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The pathogenesis of Kaposi's sarcoma (KS) is suggested to be related to an infectious agent transmitted by sexual contact. Recently, DNA sequences homologous to gamma herpesviridae have been identified in AIDS-associated KS, but no information is available concerning non-AIDS-associated KS. Five classic, 12 endemic, and 17 AIDS-associated KS skin lesion specimens were analyzed and all were positive for these novel DNA sequences. Twenty-four specimens from normal skin were negative. These novel sequences were also found in lymph nodes from human immunodeficiency virus-positive patients with AIDS or lymphadenopathy syndrome but not in peripheral blood mononuclear cells from normal subjects. Therefore, the data support the view that a novel gamma herpesvirus might be specifically associated with KS etiopathogenesis. [ABSTRACT FROM AUTHOR]

The study investigated the application of Wastewater-Based Epidemiology (WBE) as a tool for monitoring the SARS-CoV-2 prevalence in a city in northern Italy from October 2021 to May 2023. Based on a previously used deterministic model, this study proposed a variation to account for the population characteristics and virus biodegradation in the sewer network. The model calculated virus loads and corresponding COVID-19 cases over time in different areas of the city and was validated using healthcare data while considering viral mutations, vaccinations, and testing variability. The correlation between the predicted and reported cases was high across the three waves that occurred during the period considered, demonstrating the ability of the model to predict the relevant fluctuations in the number of cases. The population characteristics did not substantially influence the predicted and reported infection rates. Conversely, biodegradation significantly reduced the virus load reaching the wastewater treatment plant, resulting in a 30% reduction in the total virus load produced in the study area. This approach can be applied to compare the virus load values across cities with different population demographics and sewer network structures, improving the comparability of the WBE data for effective surveillance and intervention strategies.

Pancreatic cancer (PC) has a poor prognosis and displays resistance to immunotherapy. A better understanding of tumor-derived extracellular vesicle (EV) effects on immune responses might contribute to improved immunotherapy. EVs derived from Capan-2 and BxPC-3 PC cells isolated by ultracentrifugation were characterized by atomic force microscopy, Western blot (WB), nanoparticle tracking analysis, and label-free proteomics. Fresh PBMCs from healthy donors were treated with PC- or control-derived heterologous EVs, followed by flow cytometry analysis of CD8+ and CD4+ lymphocytes. The proteomics of lymphocytes sorted from EV-treated or untreated PBMCs was performed, and the IFN-γ concentration was measured by ELISA. Notably, most of the proteins identified in Capan-2 and BxPC-3 EVs by the proteomic analysis were connected in a single functional network ( p = 1 × 10 -16 ) and were involved in the "Immune System" (FDR: 1.10 × 10 -24 and 3.69 × 10 -19 , respectively). Interestingly, the treatment of healthy donor-derived PBMCs with Capan-2 EVs but not with BxPC-3 EVs or heterologous control EVs induced early activation of CD8+ and CD4+ lymphocytes. The proteomics of lymphocytes sorted from EV-treated PBMCs was consistent with their activation by Capan-2 EVs, indicating IFN-γ among the major upstream regulators, as confirmed by ELISA. The proteomic and functional analyses indicate that PC-EVs have pleiotropic effects, and some may activate early immune responses, which might be relevant for the development of highly needed immunotherapeutic strategies in this immune-cold tumor.

