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MicroRNAs are small non-coding regulatory molecules exerting pleiotropic action in different biological processes such as proliferation, differentiation, apoptosis, migration and metastasis. Deregulation of miRNA expression has been observed in various cancers, and accumulating data suggest that miRNAs can display an oncogenic, antioncogenic or an ambiguous behavior in relationship to tumor environment. In a previous research we showed that the synthetic cannabinoid WIN55,212-2 is able to reduce the migratory activity of osteosarcoma MG63 cells analyzed by means of wound healing assay. So we undertook a study to evaluate the biochemical mechanism through which WIN plays this action. To this purpose we evaluated the levels of miR-29b1, a member of miR-29 family which has been shown to impact critical steps in the migratory and metastatic cascade, such as EMT, apoptosis and angiogenesis. RT-PCR experiments showed that in MG63 cells 5 M WIN increased the level of miR-29b1 of about 700-fold. This effect was accompanied by the reduction in its putative targets MMP-2, PDGF-B and N-MYC, thus indicating that the miRNA is functionally active. Moreover, cells stably overexpressing miR-29b1 did not close the wound after 48 h, mimicking the effect of WIN in untransfected control cells. Notably, ERα(+) MCF-7 and triple negative MDA-MB-231 cells, two different breast cancer models, treated with the cannabinoid migrated into the scratched area significantly faster than the respective control cells. In these cells WIN also increased the level of miR-29b1 targets. Therefore, differently from osteosarcoma cells, these preliminary observations seem to indicate that WIN promotes migration ability in breast cancer cells. The reasons for this diverse behaviour could rely on miR-29b1, whose expression can change in different cell types or show temporal differences dictated by cell physiology and tumor microenvironment impact. Studies are in progress to shed light on the molecular mechanisms underlying this different response.

Caspase-8 (casp-8) is well known as an initiator caspase involved in cell death signalling, although its activity in many cancer cell types seems to work under non-apoptotic conditions. Moreover, in several types of cancer, casp-8 is only rarely mutated and often its expression is very elevated. Since cancer cell growth also depends on evasion of apoptosis, the upregulation of casp-8 in tumours may suggest one or more non-apoptotic roles (1). Here we report our recent studies carried out in MDA-MB-231 cells, derived from clinically aggressive forms of Triple-Negative Breast Cancer, where we have assessed the non-canonical roles of casp-8. Firstly, we evaluated casp-8 mRNA and protein levels in MDA-MB-231 cells, demonstrating that they were upregulated with respect to HMEC (normal Human Mammalian Epithelial Cells). Thereafter, to assess the role of casp-8, we silenced it by small interfering-RNA. Interestingly casp-8-knockdown, strongly decreased MDA-MB-231 cell growth by delaying G0/G1- to S-phase transition and increasing p21, p27 and hypophosphorylated/active form of pRb levels. No effects were evidenced on cell viability. To assess the metastatic capacity of MDA-MB-231 cells, the gene expression profiles of the relative markers after casp-8 knockdown were also measured. Surprisingly the expression of a number of genes and/or proteins such as VEGFA, C-MYC, CTNNB1, HMGA2, CXCR4, KLF4, VERSICAN V1 and MMP2 potently increased accompanied by migratory and metastatic capacities of cells, as shown by wound healing and matrigel assays. We suggest that among these genes, KLF4, a transcriptional factor with a dual role (activator and repressor), and responsible for p21 and p27 induction, could play critical roles (2). Casp-8 through KLF4 down-regulation, could manage the expression of critical proliferative and migratory/invasive genes. We suggest that these unusual roles played by casp-8 in MDA-MB-231 cells, should be better explored, in order to identify it as a molecular therapeutic target. References 1. Stupack DG. Caspase-8 as a Therapeutic Target in Cancer. Cancer Lett 332:133–140, 2013. 2. Tiwari N et al. Klf4 Is a Transcriptional Regulator of Genes Critical for EMT, Including Jnk1 (Mapk8). PLoS One 8, 2013.

Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/ mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology. © The Author 2015.

