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2.  Glioblastoma Stem Cells Microenvironment: The Paracrine Roles of the Niche in Drug and Radioresistance 
Stem Cells International  2016;2016:6809105.
Among all solid tumors, the high-grade glioma appears to be the most vascularized one. In fact, “microvascular hyperplasia” is a hallmark of GBM. An altered vascular network determines irregular blood flow, so that tumor cells spread rapidly beyond the diffusion distance of oxygen in the tissue, with the consequent formation of hypoxic or anoxic areas, where the bulk of glioblastoma stem cells (GSCs) reside. The response to this event is the induction of angiogenesis, a process mediated by hypoxia inducible factors. However, this new capillary network is not efficient in maintaining a proper oxygen supply to the tumor mass, thereby causing an oxygen gradient within the neoplastic zone. This microenvironment helps GSCs to remain in a “quiescent” state preserving their potential to proliferate and differentiate, thus protecting them by the effects of chemo- and radiotherapy. Recent evidences suggest that responses of glioblastoma to standard therapies are determined by the microenvironment of the niche, where the GSCs reside, allowing a variety of mechanisms that contribute to the chemo- and radioresistance, by preserving GSCs. It is, therefore, crucial to investigate the components/factors of the niche in order to formulate new adjuvant therapies rendering more efficiently the gold standard therapies for this neoplasm.
doi:10.1155/2016/6809105
PMCID: PMC4736577  PMID: 26880981
3.  MicroRNA-29b-1 impairs in vitro cell proliferation, self-renewal and chemoresistance of human osteosarcoma 3AB-OS cancer stem cells 
International Journal of Oncology  2014;45(5):2013-2023.
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.
doi:10.3892/ijo.2014.2618
PMCID: PMC4432724  PMID: 25174983
osteosarcoma; cancer stem cells; microRNA; microRNA-29b-1; multidrug resistance; 3AB-OS cells
4.  Triple negative breast cancer: looking for the missing link between biology and treatments 
Oncotarget  2015;6(29):26560-26574.
The so called “Triple Negative Breast Cancer” (TNBC) represents approximately 15-20% of breast cancers. This acronym simply means that the tumour does not express oestrogen receptor (ER) and progesterone receptor (PR) and does not exhibit amplification of the human epidermal growth factor receptor 2 (HER2) gene. Despite this unambiguous definition, TNBCs are an heterogeneous group of tumours with just one common clinical feature: a distinctly aggressive nature with higher rates of relapse and shorter overall survival in the metastatic setting compared with other subtypes of breast cancer. Because of the absence of well-defined molecular targets, cytotoxic chemotherapy is currently the only treatment option for TNBC.
In the last decades, the use of more aggressive chemotherapy has produced a clear improvement of the prognosis in women with TNBC, but this approach results in an unacceptable deterioration in the quality of life, also if some support therapies try to relieve patients from distress. In addition, there is the general belief that it is impossible to further improve the prognosis of TNBC patients with chemotherapy alone. In view of that, there is a feverish search for new “clever drugs” able both to rescue chemo-resistant, and to reduce the burden of chemotherapy in chemo-responsive TNBC patients.
A major obstacle to identifying actionable targets in TNBC is the vast disease heterogeneity both inter-tumour and intra-tumour and years of study have failed to demonstrate a single unifying alteration that is targetable in TNBC. TNBC is considered the subtype that best benefits from the neoadjuvant model, since the strong correlation between pathological Complete Response and long-term Disease-Free-Survival in these patients.
In this review, we discuss the recent discoveries that have furthered our understanding of TNBC, with a focus on the subtyping of TNBC. We also explore the implications of these discoveries for future treatments and highlight the need for a completely different type of clinical trials.
PMCID: PMC4694936  PMID: 26387133
breast cancer; triple negative; oncology; treatments; biology
5.  A decade of EGFR inhibition in EGFR-mutated non small cell lung cancer (NSCLC): Old successes and future perspectives 
Oncotarget  2015;6(29):26814-26825.
The discovery of Epidermal Growth Factor Receptor (EGFR) mutations in Non Small Cell Lung Cancer (NSCLC) launched the era of personalized medicine in advanced NSCLC, leading to a dramatic shift in the therapeutic landscape of this disease. After ten years from the individuation of activating mutations in the tyrosine kinase domain of the EGFR in NSCLC patients responding to the EGFR tyrosine kinase inhibitor (TKI) Gefitinib, several progresses have been done and first line treatment with EGFR TKIs is a firmly established option in advanced EGFR-mutated NSCLC patients. During the last decade, different EGFR TKIs have been developed and three inhibitors have been approved so far in these selected patients. However, despite great breakthroughs have been made, treatment of these molecularly selected patients poses novel therapeutic challenges, such as emerging of acquired resistance, brain metastases development or the need to translate these treatments in earlier clinical settings, such as adjuvant therapy.