Sex might influence prognosis in patients affected by colorectal cancer. We retrospectively studied a cohort of patients affected by metastatic colon cancer (mCC) stratified by sex and primary tumor location. RAS mutational status was also included in the analysis. Overall, 616 patients met the eligibility criteria, 261 women and 355 men. Neither gender, nor RAS mutational status influenced overall survival (OS) in the entire population. As expected, patients with right-sided colon cancer (RCC) had a significant shorter OS compared to those with left-sided colon cancer (LCC) (21.3 vs 33.1 months, p= 0.002). When the analysis was performed stratifying for gender, RCC retained worse prognosis among men (OS 20.5 vs 33.9 months, p= 0.008), but not among women (p= 0.132). Similarly, the presence of RAS mutations had no prognostic effect in women, but was significantly associate with shorter survival in men (OS 29.5 vs 33.7 months, p= 0.046). In addition, when comparing clinical outcome of women or men according to sidedness and RAS mutational status, RCC was associated with dismal prognosis only in men with RAS mutated tumor (OS 17.2 vs 32.3 months, p= 0.008). Our study highlights the importance of gender in the outcome of patients with mCC., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Extracellular vesicles (EVs) are a heterogenous population of plasma membrane-surrounded particles that are released in the extracellular milieu by almost all types of living cells. EVs are key players in intercellular crosstalk, both locally and systemically, given that they deliver their cargoes (consisting of proteins, lipids, mRNAs, miRNAs, and DNA fragments) to target cells, crossing biological barriers. Those mechanisms further trigger a wide range of biological responses. Interestingly, EV phenotypes and cargoes and, therefore, their functions, stem from their specific parental cells. For these reasons, EVs have been proposed as promising candidates for EV-based, cell-free therapies. One of the new frontiers of cell-based immunotherapy for the fight against refractory neoplastic diseases is represented by genetically engineered chimeric antigen receptor T (CAR-T) lymphocytes, which in recent years have demonstrated their effectiveness by reaching commercialization and clinical application for some neoplastic diseases. CAR-T-derived EVs represent a recent promising development of CAR-T immunotherapy approaches. This crosscutting innovative strategy is designed to exploit the advantages of genetically engineered cell-based immunotherapy together with those of cell-free EVs, which in principle might be safer and more efficient in crossing biological and tumor-associated barriers. In this review, we underlined the potential of CAR-T-derived EVs as therapeutic agents in tumors.

Pancreatic cancer (PC) is one of the deadliest malignancies, with an increasing incidence and limited response to current therapeutic options. Therefore, more effective and low-toxic agents are needed to improve PC patients' outcomes. Resveratrol (RSV) is a natural polyphenol with multiple biological properties, including anticancer effects. In this study, we explored the antiproliferative activities of newly synthetized RSV analogues in a panel of PC cell lines and evaluated the physicochemical properties of the most active compound. This derivative exhibited marked antiproliferative effects in PC cells through mechanisms involving DNA damage, apoptosis induction, and interference in cell cycle progression, as assessed using flow cytometry and immunoblot analysis of cell cycle proteins, PARP cleavage, and H2AX phosphorylation. Notably, the compound induced a consistent reduction in the PC cell subpopulation with a CD133 + EpCAM + stem-like phenotype, paralleled by dramatic effects on cell clonogenicity. Moreover, the RSV derivative had negligible toxicity against normal HFF-1 cells and, thus, good selectivity index values toward PC cell lines. Remarkably, its higher lipophilicity and stability in human plasma, as compared to RSV, might ensure a better permeation along the gastrointestinal tract. Our results provide insights into the mechanisms of action contributing to the antiproliferative activity of a synthetic RSV analogue, supporting its potential value in the search for effective and safe agents in PC treatment.

The use of anthracycline derivatives was approved for the treatment of a broad spectrum of human tumors (i.e., breast cancer). The need to test these drugs on cancer models has pushed the basic research to apply many types of in vitro assays, and, among them, the study of anthracycline-induced apoptosis was mainly based on the application of flow cytometry protocols. However, the chemical structure of anthracycline derivatives gives them a strong autofluorescence effect that must be considered when flow cytometry is used. Unfortunately, the guidelines on the analysis of anthracycline effects through flow cytometry are lacking. Therefore, in this study, we optimized the flow cytometry detection of doxorubicin and epirubicin-treated breast cancer cells. Their autofluorescence was assessed both by using conventional and imaging flow cytometry; we found that all the channels excited by the 488 nm laser were affected. Anthracycline-induced apoptosis was then measured via flow cytometry using the optimized setting. Consequently, we established a set of recommendations that enable the development of optimized flow cytometry settings when the in vitro assays of anthracycline effects are analyzed, with the final aim to reveal a new perspective on the use of those in vitro tests for the further implementation of precision medicine strategies in cancer.

Advances in the management of gastric cancer have improved patient survival in the last decade. Nonetheless, the number of patients relapsing and dying after a diagnosis of localized gastric cancer is still too high, even in early stages (10% in stage I). Adjuvant systemic chemotherapy has been proven to significantly improve outcomes. In the present article we have critically reviewed the clinical trials that guide the current clinical practice in the adjuvant treatment of patients affected by resectable gastric cancer, focusing on the different approaches worldwide, i.e., adjuvant chemotherapy, adjuvant chemoradiotherapy, and perioperative chemotherapy. We also delineate the clinical-pathological characteristics that are commonly taken into account to identify patients at a higher risk of recurrence and requiring adjuvant chemotherapy, and also describe novel biomarkers and therapeutic agents that might allow personalization of the treatment.