Goodson, William H. et al., © The Author 2015. Lifestyle factors are responsible for a considerable portion of cancer incidence worldwide, but credible estimates from the World Health Organization and the International Agency for Research on Cancer (IARC) suggest that the fraction of cancers attributable to toxic environmental exposures is between 7% and 19%. To explore the hypothesis that low-dose exposures to mixtures of chemicals in the environment may be combining to contribute to environmental carcinogenesis, we reviewed 11 hallmark phenotypes of cancer, multiple priority target sites for disruption in each area and prototypical chemical disruptors for all targets, this included dose-response characterizations, evidence of low-dose effects and cross-hallmark effects for all targets and chemicals. In total, 85 examples of chemicals were reviewed for actions on key pathways/ mechanisms related to carcinogenesis. Only 15% (13/85) were found to have evidence of a dose-response threshold, whereas 59% (50/85) exerted low-dose effects. No dose-response information was found for the remaining 26% (22/85). Our analysis suggests that the cumulative effects of individual (non-carcinogenic) chemicals acting on different pathways, and a variety of related systems, organs, tissues and cells could plausibly conspire to produce carcinogenic synergies. Additional basic research on carcinogenesis and research focused on low-dose effects of chemical mixtures needs to be rigorously pursued before the merits of this hypothesis can be further advanced. However, the structure of the World Health Organization International Programme on Chemical Safety 'Mode of Action' framework should be revisited as it has inherent weaknesses that are not fully aligned with our current understanding of cancer biology., We gratefully acknowledge the support of the National Institute of Health-National Institute of Environmental Health Sciences (NIEHS) conference grant travel support (R13ES023276); Glenn Rice, Office of Research and Development, United States Environmental Protection Agency, Cincinnati, OH, USA also deserves thanks for his thoughtful feedback and inputs on the manuscript; William H.Goodson III was supported by the California Breast Cancer Research Program, Clarence Heller Foundation and California Pacific Medical Center Foundation; Abdul M.Ali would like to acknowledge the financial support of the University of Sultan Zainal Abidin, Malaysia; Ahmed Lasfar was supported by an award from the Rutgers Cancer Institute of New Jersey; Ann-Karin Olsen and Gunnar Brunborg were supported by the Research Council of Norway (RCN) through its Centres of Excellence funding scheme (223268/F50), Amancio Carnero’s lab was supported by grants from the Spanish Ministry of Economy and Competitivity, ISCIII (Fis: PI12/00137, RTICC: RD12/0036/0028) co-funded by FEDER from Regional Development European Funds (European Union), Consejeria de Ciencia e Innovacion (CTS-1848) and Consejeria de Salud of the Junta de Andalucia (PI-0306-2012); Matilde E. Lleonart was supported by a trienal project grant PI12/01104 and by project CP03/00101 for personal support. Amaya Azqueta would like to thank the Ministerio de Educacion y Ciencia (‘Juande la Cierva’ programme, 2009) of the Spanish Government for personal support; Amedeo Amedei was supported by the Italian Ministry of University and Research (2009FZZ4XM_002), and the University of Florence (ex60%2012); Andrew R.Collins was supported by the University of Oslo; Annamaria Colacci was supported by the Emilia-Romagna Region - Project ‘Supersite’ in Italy; Carolyn Baglole was supported by a salary award from the Fonds de recherche du Quebec-Sante (FRQ-S); Chiara Mondello’s laboratory is supported by Fondazione Cariplo in Milan, Italy (grant n. 2011-0370); Christian C.Naus holds a Canada Research Chair; Clement Yedjou was supported by a grant from the National Institutes of Health (NIH-NIMHD grant no. G12MD007581); Daniel C.Koch is supported by the Burroughs Wellcome Fund Postdoctoral Enrichment Award and the Tumor Biology Training grant: NIH T32CA09151; Dean W. Felsher would like to acknowledge the support of United States Department of Health and Human Services, NIH grants (R01 CA170378 PQ22, R01 CA184384, U54 CA149145, U54 CA151459, P50 CA114747 and R21 CA169964); Emilio Rojas would like to thank CONACyT support 152473, Ezio Laconi was supported by AIRC (Italian Association for Cancer Research, grant no. IG 14640) and by the Sardinian Regional Government (RAS); Eun-Yi Moon was supported by grants from the Public Problem-Solving Program (NRF-015M3C8A6A06014500) and Nuclear R&D Program (#2013M2B2A9A03051296 and 2010-0018545) through the National Research Foundation of Korea (NRF) and funded by the Ministry of Education, Science and Technology (MEST) in Korea; Fahd Al-Mulla was supported by the Kuwait Foundation for the Advancement of Sciences (2011-1302-06); Ferdinando Chiaradonna is supported by SysBioNet, a grant for the Italian Roadmap of European Strategy Forum on Research Infrastructures (ESFRI) and by AIRC (Associazione Italiana Ricerca sul Cancro; IG 15364); Francis L.Martin acknowledges funding from Rosemere Cancer Foundation; he also thanks Lancashire Teaching Hospitals NHS trust and the patients who have facilitated the studies he has undertaken over the course of the last 10 years; Gary S.Goldberg would like to acknowledge the support of the New Jersey Health Foundation; Gloria M.Calaf was supported by Fondo Nacional de Ciencia y Tecnología (FONDECYT), Ministerio de Educación de Chile (MINEDUC), Universidad de Tarapacá (UTA); Gudrun Koppen was supported by the Flemish Institute for Technological Research (VITO), Belgium; Hemad Yasaei was supported from a triennial project grant (Strategic Award) from the National Centre for the Replacement, Refinement and Reduction (NC3Rs) of animals in research (NC.K500045.1 and G0800697); Hiroshi Kondoh was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, Japan Science and Technology Agency and by JST, CREST; Hsue-Yin Hsu was supported by the Ministry of Science and Technology of Taiwan (NSC93-2314-B-320-006 and NSC94-2314-B-320-002); Hyun Ho Park was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) of the Ministry of Education, Science and Technology (2012R1A2A2A01010870) and a grant from the Korea Healthcare Technology R&D project, Ministry of Health and Welfare, Republic of Korea (HI13C1449); Igor Koturbash is supported by the UAMS/NIH Clinical and Translational Science Award (UL1TR000039 and KL2TR000063) and the Arkansas Biosciences Institute, the major research component of the Arkansas Tobacco Settlement Proceeds Act of 2000; Jan Vondráček acknowledges funding from the Czech Science Foundation (13-07711S); Jesse Roman thanks the NIH for their support (CA116812), John Pierce Wise Sr. and Sandra S.Wise were supported by National Institute of Environmental Health Sciences (ES016893 to J.P.W.) and the Maine Center for Toxicology and Environmental Health; Jonathan Whitfield acknowledges support from the FERO Foundation in Barcelona, Spain; Joseph Christopher is funded by Cancer Research UK and the International Journal of Experimental Pathology; Julia Kravchenko is supported by a philanthropic donation by Fred and Alice Stanback; Jun Sun is supported by a Swim Across America Cancer Research Award; Karine A.Cohen-Solal is supported by a research scholar grant from the American Cancer Society (116683-RSG-09-087-01-TBE); Laetitia Gonzalez received a postdoctoral fellowship from the Fund for Scientific Research–Flanders (FWO-Vlaanderen) and support by an InterUniversity Attraction Pole grant (IAP-P7-07); Laura Soucek is supported by grant #CP10/00656 from the Miguel Servet Research Contract Program and acknowledges support from the FERO Foundation in Barcelona, Spain; Liang-Tzung Lin was supported by funding from the Taipei Medical University (TMU101-AE3-Y19); Linda Gulliver is supported by a Genesis Oncology Trust (NZ) Professional Development Grant, and the Faculty of Medicine, University of Otago, Dunedin, New Zealand; Louis Vermeulen is supported by a Fellowship of the Dutch Cancer Society (KWF, UVA2011-4969) and a grant from the AICR (14–1164); Mahara Valverde would like to thank CONACyT support 153781; Masoud H. Manjili was supported by the office of the Assistant Secretary of Defense for Health Affairs (USA) through the Breast Cancer Research Program under Award No. W81XWH-14-1-0087 Neetu Singh was supported by grant #SR/FT/LS-063/2008 from the Department of Science and Technology, Government of India; Nicole Kleinstreuer is supported by NIEHS contracts (N01-ES 35504 and HHSN27320140003C); P.K. Krishnakumar is supported by the Funding (No. T.K. 11-0629) of King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia; Paola A.Marignani is supported by the Dalhousie Medical Research Foundation, The Beatrice Hunter Cancer Institute and CIHR and the Nova Scotia Lung Association; Paul Dent is the holder of the Universal Inc.Chair in Signal Transduction Research and is supported with funds from PHS grants from the NIH (R01-CA141704, R01-CA150214, R01-DK52825 and R01-CA61774); Petr Heneberg was supported by the Charles University in Prague projects UNCE 204015 and PRVOUK P31/2012, and by the Czech Science Foundation projects P301/12/1686 and 15-03834Y; Po Sing Leung was supported by the Health and Medical Research Fund of Food and Health Bureau, Hong Kong Special Administrative Region, Ref. No: 10110021; Qiang Cheng was supported in part by grant NSF IIS-1218712; R. Brooks Robey is supported by the United States Department of Veterans Affairs; Rabindra Roy was supported by United States Public Health Service Grants (RO1 CA92306, RO1 CA92306-S1 and RO1 CA113447); Rafaela Andrade-Vieira is supported by the Beatrice Hunter Cancer Research Institute and the Nova Scotia Health Research Foundation, Renza Vento was partially funded by European Regional Development Fund, European Territorial Cooperation 2007–13 (CCI 2007 CB 163 PO 037, OP Italia-Malta 2007–13) and grants from the Italian Ministry of Education, University and Research (MIUR) ex-60%, 2007; Riccardo Di Fiore was a recipient of fellowship granted by European Regional Development Fund, European Territorial Cooperation 2007–2013 (CCI 2007 CB 163 PO 037, OP Italia-Malta 2007–2013); Rita Dornetshuber-Fleiss was supported by the Austrian Science Fund (FWF, project number T 451-B18) and the Johanna Mahlke, geb.-Obermann-Stiftung; Roberta Palorini is supported by a SysBioNet fellowship; Roslida Abd Hamid is supported by the Ministry of Education, Malaysia-Exploratory Research Grant Scheme-Project no: ERGS/1-2013/5527165; Sabine A.S.Langie is the beneficiary of a postdoctoral grant from the AXA Research Fund and the Cefic-LRI Innovative Science Award 2013; Sakina Eltom is supported by NIH grant SC1CA153326; Samira A.Brooks was supported by National Research Service Award (T32 ES007126) from the National Institute of Environmental Health Sciences and the HHMI Translational Medicine Fellowship; Sandra Ryeom was supported by The Garrett B. Smith Foundation and the TedDriven Foundation; Thierry Massfelder was supported by the Institut National de la Santé et de la Recherche Médicale INSERM and Université de Strasbourg; Thomas Sanderson is supported by the Canadian Institutes of Health Research (CIHR; MOP-115019), the Natural Sciences and Engineering Council of Canada (NSERC; 313313) and the California Breast Cancer Research Program (CBCRP; 17UB-8703); Tiziana Guarnieri is supported by a grant from Fundamental Oriented Research (RFO) to the Alma Mater Studiorum University of Bologna, Bologna, Italy and thanks the Fondazione Cassa di Risparmio di Bologna and the Fondazione Banca del Monte di Bologna e Ravenna for supporting the Center for Applied Biomedical Research, S.Orsola-Malpighi University Hospital, Bologna, Italy; W.Kimryn Rathmell is supported by the V Foundation for Cancer Research and the American Cancer Society; William K.Decker was supported in part by grant RP110545 from the Cancer Prevention Research Institute of Texas; William H.Bisson was supported with funding from the NIH P30 ES000210; Yon Rojanasakul was supported with NIH grant R01-ES022968; Zhenbang Chen is supported by NIH grants (MD004038, CA163069 and MD007593); Zhiwei Hu is grateful for the grant support from an institutional start-up fund from The Ohio State University College of Medicine and The OSU James Comprehensive Cancer Center (OSUCCC) and a Seed Award from the OSUCCC Translational Therapeutics Program.