The aim of this paper is to provide a comprehensive review of the major progresses reported so far in the EGFR inhibition in this molecularly-selected subgroup of NSCLC patients, from the early successes with first generation EGFR TKIs, Erlotinib and Gefitinib, to the novel irreversible and mutant-selective inhibitors and ultimately the emerging challenges that we, in the next future, are called to deal with.
PMCID: PMC4694955  PMID: 26308162
EGFR mutations; third generation EGFR TKIs; non small cell lung cancer; tyrosine kinase inhibitors; targeted therapy
6.  Inhibition of miR-21 restores RANKL/OPG ratio in multiple myeloma-derived bone marrow stromal cells and impairs the resorbing activity of mature osteoclasts 
Oncotarget  2015;6(29):27343-27358.
miR-21 is an oncogenic microRNA (miRNA) with an emerging role as therapeutic target in human malignancies, including multiple myeloma (MM). Here we investigated whether miR-21 is involved in MM-related bone disease (BD). We found that miR-21 expression is dramatically enhanced, while osteoprotegerin (OPG) is strongly reduced, in bone marrow stromal cells (BMSCs) adherent to MM cells. On this basis, we validated the 3′UTR of OPG mRNA as miR-21 target. Constitutive miR-21 inhibition in lentiviral-transduced BMSCs adherent to MM cells restored OPG expression and secretion. Interestingly, miR-21 inhibition reduced RANKL production by BMSCs. Overexpression of protein inhibitor of activated STAT3 (PIAS3), which is a direct and validated target of miR-21, antagonized STAT3-mediated RANKL gene activation. Finally, we demonstrate that constitutive expression of miR-21 inhibitors in BMSCs restores RANKL/OPG balance and dramatically impairs the resorbing activity of mature osteoclasts. Taken together, our data provide proof-of-concept that miR-21 overexpression within MM-microenviroment plays a crucial role in bone resorption/apposition balance, supporting the design of innovative miR-21 inhibition-based strategies for MM-related BD.
PMCID: PMC4694994  PMID: 26160841
miR-21; miRNAs; multiple myeloma bone disease; OPG; RANKL
7.  New findings on primary and acquired resistance to anti-EGFR therapy in metastatic colorectal cancer: do all roads lead to RAS? 
Oncotarget  2015;6(28):24780-24796.
Anti-epidermal growth factor receptor therapy with the monoclonal antibodies cetuximab and panitumumab is the main targeted treatment to combine with standard chemotherapy for metastatic colorectal cancer. Many clinical studies have shown the benefit of the addition of these agents for patients without mutations in the EGFR pathway. Many biomarkers, including KRAS and NRAS mutations, BRAF mutations, PIK3CA mutations, PTEN loss, AREG and EREG expression, and HER-2 amplification have already been identified to select responders to anti-EGFR agents. Among these alterations KRAS and NRAS mutations are currently recognized as the best predictive factors for primary resistance. Liquid biopsy, which helps to isolate circulating tumor DNA, is an innovative method to study both primary and acquired resistance to anti-EGFR monoclonal antibodies. However, high-sensitivity techniques should be used to enable the identification of a wide set of gene mutations related to resistance.
PMCID: PMC4694794  PMID: 26318427
RAS; colorectal cancer; epidermal growth factor receptor; cetuximab; panitumumab
8.  Clinical Features and Outcomes of Pasteurella multocida Infection 
Medicine  2015;94(36):e1285.
Abstract
Pasteurella multocida, a zoonotic infectious organism, has most often been described in patients after an animal bite. Here, we characterize the clinical features and outcomes of P multocida infection in a large cohort of patients according to the presence or absence of an animal bite.
We retrospectively searched MUSC's laboratory information system for all patients with positive P multocida cultures from 2000 to 2014. Extensive data were abstracted, including clinical and outcome data. The Charlson comorbidity index (CCI) was used to assess comorbidities among patients.
We identified 44 patients with P multocida infections, including 25 with an animal bite. The average age was 64 years and the majority of patients were women (N = 30). There was no difference in age and sex distribution among those with and without a bite (P = 0.38 and 0.75, respectively). A CCI ≥1 was significantly associated with the absence of a bite (P = 0.006). Patients presenting without a bite were more frequently bacteremic (37% vs 4%, respectively, P = 0.001), and were hospitalized more often (84% vs 44%, respectively, P = 0.012). Of the 8 patients who required intensive care unit (ICU)-based care, 7 were non-bite-related. There were 4 deaths, all occurring in patients not bitten.
P multocida infections not associated with an animal bite were often associated with bacteremia, severe comorbidity(ies), immune-incompetent states, the need for ICU management, and were associated with substantial mortality.
doi:10.1097/MD.0000000000001285
PMCID: PMC4616664  PMID: 26356688
9.  A pilot study on the impact of known drug-drug interactions in cancer patients 
Background
When a patient concomitantly uses two or more drugs, a drug-drug interaction (DDI) can possibly occur, potentially leading to an increased or decreased clinical effect of a given treatment. Cancer patients are at high risk of such interactions because they commonly receive multiple medications. Moreover, most cancer patients are elderly and require additional medications for comorbidities. Aim of this preliminary observational study was to evaluate the incidence of well known and established DDIs in a cohort of cancer outpatients undergoing multiple treatments.
