tumor models, such as established cell lines, primary cultures, and recently developed three-dimensional tumor organoids. In this manuscript, we demonstrated that current employed parameters used by researchers to quantify in vitro growth inhibition, IC50 and GI50 , lead to a misinterpretation of results based on the exponential, and not linear, proliferation of the cells in culture. Therefore, we introduce a new parameter for the analysis of growth inhibition in cell proliferation assays, termed relative population doubling capacity, that can be employed to properly quantify the anti-proliferative activity of tested compounds and to compare drug efficacy between distinct cell models.

The role of enhancer of zeste homolog 2: From viral epigenetics to the carcinogenesis of hepatocellular carcinoma.

Nowadays, epigenetics covers a crucial role in different fields of science. The enhancer of zeste homolog 2 (EZH2), the catalytic subunit of the Polycomb Repressive Complex 2 (PRC2), is a big proponent of how epigenetic changes can affect the initiation and progression of several diseases. Through its catalytic activity, responsible for the tri-methylation of lysine 27 of the histone H3 (H3K27me3), EZH2 is a good target for both diagnosis and therapy of different pathologies. A large number of studies have demonstrated its crucial role in cancer initiation and progression. Nevertheless, only recently its function in virus diseases has been uncovered; therefore, EZH2 can be an important promoter of viral carcinogenesis. This review explores the role of EZH2 in viral epigenetics based on recent progress that demonstrated the role of this protein in virus environment. In particular, the review focuses on EZH2 behavior in Hepatitis B Virus, analyzing its role in the rise of Hepatocellular Carcinoma.

12-O-tetradecanoylphorbol-13-acetate and EZH2 inhibition: A novel approach for promoting myogenic differentiation in embryonal rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is a soft tissue sarcoma that arises from muscle precursors affecting predominately children and young adults. It can be divided into two main classes: embryonal (eRMS) and alveolar rhabodomyosarcomas (aRMS). Despite the expression of early muscle specific genes, RMS cells fail to complete myogenesis even in differentiation conditions. We previously demonstrated that Enhancer Zeste of Homolog 2 (EZH2), the catalytic subunits of PRC2 complex, contributes to inhibit muscle differentiation in eRMS and its down-regulation causes a partial recovery of myogenesis. 12-O-tetradecanoylphorbol-13-acetate (TPA) is a molecule able to induce differentiation in eRMS with a mechanism that involves the protein kinase C (PKC). In this paper we report that treatment with TPA reduces the expression of EZH2 without affecting levels of H3K27me3. The combination of TPA with GSK126, an inhibitor of the catalytic activity of EZH2, has a synergic effect on the induction of muscle differentiation in RD rhabdomyosarcoma cells, suggesting a new therapeutic combinatory approach for RMS treatment.

On the role of retinoblastoma family proteins in the establishment and maintenance of the epigenetic landscape.

RB family members are negative regulators of the cell cycle, involved in numerous biological processes such as cellular senescence, development and differentiation. Disruption of RB family pathways are linked to loss of cell cycle control, cellular immortalization and cancer. RB family, and in particular the most studied member RB/p105, has been considered a tumor suppressor gene by more than three decades, and numerous efforts have been done to understand his molecular activity. However, the epigenetic mechanisms behind Rb-mediated tumor suppression have been uncovered only in recent years. In this review, the role of RB family members in cancer epigenetics will be discussed. We start with an introduction to epigenomes, chromatin modifications and cancer epigenetics. In order to provide a clear picture of the involvement of RB family in the epigenetic field, we describe the RB family role in the epigenetic landscape dynamics based on the heterochromatin variety involved, facultative or constitutive. We want to stress that, despite dissimilar modulations, RB family is involved in both mammalian varieties of heterochromatin establishment and maintenance and that disruption of RB family pathways drives to alterations of both heterochromatin structures, thus to the global epigenetic landscape.

Sputum analysis: non-invasive early lung cancer detection.