The Bcl-2 family, which plays important roles in controlling cancer development, is divided into antiapoptotic and proapoptotic members. The change in the balance between these members governs the life and death of the cells. Mcl-1 is an antiapoptotic member of this family and its distribution in normal and cancerous tissues strongly differs from that of Bcl-2. In human cancers, where upregulation of antiapoptotic proteins is common, Mcl-1 expression is regulated independent of Bcl-2 and its inhibition promotes senescence, a major barrier to tumorigenesis. Cancer chemotherapy determines various kinds of responses, such as senescence and autophagy; however, the ideal response to chemotherapy is apoptosis. Mcl-1 is a potent oncogene that is regulated at the transcriptional, posttranscriptional, and posttranslational levels. Mcl-1 is a short-lived protein that, in the NH2 terminal region, contains sites for posttranslational regulation that can lead to proteasomal degradation. The USP9X Mcl-1 deubiquitinase regulates Mcl-1 and the levels of these two proteins are strongly correlated. Mcl-1 has three splicing variants (the antiapoptotic protein Mcl-1L and the proapoptotic proteins Mcl-1S and Mcl-1ES), each contributing toward apoptosis regulation. In cancers responsible for the most deaths in the world, the presence of Mcl-1 is associated with malignant cell growth and evasion of apoptosis. Mcl-1 is also one of the key regulators ofcancer stem cells’ self-renewal that contributes to tumor survival. A great number of indirect and selective Mcl-1 inhibitors have been produced and some of these have shown efficacy in several clinical trials. Thus, therapeutic manipulation of Mcl-1 can be a useful strategy to combat cancer.