Methods
Anamnestic and clinical data were collected for 64 adult patients in the ambulatory setting with malignant solid tumors who were receiving systemic anticancer treatment.
Patients also declared all drugs prescribed by other specialists or self-taken in the previous 2 weeks. DDIs were divided into two different groups: ‘neoplastic DDIs’ (NDDIs), involving antitumoral drugs, and ‘not neoplastic DDIs’ (nDDIs), involving all other classes of drugs. The severity of DDIs was classified as major, moderate and minor, according to the ‘Institute for Pharmacological Research Mario Negri’ definition.
Results
About 34 % of cancer outpatients within our cohort were prescribed/assumed interacting drug combinations. The most frequent major NDDIs involved the anticoagulant warfarin (33 % of total NDDIs) that, in association with tamoxifen, or capecitabine and paclitaxel, increased the risk of haemorrhage. About 60 % of nDDIs involved acetylsalicylic acid.
Conclusions
Overall, 16 % of DDIs were related to an A-level strength of recommendation to be avoided. The lack of effective communication among specialists and patients might have a role in determining therapeutic errors. Our pilot study, although limited by a small cohort size, highlights the urgent need of implementing the clinical management of cancer outpatients with new strategies to prevent or minimize potential harmful DDIs.
doi:10.1186/s13046-015-0201-2
PMCID: PMC4547416  PMID: 26303220
Drug-drug interaction (DDI); Drug toxicity; Clinical relevance; Adverse drug reactions; Oncology
10.  A gene highly expressed in tumor cells encodes novel structure proteins 
Oncogene  2004;23(58):9438-9446.
We isolated several related but distinct cDNA clones encoding novel structure proteins (NSP) when screening a cDNA library. Analysis revealed that these cDNAs and several similar ESTs in the public databases are derived from a single gene of 17 exons that span a minimum of 227-kb region. This gene is located at chromosome 17p11.2, a region frequently amplified in human gliomas and osteosarcomas, and involved in Birt–Hogg–Dube syndrome, a tumor-prone syndrome. The major coding sequences shared by all isolated transcripts are predicted to encode SMC (structural maintenance of chromosome)/SbcC ATPase motifs and coiled-coil domains commonly seen in motor or structure proteins. Two 5′-end and two 3′-end variants (type 5α/β and 3α/β, respectively) were identified, making a total of four possible transcripts. Both 5α and 5β variants were detected in human testis mRNA, but only type 5α was detectable in RNA samples extracted from HeLa cells. The unique carboxyl-terminus of 3β contains a Ca2+-dependent actin-binding domain. Immunohistochemistry studies revealed that NSPs were mostly localized to nuclei. Northern blot analysis demonstrated two major bands and the expression levels are tremendously high in testis while barely detectable in other normal tissues examined. Interestingly, NSP5α3α is highly expressed in some tumor cell lines. These results suggest that NSPs represent a new family of structure proteins with a possible role in nuclear dynamics during cell division, and that NSP5α3α may serve as a tumor marker.
doi:10.1038/sj.onc.1207988
PMCID: PMC4518866  PMID: 15602574
NSP; SMC; ATPase; cell division; actin-binding protein; coiled-coil domain
11.  Inflammation Mediated Metastasis: Immune Induced Epithelial-To-Mesenchymal Transition in Inflammatory Breast Cancer Cells 
PLoS ONE  2015;10(7):e0132710.
Inflammatory breast cancer (IBC) is the most insidious form of locally advanced breast cancer; about a third of patients have distant metastasis at initial staging. Emerging evidence suggests that host factors in the tumor microenvironment may interact with underlying IBC cells to make them aggressive. It is unknown whether immune cells associated to the IBC microenvironment play a role in this scenario to transiently promote epithelial to mesenchymal transition (EMT) in these cells. We hypothesized that soluble factors secreted by activated immune cells can induce an EMT in IBC and thus promote metastasis. In a pilot study of 16 breast cancer patients, TNF-α production by peripheral blood T cells was correlated with the detection of circulating tumor cells expressing EMT markers. In a variety of IBC model cell lines, soluble factors from activated T cells induced expression of EMT-related genes, including FN1, VIM, TGM2, ZEB1. Interestingly, although IBC cells exhibited increased invasion and migration following exposure to immune factors, the expression of E-cadherin (CDH1), a cell adhesion molecule, increased uniquely in IBC cell lines but not in non-IBC cell lines. A combination of TNF-α, IL-6, and TGF-β was able to recapitulate EMT induction in IBC, and conditioned media preloaded with neutralizing antibodies against these factors exhibited decreased EMT. These data suggest that release of cytokines by activated immune cells may contribute to the aggressiveness of IBC and highlight these factors as potential target mediators of immune-IBC interaction.
doi:10.1371/journal.pone.0132710
PMCID: PMC4514595  PMID: 26207636
12.  RB1 dual role in proliferation and apoptosis: Cell fate control and implications for cancer therapy 
Oncotarget  2015;6(20):17873-17890.