Lung cancer is the leading cause of cancer-related deaths over the world, characterized by a very high mortality rate. Molecular technique development tries to focus on early detection of cancers by studying molecular alterations that characterize cancer cells. Worldwide lung cancer research has focused on an ever-increasing number of molecular elements of carcinogenesis at genetic, epigenetic and protein levels. The non-invasiveness is the characteristic that all clinical trials on cancer detection should have. Abnormal chest imaging and/or non-specific symptoms are initial signals of lung cancer that appear in an advanced stage of disease. This fact represents the cause of the low 5-year survival rate: over 90% of patients dying within 5 years of diagnosis. Since smokers have higher quantity of sputum containing exfoliated cells from the bronchial tree, and the sputum represents the most easily accessible biological fluid and its collection is non-invasive, analysis of this sample represents a good area of research in early lung cancer diagnosis. Continued cigarette smoking is the cause of chronic obstructive pulmonary disease (COPD), with an estimated attributable risk factor exceeding 80% in smoking affected individuals. Lung cancer is found in 40-70% of patients with COPD, particularly in severe disease, and it is a common cause of death in these patients. A large prospective trial of almost half a million non-smokers showed as lung cancer is also common in patients with COPD who have never smoked. This review describes issues related to early lung cancer screening using non-invasive methods.

Activation and function of murine Cyclin T2A and Cyclin T2B during skeletal muscle differentiation.

Cyclin-dependent kinase 9 (Cdk9) is a serine-threonine kinase, involved in many cellular processes. The regulatory units of Cdk9 are the T family Cyclins (T1, T2) and Cyclin K. Cyclin T2 has two forms termed Cyclin T2a and Cyclin T2b that arise by an alternative splicing of the primary transcript. Upon induction of muscle differentiation, MyoD recruits Cdk9/Cyclin T2 on muscle-specific gene promoter sequences. This complex is able to phosphorylate the C-terminal domain of RNA polymerase II, enhancing MyoD function and promoting myogenic differentiation. This work focuses on the characterization of two murine Cyclin T2 isoforms and the evaluation of the role of Cdk9/Cyclin T2 complexes during the skeletal muscle differentiation. This study demonstrated a predominant expression of isoform b in all stages of differentiation. Moreover, both isoforms of Cyclin T2 are able to activate the myogenic program but Cyclin T2b has a predominant role, in particular during the latest stages.

PRC2: an epigenetic multiprotein complex with a key role in the development of rhabdomyosarcoma carcinogenesis.

Skeletal muscle formation represents a complex of highly organized and specialized systems that are still not fully understood. Epigenetic systems underline embryonic development, maintenance of stemness, and progression of differentiation. Polycomb group proteins play the role of gene silencing of stemness markers that regulate muscle differentiation. Enhancer of Zeste EZH2 is the catalytic subunit of the complex that is able to trimethylate lysine 27 of histone H3 and induce silencing of the involved genes. In embryonal Rhabdomyosarcoma and several other tumors, EZH2 is often deregulated and, in some cases, is associated with tumor malignancy. This review explores the molecular processes underlying the failure of muscle differentiation with a focus on the PRC2 complex. These considerations could open new studies aimed at the development of new cutting-edge therapeutic strategies in the onset of Rhabdomyosarcoma.

Clarifying the molecular mechanism of tomentosin­induced antiproliferative and proapoptotic effects in human multiple myeloma via gene expression profile and genetic interaction network analysis.

Multiple myeloma (MM) is an aggressive B cell malignancy. Substantial progress has been made in the therapeutic context for patients with MM, however it still represents an incurable disease due to drug resistance and recurrence. Development of more effective or synergistic therapeutic approaches undoubtedly represents an unmet clinical need. Tomentosin is a bioactive natural sesquiterpene lactone extracted by various plants with therapeutic properties, including anti­neoplastic effects. In the present study, the potential antitumor activity of tomentosin was evaluated on the human RPMI­8226 cell line, treated with increasing tomentosin concentration for cytotoxicity screening. The data suggested that both cell cycle arrest and cell apoptosis could explain the antiproliferative effects of tomentosin and may result in the inhibition of RPMI­8226 cell viability. To assess differentially expressed genes contributing to tomentosin activity and identify its mechanism of action, a microarray gene expression profile was performed, identifying 126 genes deregulated by tomentosin. To address the systems biology and identify how tomentosin deregulates gene expression in MM from a systems perspective, all deregulated genes were submitted to enrichment and molecular network analysis. The Protein­Protein Interaction (PPI) network analysis showed that tomentosin in human MM induced the downregulation of genes involved in several pathways known to lead immune­system processes, such as cytokine­cytokine receptor interaction, chemokine or NF­κB signaling pathway, as well as genes involved in pathways playing a central role in cellular neoplastic processes, such as growth, proliferation, migration, invasion and apoptosis. Tomentosin also induced endoplasmic reticulum stress via upregulation of cyclic AMP­dependent transcription factor ATF­4 and DNA damage­inducible transcript 3 protein genes, suggesting that in the presence of tomentosin the protective unfolded protein response signaling may induce cell apoptosis. The functional connections analysis executed using the Connectivity Map tool, suggested that the effects of tomentosin on RPMI­8226 cells might be similar to those exerted by heat shock proteins inhibitors. Taken together, these data suggested that tomentosin may be a potential drug candidate for the treatment of MM.