MiR-29 family dysregulation occurs in various cancers including breast cancers. We investigated miR-29b-1 functional role in human triple negative breast cancer (TNBC) the most aggressive breast cancer subtype. We found that miR-29b-1-5p was downregulated in human TNBC tissues and cell lines. To assess whether miR- 29b-1-5p correlated with TNBC regenerative potential, we evaluated cancer stem cell enrichment in our TNBC cell lines, and found that only MDA-MB-231 and BT-20 produced primary, secondary and tertiary mammospheres, which were progressively enriched in OCT4, NANOG and SOX2 stemness genes. MiR-29b-1-5p expression inversely correlated with mammosphere stemness potential, and miR-29b-1 ectopic overexpression decreased TNBC cell growth, self-renewal, migration, invasiveness and paclitaxel resistance repressing WNT/βcatenin and AKT signaling pathways and stemness regulators. We identified SPINDLIN1 (SPIN1) among predicted miR-29b- 1-5p targets. Consistently, SPIN1 was overexpressed in most TNBC tissues and cell lines and negatively correlated with miR-29b-1-5p. Target site inhibition showed that SPIN1 seems to be directly controlled by miR-29b-1-5p. Silencing SPIN1 mirrored the effects triggered by miR-29b-1 overexpression, whereas SPIN1 rescue by SPIN1miScript protector, determined the reversal of the molecular effects produced by the mimic-miR-29b-1-5p. Overall, we show that miR-29b-1 deregulation impacts on multiple oncogenic features of TNBC cells and their renewal potential, acting, at least partly, through SPIN1, and suggest that both these factors should be evaluated as new possible therapeutic targets against TNBC., This study was partially funded by the European Regional Development Fund, European Territorial Cooperation 2007-2013, CCI 2007 CB 163 PO 037, OP Italia-Malta 2007-2013; the Italian Ministry of Education, University and Research (MIUR) ex-60%, 2013; R. Di Fiore and R. Drago-Ferrante were recipients of fellowships granted by the European Regional Development Fund, European Territorial Cooperation 2007-2013, CCI 2007 CB 163 PO 037, OP Italia-Malta 2007-2013; D. Carlisi was a recipient of a fellowship granted by MIUR (contract no. 82, January 23, 2014)., peer-reviewed

Osteosarcoma (OS), an aggressive highly invasive and metastatic bone-malignancy, shows therapy resistance and recurrence, two features that likely depend on cancer stem cells (CSCs), which hold both self-renewing and malignant potential. So, effective anticancer therapies against OS should specifically target and destroy CSCs. We previously found that the let-7d microRNA was downregulated in the 3AB-OS-CSCs, derived from the human OS-MG63 cells. Here, we aimed to assess whether let-7d modulation affected tumorigenic and stemness properties of these OS-CSCs. We found that let-7d-overexpression reduced cell proliferation by decreasing CCND2 and E2F2 cell-cycle-activators and increasing p21 and p27 CDK-inhibitors. Let-7d also decreased sarcosphere-and-colony forming ability, two features associated with self-renewing, and it reduced the expression of stemness genes, including Oct3/4, Sox2, Nanog, Lin28B, and HMGA2. Moreover, let-7d induced mesenchymal-to-epithelial-transition, as shown by both N-Cadherin-E-cadherin-switch and decrease in vimentin. Surprisingly, such switch was accompanied by enhanced migratory/invasive capacities, with a strong increase in MMP9, CXCR4 and VersicanV1. Let-7d- overexpression also reduced cell sensitivity to apoptosis induced by both serum-starvation and various chemotherapy drugs, concomitant with decrease in caspase-3 and increase in BCL2 expression. Our data suggest that let-7d in 3AB-OS-CSCs could induce plastic-transitions from CSCs-to-non-CSCs and vice-versa. To our knowledge this is the first study to comprehensively examine the expression and functions of let-7d in OS-CSCs. By showing that let-7d has both tumor suppressor and oncogenic functions in this context, our findings suggest that, before prospecting new therapeutic strategies based on let-7d modulation, it is urgent to better define its multiple functions. J. Cell. Physiol. 231: 1832-1841, 2016. © 2015 Wiley Periodicals, Inc.