Inactivation of the retinoblastoma (RB1) tumor suppressor is one of the most frequent and early recognized molecular hallmarks of cancer. RB1, although mainly studied for its role in the regulation of cell cycle, emerged as a key regulator of many biological processes. Among these, RB1 has been implicated in the regulation of apoptosis, the alteration of which underlies both cancer development and resistance to therapy. RB1 role in apoptosis, however, is still controversial because, depending on the context, the apoptotic cues, and its own status, RB1 can act either by inhibiting or promoting apoptosis. Moreover, the mechanisms whereby RB1 controls both proliferation and apoptosis in a coordinated manner are only now beginning to be unraveled. Here, by reviewing the main studies assessing the effect of RB1 status and modulation on these processes, we provide an overview of the possible underlying molecular mechanisms whereby RB1, and its family members, dictate cell fate in various contexts. We also describe the current antitumoral strategies aimed at the use of RB1 as predictive, prognostic and therapeutic target in cancer. A thorough understanding of RB1 function in controlling cell fate determination is crucial for a successful translation of RB1 status assessment in the clinical setting.
PMCID: PMC4627222  PMID: 26160835
RB family; apoptosis; E2F; cancer therapy; CDK inhibitors
13.  Antineoplastic activity of povidone-iodine on different mesothelioma cell lines: results of in vitro study† 
OBJECTIVE
Povidone-iodine (PVP-I) or Betadine, owing to its antineoplastic activity, is also used as an adjuvant during intra-abdominal or intrathoracic surgery. However, the protocol of PVP-I administration has not been optimized to achieve the best antitumoural efficacy. We aimed to determine the optimal concentration of PVP-I, the time of incubation and the mechanism of cell death by analysing the effect of different doses and time of administration of PVP-I on the cell viability of different mesothelioma cell lines.
METHODS
Four different cell lines (MET 5A/normal mesothelium; H2052/sarcomatoid mesothelioma; ISTMES2/epithelial mesothelioma; MSTO/biphasic mesothelioma) were incubated with increasing concentrations of diluted PVP-I (0.0001; 0.001; 0.01; 0.1; 1%) for 5, 10, 30, 60 min and 24 h, respectively. Cell viability was determined using cell direct cytotoxicity assay and cell death was determined through flow cytometry assay analysis. The superoxide dismutase activity was assessed functionally through a specific inhibitor to evaluate the mechanism of cell death.
RESULTS
The antiproliferative effect of PVP-I varied largely among different cell lines in a dose- and time-dependent manner. At 0.1% concentration for 10 min of incubation, the percentage of viable cells was 0.5 ± 0.1; 0.8 ± 0.5 and 0% (P < 0.01) for MET5A, ISTMES2 and MSTO, respectively. Conversely, the same concentration did not significantly affect the H2052 cell line which was completely suppressed at a 1% concentration of PVP-I. Double staining of Annexin V and DNA showed that PVP-I induced cell death in all four cell lines via necrosis depending on PVP-I concentration. However, H2052 was found to be more resistant than MSTO, ISTMES2 and MET 5A cells lines. The activity of superoxide dismutase was significantly inhibited in all cell lines.
CONCLUSIONS
Our results confirmed the anti-neoplastic activity of PVP-I especially on ISTMES2 and MSTO cell lines. With respect to chemotherapy pleural irrigation, washing with PVP-I is cost-effective and easy. If confirmed by larger studies, our findings suggest that the intrapleural irrigation with PVP-I (0.1% concentration for 10 min) in patients with epithelial or biphasic mesothelioma undergoing cytoreductive surgery might be applied in thoracic surgery practice to prevent neoplastic cell growth.
doi:10.1093/ejcts/ezt534
PMCID: PMC4038782  PMID: 24394552
Povidone-iodide; Betadine; Mesothelioma
14.  SRC family kinase (SFK) inhibition reduces rhabdomyosarcoma cell growth in vitro and in vivo and triggers p38 MAP kinase-mediated differentiation 
Oncotarget  2015;6(14):12421-12435.
Recent data suggest that SRC family kinases (SFKs) could represent potential therapeutic targets for rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in children. Here, we assessed the effect of a recently developed selective SFK inhibitor (a pyrazolo[3,4-d]pyrimidine derivative, called SI221) on RMS cell lines. SI221, which showed to be mainly effective against the SFK member YES, significantly reduced cell viability and induced apoptosis, without affecting non-tumor cells, such as primary human skin fibroblasts and differentiated C2C12 cells. Moreover, SI221 decreased in vitro cell migration and invasion and reduced tumor growth in a RMS xenograft model. SFK inhibition also induced muscle differentiation in RMS cells by affecting the NOTCH3 receptor-p38 mitogen-activated protein kinase (MAPK) axis, which regulates the balance between proliferation and differentiation. Overall, our findings suggest that SFK inhibition, besides reducing RMS cell growth and invasive potential, could also represent a differentiation therapeutic strategy for RMS.