Tomentosin a Sesquiterpene Lactone Induces Antiproliferative and Proapoptotic Effects in Human Burkitt Lymphoma by Deregulation of Anti- and Pro-Apoptotic Genes.

(1) Tomentosin is the most representative sesquiterpene lactone extracted by I. viscosa. Recently, it has gained particular attention in therapeutic oncologic fields due to its anti-tumor properties. (2) In this study, the potential anticancer features of tomentosin were evaluated on human Burkitt's lymphoma (BL) cell line, treated with increasing tomentosin concentration for cytotoxicity screening. (3) Our data showed that both cell cycle arrest and cell apoptosis induction are responsible of the antiproliferative effects of tomentosin and may end in the inhibition of BL cell viability. Moreover, a microarray gene expression profile was performed to assess differentially expressed genes contributing to tomentosin activity. Seventy-five genes deregulated by tomentosin have been identified. Downregulated genes are enriched in immune-system pathways, and PI3K/AKT and JAK/STAT pathways which favor proliferation and growth processes. Importantly, different deregulated genes identified in tomentosin-treated BL cells are prevalent in molecular pathways known to lead to cellular death, specifically by apoptosis. Tomentosin-treatment in BL cells induces the downregulation of antiapoptotic genes such as BCL2A1 and CDKN1A and upregulation of the proapoptotic PMAIP1 gene. (4) Overall, our results suggest that tomentosin could be taken into consideration as a potential natural product with limited toxicity and relevant anti-tumoral activity in the therapeutic options available to BL patients.

Synthesis and cytotoxicity of novel hexahydrothienocycloheptapyridazinone derivatives.

Designed as a new group of tricyclic molecules containing the thienocycloheptapyridazinone ring system, a number of 2N-substituted-hexahydrothienocycloheptapyridazinone derivatives were synthesized and their biological activity evaluated. Among the synthesized compounds, derivatives 7d and 7h were found to possess cytotoxic activity against non-small cell lung cancer and central nervous system cancer cell lines, respectively.

"Verteporfin exhibits anti-proliferative activity in embryonal and alveolar rhabdomyosarcoma cell lines".

Rhabdomyosarcoma (RMS) is a pediatric tumor, which arises from muscle precursor cells. Recently, it has been demonstrated that Hippo Pathway (Hpo), a pathway that regulates several physiological and biological features, is involved in RMS tumorigenesis. For instance, an upregulation of the Hpo downstream effector Yes-Associated Protein 1 (YAP) leads to the development of embryonal rhabdomyosarcoma (eRMS) in murine activated muscle satellite cells. On the other hand, the YAP paralog transcriptional co-activator with PDZ-binding motif (TAZ) is overexpressed in alveolar rhabdomyosarcoma (aRMS) patients with poor survival. YAP and TAZ exhibit both cytoplasmic and nuclear functions. In the nucleus, YAP binds TEADs (TEA domain family members) factors and together they constitute a complex that is able either to activate the transcription of several genes such as MYC, Tbx5 and PAX8 or to maintain the stability of others like p73. Due to the key role of YAP and TAZ in cancer, the identification and/or development of new compounds able to block their activity might be an effective antineoplastic strategy. Verteporfin (VP) is a molecule able to stop the formation of YAP/TEAD complex in the nucleus. The aim of this study is to evaluate the action of VP on RMS cell lines. This work shows that VP has an anti-proliferative activity on all RMS cell lines analyzed. Depending on RMS cell lines, VP affects cell cycle differently. Moreover, VP is able to decrease YAP protein levels, and to induce the activation of apoptosis mechanism through the cleavage of PARP-1. In addition, Annexin V assay showed the activation of apoptosis and necrosis after VP treatment. In summary, the ability of VP to disrupt RMS cell proliferation could be a novel and valuable strategy to improve the therapeutic approaches in treating rhabdomyosarcoma.

Targeting Enhancer of Zeste Homolog 2 as a promising strategy for cancer treatment.