Triple-negative breast cancer (TNBC) is a clinically aggressive form of breast cancer that is unresponsive to endocrine agents or trastuzumab. TNBC accounts for ~10-20% of all breast cancer cases and represents the form with the poorest prognosis. Patients with TNBC are at higher risk of early recurrence, mainly in the lungs, brain and soft tissue, therefore, there is an urgent need for new therapies. The present study was carried out in MDA-MB-231 cells, where we assessed the role of caspase-8 (casp-8), a critical effector of death receptors, also involved in non‑apoptotic functions. Analysis of casp-8 mRNA and protein levels indicated that they were up-regulated with respect to the normal human mammalian epithelial cells. We demonstrated that silencing of casp-8 by small interfering-RNA, strongly decreased MDA-MB-231 cell growth by delaying G0/G1- to S-phase transition and increasing p21, p27 and hypo-phosphorylated/active form of pRb levels. Surprisingly, casp-8-knockdown, also potently increased both the migratory and metastatic capacity of MDA-MB‑231 cells, as shown by both wound healing and Matrigel assay, and by the expression of a number of related-genes and/or proteins such as VEGFA, C-MYC, CTNNB1, HMGA2, CXCR4, KLF4, VERSICAN V1 and MMP2. Among these, KLF4, a transcriptional factor with a dual role (activator and repressor), seemed to play critical roles. We suggest that in MDA-MB‑231 cells, the endogenous expression of casp-8 might keep the cells perpetually cycling through downregulation of KLF4, the subsequent lowering of p21 and p27, and the inactivation by hyperphosphorylation of pRb. Simultaneously, by lowering the expression of some migratory and invasive genes, casp-8 might restrain the metastatic ability of the cells. Overall, our findings showed that, in MDA-MB-231 cells, casp-8 might play some unusual roles which should be better explored, in order to understand whether it might be identified as a molecular therapeutic target.

The sesquiterpene lactone Parthenolide (PN) exerted a cytotoxic effect on MDA-MB231 cells, a triple-negative breast cancer (TNBC) cell line, but its effectiveness was scarce when employed at low doses. This represents an obstacle for a therapeutic utilization of PN. In order to overcome this difficulty we associated to PN the suberoylanilide hydroxamic acid (SAHA), an histone deacetylase inhibitor. Our results show that SAHA synergistically sensitized MDA-MB231 cells to the cytotoxic effect of PN. It is noteworthy that treatment with PN alone stimulated the survival pathway Akt/mTOR and the consequent nuclear translocation of Nrf2, while treatment with SAHA alone induced autophagic activity. However, when the cells were treated with SAHA/PN combination, SAHA suppressed PN effect on Akt/mTOR/Nrf2 pathway, while PN reduced the prosurvival autophagic activity of SAHA. In addition SAHA/PN combination induced GSH depletion, fall in m, release of cytochrome c, activation of caspase 3 and apoptosis. Finally we demonstrated that combined treatment maintained both hyperacetylation of histones H3 and H4 induced by SAHA and down-regulation of DNMT1 expression induced by PN. Inhibition of the DNA-binding activity of NF-kB, which is determined by PN, was also observed after combined treatment. In conclusion, combination of PN to SAHA inhibits the cytoprotective responses induced by the single compounds, but does not alter the mechanisms leading to the cytotoxic effects. Taken together our results suggest that this combination could be a candidate for TNBC therapy. J. Cell. Physiol. 230: 1276-1289, 2015. (c) 2014 Wiley Periodicals, Inc., A Wiley Company

Triple-negative breast cancers (TNBCs) are aggressive forms of breast carcinoma associated with a high rate of recidivism. It is known that a small proportion of tumour cells, termed cancer stem cells (CSCs), is responsible for tumour formation, progression and recurrence. The sesquiterpene lactone parthenolide (PN) was identified as the first small molecule capable of killing CSCs.1 Previously we have shown2 that PN and its soluble analogue DMAPT induce a strong cytotoxic effect in MDA-MB231 cells, the most studied line of TNBCs. In the present research we investigated about the effects exerted by both PN and DMAPT on breast cancer stem-like cells derived from three lines of TNBCs (MDA-MB231, BT20 and MDA-MB436). The two compounds inhibited both the production of mammospheres from the three lines of cells and the viability of breast cancer stem-like cells derived from dissociation of mammospheres. This effect was suppressed by NAC, while z-VAD, a general inhibitor of caspase activity, was ineffective. PN and DMAPT induced in stem-like cells, in the first hours of treatment, a strong production of hydrogen peroxide. Prolonging the time of treatment (12-24h) the levels of both superoxide anion and hROS (hydroxyl radicals and peroxynitrite) increased in concomitance with down-regulation of MnSOD and catalase, dissipation of mitochondrial membrane potential and cell necrosis. It is noteworthy that treatment with PN and DMAPT also caused a rapid and remarkable decrement of the level of Nrf-2, which is a critical regulator of the intracellular antioxidant response. In conclusion PN and DMAPT markedly inhibited viability of stem-like cells derived from three lines of TNBCs by inducing ROS generation, mitochondrial dysfunction and cell necrosis.

Osteosarcoma (OS) is the most common malignancy of bone in children and adolescent. It is a highly invasive and metastatic bone-malignancy because of which, despite therapeutic advances, 30%-50% of patients still die of pulmonary metastasis. As a consequence, there is an urgent need to identify new therapeutic strategies to improve the clinical outcome of the patients. Advances in OS treatment are inconceivable without better understanding of molecular mechanism of osteosarmagenesis and, especially, metastatic processes. Growing evidence suggests that cancer stem cells (CSCs), which have self-renewing and malignant potential, are at the root of tumor growth and relapse. Thus, a challenge for innovative therapy is their identification and eradication. Here, we have used the 3AB-OS CSCs, a cell line previously produced in our laboratory from the OS-MG63 cells, which was genetically, molecularly and functionally characterized. This study was focused on the role of let-7d miRNA-previously found by us to be downregulated in 3AB-OS-CSCs- in managing their stemness properties. We have found that let-7d-overesperession reduces cell proliferation by both decreasing CCND2 and E2F2 cell-cycle-activators and increasing p21 and p27 CDK-inhibitors. Let-7d also reduces sarcosphere and colony-forming ability and the expression of Oct3/4, Sox2, Nanog, Lin28B and HMGA2, key regulators of cancer cell stemness. Moreover, let-7d induces mesenchymal-to-epithelium-transition, as shown by both N-Cadherin-E-cadherin-switch and vimentin decrease. Surprisingly, this swich was accompanied by enhanced migratory/invasive capacities and by increases in MMP9, CXCR4 and VersicanV1. Let-7d also reduced the resistance to serum starvation and chemotherapy. A decrease in caspase-3 with an increase in Bcl-2 was also observed. Overall, this study shows that let-7d displaying both suppressor and oncogenic functions behaves as a Janus-Faced miRNA. Thus, we suggest that, before prospecting new therapeutic strategies by let-7d modulation, it is urgent to better understand its functions.