PMCID: PMC4494948  PMID: 25762618
rhabdomyosarcoma; SRC family inhibition; NOTCH3; muscle differentiation; p38 MAPK; YES
15.  Deregulation of dicer and mir-155 expression in liposarcoma 
Oncotarget  2015;6(12):10586-10591.
Liposarcoma (LPS) is the most common soft tissue sarcoma. It has been demonstrated that mir-155 was the most overexpressed miRNA in well-differentiated LPS(WDLPS)/dedifferentiated LPS (DDLPS). The aim of this study is to evaluate the involvement of Dicer, Drosha and mir-155 in development of LPS and their possible role in stratification of different histological subtypes. Dicer, Drosha and mir-155 mRNA levels were analyzed in formalin-fixed paraffin-embedded specimens from patients diagnosed with 62 LPS and compared with samples of adipose tissues of healthy donors. The experimental data were obtained using qRT-PCR comparing Dicer, Drosha and mir-155 expression levels in tumor samples versus normal fat. The tumor samples from LPS patients showed a significantly lower Dicer expression versus normal adipose tissue, while Drosha levels did not differ. Concerning mir155 expression levels, our results demonstrated a significant mir-155 up-regulation in all LPS subtypes versus normal adipose tissue except for WDLS. These findings demonstrate for the first time that Dicer is deregulated in LPS and show that mir-155 is differentially expressed in LPS subgroups and it could be a promising tool to improve LPS disease stratification and differential diagnosis.
PMCID: PMC4496377  PMID: 25888631
Dicer; Drosha; mir-155; liposarcoma
16.  Involvement of peroxisome proliferator-activated receptor β/δ (PPAR β/δ) in BDNF signaling during aging and in Alzheimer disease 
Cell Cycle  2014;13(8):1335-1344.
Aging and many neurological disorders, such as AD, are linked to oxidative stress, which is considered the common effector of the cascade of degenerative events. In this phenomenon, reactive oxygen species play a fundamental role in the oxidative decomposition of polyunsaturated fatty acids, resulting in the formation of a complex mixture of aldehydic end products, such as malondialdehyde, 4-hydroxynonenal, and other alkenals. Interestingly, 4-HNE has been indicated as an intracellular agonist of peroxisome proliferator-activated receptor β/δ. In this study, we examined, at early and advanced AD stages (3, 9, and 18 months), the pattern of 4-HNE and its catabolic enzyme glutathione S-transferase P1 in relation to the expression of PPARβ/δ, BDNF signaling, as mRNA and protein, as well as on their pathological forms (i.e., precursors or truncated forms). The data obtained indicate a novel detrimental age-dependent role of PPAR β/δ in AD by increasing pro-BDNF and decreasing BDNF/TrkB survival pathways, thus pointing toward the possibility that a specific PPARβ/δ antagonist may be used to counteract the disease progression.
doi:10.4161/cc.28295
PMCID: PMC4049970  PMID: 24621497
aging; neurodegenerative disease; transcription factors; oxidative stress; BDNF; TrkB; p75; JNK
17.  Abrogating G2/M checkpoint through WEE1 inhibition in combination with chemotherapy as a promising therapeutic approach for mesothelioma 
Cancer Biology & Therapy  2014;15(4):380-388.
Malignant mesothelioma (MM) is a very aggressive asbestos-related neoplasm of the serous membranes, whose incidence is increasing worldwide. Although the introduction of new drug combinations, such as cisplatin plus pemetrexed/gemcitabine, has determined an improvement in the patient quality of life, MM remains a universally fatal disease. The observation that key G1/S checkpoint regulators are often functionally inactivated in MM prompted us to test whether the use of G2/M checkpoint inhibitors, able to sensitize G1/S checkpoint-defective cancer cells to DNA-damaging agents, could be successful in MM. We treated six MM cell lines, representative of different histotypes (epithelioid, biphasic, and sarcomatoid), with cisplatin in combination with MK-1775, an inhibitor of the G2/M checkpoint kinase WEE1. We observed that MK-1775 enhanced the cisplatin cytotoxic effect in all MM cell lines, except the sarcomatoid cell line, which is representative of the most aggressive histotype. As expected, the enhancement in cisplatin toxicity was accompanied by a decrease in the inactive phosphorylated form of cyclin-dependent kinase 1 (CDK1), a key substrate of WEE1, which is indicative of G2/M checkpoint inactivation. Consistently, we also observed a decrease in G2/M accumulation and an increase in mitotic entry of DNA-damaged cells and apoptosis, probably due to the loss of the cell ability to arrest cell cycle in response to DNA damage, irrespectively of p53 mutational status. Notably, this treatment did not increase cisplatin cytotoxicity on normal cells, thus suggesting a possible use of MK-1775 in combination with cisplatin for a safe and efficient treatment of epithelioid and biphasic MM.