The antiproliferative effects played by benzothiazoles in different cancers have aroused the interest for these molecules as promising antitumor agents. In this work, a library of phenylacetamide derivatives containing the benzothiazole nucleus was synthesized and compounds were tested for their antiproliferative activity in paraganglioma and pancreatic cancer cell lines. The novel synthesized compounds induced a marked viability reduction at low micromolar concentrations both in paraganglioma and pancreatic cancer cells. Derivative 4l showed a greater antiproliferative effect and higher selectivity index against cancer cells, as compared to other compounds. Notably, combinations of derivative 4l with gemcitabine at low concentrations induced enhanced and synergistic effects on pancreatic cancer cell viability, thus supporting the relevance of compound 4l in the perspective of clinical translation. A target prediction analysis was also carried out on 4l by using multiple computational tools, identifying cannabinoid receptors and sentrin-specific proteases as putative targets contributing to the observed antiproliferative activity.

Colorectal cancer (CRC) is one of the most incident and lethal malignancies worldwide. Recent treatment advances prolonged survival in patients with metastatic colorectal cancer (mCRC). However, there are still few biomarkers to guide clinical management and treatment selection in mCRC. In this study, we applied an optimized flow cytometry protocol for EV identification, enumeration, and subtyping in blood samples of 54 patients with mCRC and 48 age and sex-matched healthy controls (HCs). The overall survival (OS) and overall response rate (ORR) were evaluated in mCRC patients enrolled and treated with a first line fluoropyrimidine-based regimen. Our findings show that patients with mCRC presented considerably higher blood concentrations of total EVs, as well as CD133+ and EPCAM+ EVs compared to HCs. Overall survival analysis revealed that increased blood concentrations of total EVs and CD133+ EVs before treatment were significantly associated with shorter OS in mCRC patients ( p = 0.001; and p = 0.0001, respectively). In addition, we observed a correlation between high blood levels of CD133+ EVs at baseline and reduced ORR to first-line systemic therapy ( p = 0.045). These findings may open exciting perspectives into the application of novel blood-based EV biomarkers for improved risk stratification and optimized treatment strategies in mCRC.

Pancreatic cancer (PC) is one of the deadliest malignancies worldwide, since patients rarely display symptoms until an advanced and unresectable stage of the disease. Current chemotherapy options are unsatisfactory and there is an urgent need for more effective and less toxic drugs to improve the dismal PC therapy. Repurposing of non-oncology drugs in PC treatment represents a very promising therapeutic option and different compounds are currently being considered as candidates for repurposing in the treatment of this tumor. In this review, we provide an update on some of the most promising FDA-approved, non-oncology, repurposed drug candidates that show prominent clinical and preclinical data in pancreatic cancer. We also focus on proposed mechanisms of action and known molecular targets that they modulate in PC. Furthermore, we provide an explorative bioinformatic analysis, which suggests that some of the PC repurposed drug candidates have additional, unexplored, oncology-relevant targets. Finally, we discuss recent developments regarding the immunomodulatory role displayed by some of these drugs, which may expand their potential application in synergy with approved anticancer immunomodulatory agents that are mostly ineffective as single agents in PC.

The 90K protein, also known as Mac-2 BP or LGALS3BP, can activate the immune response in part by increasing major histocompatibility (MHC) class I levels. In studies on a non-immune cell model, the rat FRTL-5 cell line, we observed that transforming growth factor (TGF)-β1, like γ-interferon (IFN), increased 90K levels, despite its immunosuppressive functions and the ability to decrease MHC class I. To explain this paradoxical result, we investigated the mechanisms involved in the TGF-β1 regulation of 90K expression with the aim to demonstrate that TGF-β1 utilizes different molecular pathways to regulate the two genes. We found that TGF-β1 was able to increase the binding of Upstream Stimulatory Factors, USF1 and USF2, to an E-box element, CANNTG, at -1926 to -1921 bp, upstream of the interferon response element (IRE) in the 90K promoter. Thyrotropin (TSH) suppressed constitutive and γ-IFN-induced 90K expression by decreasing USF binding to the E-box. TGF-β1 was able to overcome TSH suppression at the transcriptional level by increasing USF binding to the E-box. We suggest that the ability of TGF-β1 to increase 90K did not result in an increase in MHC class I because of a separate suppressive action of TGF-β1 directly on the MHC class I gene. We propose that the increased levels of 90K may play a role, rather than in immune response, in the context of the TGF-β1-induced changing of the cellular microenvironment that predisposes to cell motility and cancer progression. Consistently, analyzing the publicly available cancer patient data sets cBioPortal, we found that 90K expression directly correlated with TGF-β1 and USFs and that high levels of 90K were significantly associated with increased mortality in patients affected by different types of cancer.