The synthetic cannabinoid WIN 55,212-2 is a potent cannabinoid receptor agonist with anticancer potential. Experiments were performed to determine the effects of WIN on proliferation, cell cycle distribution, and programmed cell death in human osteosarcoma MG63 and Saos-2 cells. Results show that WIN induced G2/M cell cycle arrest, which was associated with the induction of the main markers of ER stress (GRP78, CHOP and TRB3). In treated cells we also observed the conversion of the cytosolic form of the autophagosome marker LC3-I into LC3-II (the lipidated form located on the autophagosome membrane) and the enhanced incorporation of monodansylcadaverine and acridine orange, two markers of the autophagic compartments such as autolysosomes. WIN also induced morphological effects in MG63 cells consisting in an increase in cell size and a marked cytoplasmic vacuolization. However, WIN effects were not associated with a canonical apoptotic pathway, as demonstrated by the absence of specific features, and only the addition of TRAIL to WIN-treated cells led to apoptotic death probably mediated by up-regulation of the tumor suppressor factor PAR-4, whose levels increased after WIN treatment, and by the translocation of GRP78 on cell surface.

The first description of a tumor resembling retinoblastoma (RB) was provided on 1597 by Pieter Pauw, who described a malignancy invading the orbit, the temporal region, and the cranium, filled with a "substance similar to brain tissue mixed with thick blood and like crushed stone". Since then, a number of retinal tumors were described and named until the 1922 when Verhoeff called these tumors RB, a term that the American Ophthalmological Society adopted in 1926. In 1971 Knudson focused on RB, and proposed his ‘two-hit’ theory of the molecular etiology of RB. In 1986, the RB1 gene was identified and the ‘two-hit’ theory of Knudson was validated. Successively, new studies in developing retinal cells suggested that two mutational events are not enough for malignant transformation and that other hits are associated with RB development. Since RB was discovered it has gone from a state in which near all children inevitably die, to the current condition in which, in developed world, more than 99% of children survive. Overall, RB can be defined as a curable cancer, provided it does not happen events which make the therapy a palliative, as metastasis, trilateral RB or secondary tumors. To establish plan treatments it is required to stage RB. This is possible by the “International Classification for Intraocular Retinoblastoma” which has divided intraocular retinoblastomas into 5 groups (A-E) based on the chances to save the eye using current treatment options. This staging system can provide information about the most effective current treatments. The current available therapeutic options for RB are numerous, with the indications to use specific modality or a combination of modalities varying according to the extent of the disease.

Invasion of cancer cells into surrounding tissue is an initial step in tumor metastasis. This event, which requires migration of cancer cells and attachment to extracellular matrix (ECM), is regulated by elements of the local microenvironment, including ECM architecture. After having demonstrated the ability of the synthetic cannabinoid WIN55,512 to induce osteosarcoma MG63 cell death (1), we studied the effects of WIN on MG63 cell migration. Wound healing assay was performed to measure the ability of cells to migrate and fill the gap obtained by physical disruption of cell monolayer (2). We observed a significant delay in wound closure in 5 M WIN treated cells compared to untreated cells that almost completed the healing in 24 hours (20% vs 87% of wound closure). The addition of conditioned medium obtained by confluent control cells to WIN treated cultures largely reverted the delaying WIN action. To evaluate the influence of matrix metalloproteinases (MMPs) in the migratory ability, we analyzed MMP-2 and MMP-9 activities by zymography in WIN-treated culture medium. MMP-9 zymographic activity was strongly lower in WIN-treated culture medium in comparison with medium from control cells, suggesting that WIN inhibits MMP-9 activity. Since it is known that cell/ECM interactions are mediated by SPARC (Secreted Protein Acidic and Rich in Cysteine) that also modulates MMPs activity (3-4), we evaluated intra- and extracellular levels of SPARC in our experimental conditions. RT-PCR and western blotting analysis showed in WIN-treated cells an increase both in mRNA and protein expression of intracellular SPARC, while a decrease in extracellular protein level was observed. Studies are in progress to study the possible involvement of SPARC in MMPs activation and MG63 cell migration.

It has been shown that the sesquiterpene lactone parthenolide lowers the viability of MDA-MB-231 breast cancer cells, in correlation with oxidative stress. The present report examined the different radical species produced during parthenolide treatment and their possible role in the toxicity caused by the drug. Time course experiments showed that in the first phase of treatment (0-8 h), and in particular in the first 3 h, parthenolide induced dichlorofluorescein (DCF) signal in a large percentage of cells, while dihydroethidium (DHE) signal was not stimulated. Since the effect on DCF signal was suppressed by apocynin and diphenyleneiodonium (DPI), two inhibitors of NADPH oxidase (NOX), we suggest that parthenolide rapidly stimulated NOX activity with production of superoxide anion (O2•-), which was converted by superoxide dismutase 1 (SOD1) into hydrogen peroxide (H2O2). In the second phase of treatment (8-16 h), parthenolide increased the number of positive cells to DHE signal. Since this event was not prevented by apocynin and DPI and was associated with positivity of cells to MitoSox Red, a fluorochrome used to detect mitochondrial production of O2•-, we suggest that parthenolide induced production of O2•- at the mitochondrial level independently by NOX activity in the second phase of treatment. Finally, in this phase, most cells became positive to hydroxyphenyl fluorescein (HPF) signal, a fluorescent probe to detect highly reactive oxygen species (hROS), such as hydroxyl radical and peroxynitrite. Therefore, parthenolide between 8-16 h of treatment induced generation of O2•- and hROS, in close correlation with a marked reduction in cell viability.