doi:10.4161/cbt.27623
PMCID: PMC3979815  PMID: 24365782
WEE1; MK-1775; cisplatin; mesothelioma; CDK1; G2/M checkpoint; apoptosis
18.  Extreme Thermal Noxious Stimuli Induce Pain Responses in Zebrafish Larvae 
Journal of cellular physiology  2014;229(3):300-308.
Exposing tissues to extreme high or low temperature leads to burns. Burned animals sustain several types of damage, from the disruption of the tissue to degeneration of axons projecting through muscle and skin. Such damage causes pain due to both inflammation and axonal degeneration (neuropathic-like pain). Thus, the approach to cure and alleviate the symptoms of burns must be twofold: rebuilding the tissue that has been destroyed and alleviating the pain derived from the burns. While tissue regeneration techniques have been developed, less is known on the treatment of the induced pain. Thus, appropriate animal models are necessary for the development of the best treatment for pain induced in burned tissues. We have developed a methodology in the zebrafish aimed to produce a new animal model for the study of pain induced by burns. Here we show that two events linked to the onset of burn-induced inflammation and neuropathic-like pain in mammals, degeneration of axons innervating the affected tissues and over-expression of specific genes in sensory tissues, are conserved from zebrafish to mammals.
doi:10.1002/jcp.24447
PMCID: PMC4106021  PMID: 23929528
thermal nociception; inflammation; neuropathic pain; burns; markers genes; animal model; zebrafish
19.  Circulating tumor cells in newly diagnosed inflammatory breast cancer 
Introduction
Circulating tumor cells (CTCs) are an independent prognostic factor for progression-free survival (PFS) and overall survival (OS) in patients with metastatic breast cancer. Inflammatory breast cancer (IBC) is one of the most aggressive forms of breast cancer. The prognostic value of a CTC count in newly diagnosed IBC has not been established. The aim of this study was to assess the prognostic value of a baseline CTC count in patients with newly diagnosed IBC.
Methods
This retrospective study included 147 patients with newly diagnosed IBC (77 with locally advanced and 70 with metastatic IBC) treated with neoadjuvant therapy or first-line chemotherapy during the period from January 2004 through December 2012 at The University of Texas MD Anderson Cancer Center. CTCs were detected and enumerated by using the CellSearch system before patients were started with chemotherapy.
Results
The proportion of patients with ≥1 CTC was lower among patients with stage III than among patients with metastatic IBC (54.5% versus 84.3%; P = 0.0002); the proportion of patients with ≥5 CTCs was also lower for stage III than for metastatic IBC (19.5% versus 47.1%; P = 0.0004). Patients with fewer than five CTCs had significantly better progression-free survival (PFS) (hazard ratio (HR) = 0.60; P = 0.02) and overall survival (HR = 0.59; P = 0.03) than patients with five or more CTCs. Among patients with stage III IBC, there was a nonsignificant difference in PFS (HR = 0.66; 95% confidence interval (CI), 0.31 to 1.39; P = 0.29) and OS (HR = 0.54; 95% CI, 0.24 to 1.26; P = 0.48) in patients with no CTCs compared with patients with one or more CTCs. In multivariate analysis, CTC was prognostic for PFS and OS independent of clinical stage.
Conclusions
CTCs can be detected in a large proportion of patients with newly diagnosed IBC and are a strong predictor of worse prognosis in patients with newly diagnosed IBC.
doi:10.1186/s13058-014-0507-6
PMCID: PMC4318180  PMID: 25572591
20.  miR-29b induces SOCS-1 expression by promoter demethylation and negatively regulates migration of multiple myeloma and endothelial cells 
Cell Cycle  2013;12(23):3650-3662.
Epigenetic silencing of tumor suppressor genes frequently occurs and may account for their inactivation in cancer cells. We previously demonstrated that miR-29b is a tumor suppressor microRNA (miRNA) that targets de novo DNA methyltransferases and reduces the global DNA methylation of multiple myeloma (MM) cells. Here, we provide evidence that epigenetic activity of miR-29b leads to promoter demethylation of suppressor of cytokine signaling-1 (SOCS-1), a hypermethylated tumor suppressor gene. Enforced expression of synthetic miR-29b mimics in MM cell lines resulted in SOCS-1 gene promoter demethylation, as assessed by Sequenom MassARRAY EpiTYPER analysis, and SOCS-1 protein upregulation. miR-29b-induced SOCS-1 demethylation was associated with reduced STAT3 phosphorylation and impaired NFκB activity. Downregulation of VEGF-A and IL-8 mRNAs could be detected in MM cells transfected with miR-29b mimics as well as in endothelial (HUVEC) or stromal (HS-5) cells treated with conditioned medium from miR-29b-transfected MM cells. Notably, enforced expression of miR-29b mimics increased adhesion of MM cells to HS-5 and reduced migration of both MM and HUVEC cells. These findings suggest that miR-29b is a negative regulator of either MM or endothelial cell migration. Finally, the proteasome inhibitor bortezomib, which induces the expression of miR-29b, decreased global DNA methylation by a miR-29b-dependent mechanism and induced SOCS-1 promoter demethylation and protein upregulation. In conclusion, our data indicate that miR-29b is endowed with epigenetic activity and mediates previously unknown functions of bortezomib in MM cells.