Polycomb group proteins represent a global silencing system involved in development regulation. In specific, they regulate the transition from proliferation to differentiation, contributing to stem-cell maintenance and inhibiting an inappropriate activation of differentiation programs. Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of Polycomb repressive complex 2, which induces transcriptional inhibition through the tri-methylation of histone H3, an epigenetic change associated with gene silencing. EZH2 expression is high in precursor cells while its level decreases in differentiated cells. EZH2 is upregulated in various cancers with high levels associated with metastatic cancer and poor prognosis. Indeed, aberrant expression of EZH2 causes the inhibition of several tumor suppressors and differentiation genes, resulting in an uncontrolled proliferation and tumor formation. This editorial explores the role of Polycomb repressive complex 2 in cancer, focusing in particular on EZH2. The canonical function of EZH2 in gene silencing, the non-canonical activities as the methylation of other proteins and the role in gene transcriptional activation, were summarized. Moreover, mutations of EZH2, responsible for an increased methyltransferase activity in cancer, were recapitulated. Finally, various drugs able to inhibit EZH2 with different mechanism were described, specifically underscoring the effects in several cancers, in order to clarify the role of EZH2 and understand if EZH2 blockade could be a new strategy for developing specific therapies or a way to increase sensitivity of cancer cells to standard therapies.

Synthesis and Antineoplastic Evaluation of Novel Unsymmetrical 1,3,4-Oxadiazoles.

A series of novel 1,3,4-oxadiazoles was synthesized and evaluated for their cytotoxic activity in in vitro tumor models. Four of the new compounds (2d, 2j, 2k, and 2n) showed growth inhibition in the XTT dye assay. The most active agent, 2j, showed high potency against human cancer cells with IC50s ranging from 0.05 to 1.7 µM. Preliminary SAR correlations suggested that the nature of chains on the oxadiazole is important for antitumor potency in vitro. Compound 2j determined a G2/M arrest of the cell cycle and also activated a strong apoptotic response. The ß-tubulin immunofluorescence analysis indicated that compound 2j effectively inhibited the microtubule organization in all cancer cell lines, causing the formation of abnormal spindle, which did not affect the normal human fibroblast cells NB1, Mrc-5 and IBR3. For all these reasons, compound 2j could be a good candidate in chemopreventive or chemotherapeutic strategies.

Roles of enhancer of zeste homolog 2: from skeletal muscle differentiation to rhabdomyosarcoma carcinogenesis.

Polycomb group proteins represent a global silencing system involved in embryonic development and stem-cell maintenance that regulates the transition from proliferation to differentiation during organogenesis. Two main complexes have been discovered: the polycomb repressive complex (PRC) 1 and 2, able to induce gene silencing by a synergistic mechanism or independently by each other. Enhancer of zeste homolog 2 (EZH2), the catalytic subunit of PRC2, represses gene transcription through the tri-methylation of histone H3 lysine 27. EZH2 deregulation is frequently associated with tumorigenesis, metastatic character, and poor prognosis in various cancer types. This review explores the role of EZH2 in normal development and in carcinogenesis. We reviewed the polycomb-mediated silencing mechanisms, the regulation of EZH2 activity and its recruitment to target genes. We also analyzed the role of EZH2 in normal muscle differentiation and in rhabdomyosarcoma, considering EZH2 blockade as a new strategy for developing specific therapies.

The ablation of EZH2 uncovers its crucial role in rhabdomyosarcoma formation.

Rhabdomyosarcoma (RMS) is a pediatric tumor that arises from muscle precursor cells. RMS cells express several markers of early myogenic differentiation, but they fail to complete both differentiation program and cell cycle arrest, resulting in uncontrolled proliferation and incomplete myogenesis. Previous studies showed that EZH2, which is involved in both differentiation and cancer progression, is overexpressed in RMS, but a functional binding between its expression and its functional role in tumor formation or progression has not yet been demonstrated. We hypothesized that EZH2 is a key regulator of muscular differentiation program in RMS cells. In this study, we demonstrated that EZH2 directly binds muscle specific genes in RD cells. Silencing of EZH2 promotes the recruitment of a multiprotein complex at muscle-specific promoters, their transcriptional activation and protein expression. Moreover, we demonstrated that EZH2 is directly involved in transcriptional repression of MyoD, the main factor promoting myogenesis. EZH2 ablation induces MyoD activation the recovery of its binding on muscle-specific genes.