Emerging evidence suggests that treatments targeting cancer stem cells (CSCs) within a tumor can halt cancer and improve patient survival. Moreover, identification of CSC-related MicroRNAs (miRNAs) would provide information for a better understanding of CSCs. miR-29 family is a class of miRNAs aberrantly expressed in multiple cancers. They are frequently down-regulated in osteosarcoma (OS), the most common form of childhood cancer with a potent metastasizing potential. 3AB-OS CSC, a human pluripotent CSC line by us produced from the human osteosarcoma MG63 cells (1) is a useful model to study CSC origin and roles (2). Previously, we have shown that in 3AB-OS CSCs miR-29b is potently down-regulated (2). Here, after stable transfection of 3AB-OS cells with miR-29b-1, we investigated its role in regulating cell proliferation, sarcosphere-forming ability, clonogenic growth, chemosensitivity, migration and invasive ability of 3AB-OS CSCs, in vitro. We found that miR-29b-1 overexpression consistently reduced both, 3AB-OS CSCs growth in two- and three-dimensional culture systems and their sarcosphere- and colony- forming ability. It also sensitized 3AB-OS cells to chemotherapeutic drug-induced apoptosis. Using publicly available databases, we proceeded to identify potential miR-29b target genes, known to play a role in the above reported functions. Among these targets we analyzed CD133, N-Myc, CCND2, E2F1 and E2F2, Bcl-2 and IAP-2. We even analyzed the most important stemness markers as Oct3/4, Sox2 and Nanog. Real-time RT-PCR and western-blot analyses showed that miR-29b-1 negatively regulated the expression of these markers. Overall, the results show that miR-29b-1 suppresses stemness properties of 3AB-OS CSCs and suggest that developing miR-29b- 1 as a novel therapeutic agent might offer benefits for OS treatment.

Osteosarcoma (OS) is the most common type of bone cancer, with a peak incidence in the early childhood. Emerging evidence suggests that treatments targeting cancer stem cells (CSCs) within a tumor can halt cancer and improve patient survival. MicroRNAs (miRNAs) have been implicated in the maintenance of the CSC phenotype, thus, identification of CSC-related miRNAs would provide information for a better understanding of CSCs. Downregulation of miRNA-29 family members (miR-29a/b/c; miR‑29s) was observed in human OS, however, little is known about the functions of miR-29s in human OS CSCs. Previously, during the characterization of 3AB-OS cells, a CSC line selected from human OS MG63 cells, we showed a potent downregulation of miR-29b. In this study, after stable transfection of 3AB-OS cells with miR-29b-1, we investigated the role of miR-29b-1 in regulating cell proliferation, sarcosphere-forming ability, clonogenic growth, chemosensitivity, migration and invasive ability of 3AB-OS cells, in vitro. We found that, miR-29b-1 overexpression consistently reduced both, 3AB-OS CSCs growth in two- and three-dimensional culture systems and their sarcosphere- and colony-forming ability. In addition, while miR-29b-1 overexpression sensitized 3AB-OS cells to chemotherapeutic drug-induced apoptosis, it did not influence their migratory and invasive capacities, thus suggesting a context-depending role of miR-29b-1. Using publicly available databases, we proceeded to identify potential miR-29b target genes, known to play a role in the above reported functions. Among these targets we analyzed CD133, N-Myc, CCND2, E2F1 and E2F2, Bcl-2 and IAP-2. We also analyzed the most important stemness markers as Oct3/4, Sox2 and Nanog. Real-time RT-PCR and western-blot analyses showed that miR-29b-1 negatively regulated the expression of these markers. Overall, the results show that miR-29b-1 suppresses stemness properties of 3AB-OS CSCs and suggest that developing miR-29b-1 as a novel therapeutic agent might offer benefits for OS treatment.

A number of solid tumors contain a distinct subpopulation of cells, termed cancer stem cells (CSCs) which represent the source for tissue renewal and hold malignant potential and which would be responsible for therapy resistance. Today, the winning goal in cancer research would be to find drugs to kill both cancer cells and cancer stem cells, while sparing normal cells. Osteosarcoma is an aggressive pediatric tumor of growing bones that, despite surgery and chemotherapy, is prone to relapse. We have recently selected from human osteosarcoma MG63 cells a cancer stem-like cell line (3AB-OS), which has unlimited proliferative potential, high levels of stemness-related markers, and in vivo tumorforming capacity in xenograft assays. Here, we have shown that 3AB-OS cells can differentiate in vitro into endoderm-, mesoderm-and ectoderm-derived lineages. Cell differentiation is morphological, molecular and functional. We propose that this model system of 3AB-OS differentiation in vitro might have a number of useful purposes, among which the study of molecular mechanisms of osteosarcoma origin, and the analysis of factors involved in specification of the various cell lineages. We still do not know either what are the shared and distinguishing characters between CSCs and normal stem cells, or what is the reason why the cancer stem cells, like the normal stem cells, have the ability to differentiate toward the derivatives of the primary germ layers. It is possible that each of the differentiation capability may be exploited by CSCs to supply their needs of growing and surviving in hostile microenvironment.