doi:10.4161/cc.26585
PMCID: PMC3903716  PMID: 24091729
miR-29b; epi-miRNA; microRNA; multiple myeloma; methylation
21.  Cancer mortality trends between 1988 and 2009 in the metropolitan area of Naples and Caserta, Southern Italy 
Cancer Biology & Therapy  2013;14(12):1113-1122.
Mortality data by geographic area and trend-based surveillance are particularly relevant in orienting public health decisions targeting specific populations. We analyzed overall and site-specific cancer mortality between 1988 and 2009 in the metropolitan area of Naples and Caserta in southern Italy. Age-standardized mortality rates (SMR) were computed for each 5-y age group, by gender, primitive cancer site and specific Province in the overall population and age-defined subgroups. Cancer mortality trends were quantified by annual percent change (APC) and 95% confidence interval (CI). From Naples and Caserta, the reduction observed between 1988 and 2009 in SMR in males, but not in females, was significantly lower compared with the decrease reported at a national level (−11.4% and −28.4%, respectively). In elderly men, differences between local and national SMR were more pronounced (+13.6% compared with −2.7%). In males, the joinpoint regression analysis showed the following APC and 95% CI: −0.9%/year (−1.2; −0.7) and −0.6%/year (−1.0; −0.2) for Naples and Caserta, respectively. In females, estimates were −0.6%/year (−0.8; −0.5) and −0.7%/year (−1.2; −0.3). The overall orientation toward declining cancer mortality trends appeared in antithesis with the slight, but significant, increase for some tumors (e.g., pancreatic cancer in both genders). A complex mixture of heterogeneous factors concurs to explain the evidence observed including lifestyle, access to screening procedures, advancements in cancer diagnosis and treatment. Further details might eventually derive from biomonitoring studies for ascertaining the causal link between exposure to potential contaminants in air, water, and soil and cancer-related outcomes in the area of interest.
doi:10.4161/cbt.26425
PMCID: PMC3912034  PMID: 24025410
cancer mortality; time trends; Naples; metropolitan area; southern Italy; joinpoint; analysis
22.  p53 status as effect modifier of the association between pre-treatment fasting glucose and breast cancer outcomes in non diabetic, HER2 positive patients treated with trastuzumab 
Oncotarget  2014;5(21):10382-10392.
Mounting evidence supports the role of p53 in metabolic processes involved in breast carcinogenesis. We investigated whether p53 status affects the association of pre-treatment fasting glucose with treatment outcomes in 106 non diabetic, HER2 positive breast cancer patients treated with trastuzumab. p53 status was validated against gene sequencing of selected codons in 49 patients. The Kaplan–Meier method and log rank test were used to compare survival by categories of fasting glucose in the overall population and separate settings. Cox models included age and body mass index. Direct sequencing confirmed the lack of mutations in 73.7% of p53 negative patients and their presence in 53.3% of p53 positive cases. At 66 months, 88.3% of patients with glucose ≤ 89.0 mg/dl (median value) did not experiment disease progression compared with 70.0% in the highest category (p=0.034), with glucose being an independent predictor (p=0.046). Stratified analysis confirmed this association in p53 negative patients only (p=0.01). In the early setting, data suggested longer disease free survival in p53 negative patients in the lowest glucose category (p=0.053). In our study, p53 status acted as effect modifier of the investigated association. This may help differentiate target sub-groups and affect outcomes interpretation in similarly characterized patients.
PMCID: PMC4279380  PMID: 25071015
p53 status; fasting glucose; HER2 positive breast cancer; trastuzumab.
23.  Expression of stemness genes in primary breast cancer tissues: the role of SOX2 as a prognostic marker for detection of early recurrence 
Oncotarget  2014;5(20):9678-9688.
The events leading to breast cancer (BC) progression or recurrence are not completely understood and new prognostic markers aiming at identifying high risk-patients and to develop suitable therapy are highly demanded. Experimental evidences found in cancer cells a deregulated expression of some genes involved in governance of stem cell properties and demonstrated a relationship between stemness genes overexpression and poorly differentiated BC subtypes.
In the present study 140 primary invasive BC specimens were collected. The expression profiles of 13 genes belonging to the OCT3/SOX2/NANOG/KLF4 core circuitry by RT-PCR were analyzed and any correlation between their expression and the BC clinic-pathological features (CPfs) and prognosis was investigated.
In our cohort (117 samples), NANOG, GDF3 and SOX2 significantly correlated with grade 2, Nodes negative status and higher KI67 proliferation index, respectively (p=0.019, p=0.029, p= 0.035). According to multivariate analysis, SOX2 expression resulted independently associated with increased risk of recurrence (HR= 2,99; p= p=0,004) as well as Nodes status (HR=2,44; p=0,009) and T-size >1 (HR=1,77; p=0,035).
Our study provides further proof of the suitable use of stemness genes in BC management. Interestingly, a prognostic role of SOX2, which seems to be a suitable marker of early recurrence irrespective of other clinicopathological features.
PMCID: PMC4259429  PMID: 25127259
Breast Cancer; Gene expression; Recurrence; SOX2; Stemness genes
24.  Cocoa Powder Triggers Neuroprotective and Preventive Effects in a Human Alzheimer's Disease Model by Modulating BDNF Signaling Pathway 
Journal of cellular biochemistry  2013;114(10):2209-2220.
The molecular mechanisms linking Aβ to the onset of neurotoxicity are still largely unknown, but several lines of evidence point to reactive oxygen species, which are produced even under the effect of nanomolar concentrations of soluble Aβ-oligomers. The consequent oxidative stress is considered as the mediator of a cascade of degenerative events in many neurological disorders. Epidemiological studies indicate that dietary habits and antioxidants from diet can influence the incidence of neurodegenerative disorders such as Alzheimer's and Parkinson's diseases. In the recent years, a number of reviews have reported on neuroprotective effects of polyphenols in cell and animal models. However, the majority of these studies have focused only on the anti-oxidant properties of these compounds and less on the mechanism/s of action at cellular level. In this work we investigated the effect of cocoa polyphenolic extract on a human AD in vitro model. The results obtained, other than confirming the anti-oxidant properties of cocoa, demonstrate that cocoa polyphenols triggers neuroprotection by activating BDNF survival pathway, both on Aβ plaque treated cells and on Aβ oligomers treated cells, resulting in the counteraction of neurite dystrophy. On the light of the results obtained the use of cocoa powder as preventive agent for neurodegeneration is further supported.
doi:10.1002/jcb.24548
PMCID: PMC4170833  PMID: 23554028
Neurodegenerative Diseases; Anti-Oxidant; BDNF Signaling; Neurons; Neurites; Cytoskeletric Proteins
25.  Circulating tumor cells as early predictors of metastatic spread in breast cancer patients with limited metastatic dissemination 
Introduction
Traditional factors currently used for prognostic stratification do not always adequately predict treatment response and disease evolution in advanced breast cancer patients. Therefore, the use of blood-based markers, such as circulating tumor cells (CTCs), represents a promising complementary strategy for disease monitoring. In this retrospective study, we explored the role of CTC counts as predictors of disease evolution in breast cancer patients with limited metastatic dissemination.
Methods
A total of 492 advanced breast cancer patients who had a CTC count assessed by CellSearch prior to starting a new line of systemic therapy were eligible for this analysis. Using the threshold of 5 CTCs/7.5 ml of blood, pretreatment CTC counts were correlated in the overall population with metastatic site distribution, evaluated at baseline and at the time of treatment failure, using Fisher’s exact test. Time to visceral progression and time to the development of new metastatic lesions and sites were estimated in patients with nonvisceral metastases and with single-site metastatic disease, respectively, by the Kaplan-Meier method. Survival times were compared between groups according to pretreatment CTC count by logrank test.
Results
In the overall population, a pretreatment level ≥5 CTCs/7.5 ml was associated with an increased baseline number of metastatic sites compared with <5 CTCs/7.5 ml (P = 0.0077). At the time of treatment failure, patients with ≥5 CTCs/7.5 ml more frequently developed new metastatic lesions and sites compared with those with <5 CTCs/7.5 ml (development of new lesions: P = 0.0002; development of new sites: P = 0.0031). Among patients with disease originally confined to nonvisceral sites, ≥5 CTCs/7.5 ml was associated with remarkably shorter time to visceral metastases (P = 0.0021) and overall survival (P = 0.0006) compared with <5 CTCs/7.5 ml. In patients with single-site metastatic disease, ≥5 CTCs/7.5 ml was associated with a significant reduction of the time to development of new metastatic sites (P = 0.0051) and new lesions (P = 0.0002) and with worse overall survival (P = 0.0101).
Conclusion
Our results suggest that baseline CTC counts can be used as an early predictor of metastatic potential in breast cancer patients with limited metastatic dissemination.
Electronic supplementary material
The online version of this article (doi:10.1186/s13058-014-0440-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s13058-014-0440-8
PMCID: PMC4303121  PMID: 25223629

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