Finding new treatments targeting cancer stem cells (CSCs) within a tumor seems to be critical to halt cancer and improve patient survival. Osteosarcoma is an aggressive tumor affecting adolescents, for which there is no second-line chemotherapy. Uncovering new molecular mechanisms underlying the development of osteosarcoma and origin of CSCs is crucial to identify new possible therapeutic strategies. Here, we aimed to characterize genetically and molecularly the human osteosarcoma 3AB-OS CSC line, previously selected from MG63 cells and which proved to have both in vitro and in vivo features of CSCs. Classic cytogenetic studies demonstrated that 3AB-OS cells have hypertriploid karyotype with 71-82 chromosomes. By comparing 3AB-OS CSCs to the parental cells, array CGH, Affymetrix microarray, and TaqMan® Human MicroRNA array analyses identified 49 copy number variations (CNV), 3,512 dysregulated genes and 189 differentially expressed miRNAs. Some of the chromosomal abnormalities and mRNA/miRNA expression profiles appeared to be congruent with those reported in human osteosarcomas. Bioinformatic analyses selected 196 genes and 46 anticorrelated miRNAs involved in carcinogenesis and stemness. For the first time, a predictive network is also described for two miRNA family (let-7/98 and miR-29a,b,c) and their anticorrelated mRNAs (MSTN, CCND2, Lin28B, MEST, HMGA2, and GHR), which may represent new biomarkers for osteosarcoma and may pave the way for the identification of new potential therapeutic targets.

Osteosarcoma (OS) is the most common type of bone cancer, with a peak incidence in the early childhood. Emerging evidence suggests that treatments targeting cancer stem cells (CSCs) within a tumor can halt cancer and improve patient survival. Although microRNAs are frequently dysregulated in human cancers, if they influence OS malignancy and whether or not targeting CSC-associated microRNAs inhibit OS progression remain unclear. Recently (1), we described a predictive network for two downregulated miRNA family (let-7/98 and miR-29a,b,c) and their upregulated anticorrelated mRNAs. Here, we investigated in vitro the role of miR-29b-1 in regulating cell proliferation, clonogenic growth and chemoresistance of 3AB-OS CSCs. We found that the exogenous overexpression of miR-29b-1 in 3AB-OS CSCs reduces both cell growth in two- and three-dimensional culture systems and clonogenic growth. Furthermore, ectopic expression of miRNA-29b-1 reduced resistance to chemotherapy agents as paclitaxel, doxorubicin, cisplatin and etoposide. Next, we explored the molecular mechanisms responsible for the observed functions of miR-29b-1. Predicted target genes of miR-29b were retrieved using publicly available databases. Among these predicted target genes we selected MSTN, NMYC, CCND2, E2F1 Bcl-2 and IAP2, because they are overexpressed in 3AB-OS cells. Real-Time-PCR and western-blot analyses have also shown that enhanced expression of miR-29b-1 diminished the endogenous expression of these genes and proteins, suggesting that miR-29b-1 could negatively regulate these targets. These results support the hypothesis that miR-29b-1 might be a key negative regulator in 3AB-OS CSCs, suggesting that developing miR-29b-1 as a novel therapeutic agent may offer benefits for OS treatment.

Loss of RB1 gene is considered either a causal or an accelerating event in retinoblastoma. A variety of mechanisms inactivates RB1 gene, including intragenic mutations, loss of expression by methylation and chromosomal deletions, with effects which are species-and cell type-specific. RB1 deletion can even lead to aneuploidy thus greatly increasing cancer risk. The RB1gene is part of a larger gene family that includes RBL1 and RBL2, each of the three encoding structurally related proteins indicated as pRb, p107, and p130, respectively. The great interest in these genes and proteins springs from their ability to slow down neoplastic growth. pRb can associate with various proteins by which it can regulate a great number of cellular activities. In particular, its association with the E2F transcription factor family allows the control of the main pRb functions, while the loss of these interactions greatly enhances cancer development. As RB1 gene, also pRb can be functionally inactivated through disparate mechanisms which are often tissue specific and dependent on the scenario of the involved tumor suppressors and oncogenes. The critical role of the context is complicated by the different functions played by the RB proteins and the E2F family members. In this review, we want to emphasize the importance of the mechanisms of RB1/pRb inactivation in inducing cancer cell development. The review is divided in three chapters describing in succession the mechanisms of RB1 inactivation in cancer cells, the alterations of pRb pathway in tumorigenesis and the RB protein and E2F family in cancer

Retinoblastoma is the most common intraocular malignancy afflicting children. The incidence is higher in developing countries, where treatment is limited and long-term survival rates are low. Vincristine, etoposide, and carboplatin -the agents commonly used in the treatment of retinoblastoma- determine side effects causing significant morbidity to pediatric patients and significantly limiting dosing. Thus, identifying new drugs and molecular targets to facilitate the development of novel therapeutics, and finding natural drug combinations to kill cancer cells by synergistically acting at subtoxic doses, may be a good goal. Here, we investigated the effects of two natural compounds, okadaic acid (OKA) and parthenolide (PN), in human retinoblastoma Y79 cells. We showed that OKA/PN combination at subtoxic doses induces potent synergistic apoptotic effects accompanied by decrease in p-Akt, increase in the stabilized p53 forms and potent decrease in pS166–Mdm2. We also showed the key involvement of PTEN which, after OKA/PN treatment, potently increased before p53, suggesting that p53 activation was under PTEN action. PTEN-knockdown increased p-Akt/ pS166Mdm2 over basal levels and significantly lowered p53, while OKA/PN treatment failed both to lower p-Akt and pS166–Mdm2 and to increase p53 below/over their basal levels respectively. OKA/PN treatment potently increased ROS levels while decreased those of GSH. Reducing cellular GSH by butathionine-sulfoximine treatment significantly anticipated the cytotoxic effect exerted by OKA/PN. The effects of OKA/PN treatment on both GSH content and cell viability were less pronounced in PTEN silenced cells than in control cells. Our study reports for the first time both a synergistic apoptotic action between OKA and PN and the involvement of PTEN as key player in the apoptotic mechanism in human retinoblastoma Y79 cells. The results provide strong suggestion for combined inhibition of the PTEN/Akt/Mdm2/p53 pathway.