Repurposing of approved non-antitumor drugs represents a promising and affordable strategy that may help to increase the repertoire of effective anticancer drugs. Benzimidazole-based anthelmintics are antiparasitic drugs commonly employed both in human and veterinary medicine. Benzimidazole compounds are being considered for drug repurposing due to antitumor activities displayed by some members of the family. In this study, we explored the effects of a large series of benzimidazole-based anthelmintics (and some enantiomerically pure forms of those containing a stereogenic center) on the viability of different tumor cell lines derived from paraganglioma, pancreatic and colorectal cancer. Flubendazole, parbendazole, oxibendazole, mebendazole, albendazole and fenbendazole showed the most consistent antiproliferative effects, displaying IC 50 values in the low micromolar range, or even in the nanomolar range. In silico evaluation of their physicochemical, pharmacokinetics and medicinal chemistry properties also provided useful information related to the chemical structures and potential of these compounds. Furthermore, in view of the potential repurposing of these drugs in cancer therapy and considering that pharmaceutically active compounds may have different mechanisms of action, we performed an in silico target prediction to assess the polypharmacology of these benzimidazoles, which highlighted previously unknown cancer-relevant molecular targets.

Pancreatic cancer (PC) incidence is rising and due to late diagnosis, combined with unsatisfactory response to current therapeutic approaches, this tumor has an extremely high mortality rate. A better understanding of the mechanisms underlying pancreatic carcinogenesis is of paramount importance for rational diagnostic and therapeutic approaches. Multiple lines of evidence have showed that exosomes are actively involved in intercellular communication by transferring their cargos of bioactive molecules to recipient cells within the tumor microenvironment and systemically. Intriguingly, exosomes may exert both protumor and antitumor effects, supporting or hampering processes that play a role in the pathogenesis and progression of PC, including shifts in tumor metabolism, proliferation, invasion, metastasis, and chemoresistance. They also have a dual role in PC immunomodulation, exerting immunosuppressive or immune enhancement effects through several mechanisms. PC-derived exosomes also induce systemic metabolic alterations, leading to the onset of diabetes and weight loss. Moreover, exosomes have been described as promising diagnostic and prognostic biomarkers for PC. Their potential application in PC therapy as drug carriers and therapeutic targets is under investigation. In this review, we provide an overview of the multiple roles played by exosomes in PC biology through their specific cargo biomolecules and of their potential exploitation in early diagnosis and treatment of PC.

Immune checkpoint inhibitors (ICIs) induce durable clinical responses only in a subset of advanced non-small cell lung cancer (NSCLC) patients. There is a need to identify mechanisms of ICI resistance and immunotherapy biomarkers to improve clinical benefit. In this study, we evaluated the prognostic and predictive value of circulating endothelial and leukocyte-derived extracellular vesicles (EV) in patients with advanced NSCLC treated with anti-PD-1/PD-L1 agents. In addition, the relationship between total blood circulating EV proteome and response to ICIs was investigated. An optimized flow cytometry method was employed for the identification and subtyping of blood circulating EVs in 59 patients with advanced NSCLC. Blood samples were collected from patients receiving anti-PD-1/PD-L1 inhibitors ( n = 31) or chemotherapy ( n = 28). An exploratory proteomic analysis of sorted blood EVs was conducted in a subset of patients. Our results show that a low blood concentration of circulating endothelial-derived EVs before treatment was strongly associated to longer overall survival ( p = 0.0004) and higher disease control rate ( p = 0.045) in patients treated with ICIs. Interestingly, shotgun proteomics revealed that EVs of responders to anti-PD-1 therapy had a specific protein cargo before treatment. In addition, EV protein cargo was specifically modulated during immunotherapy. We identified a previously unknown association between circulating endothelial-derived extracellular vesicle concentration and immunotherapy-related clinical outcomes. We also observed differences in circulating extracellular vesicle proteome according to anti-PD-1-based treatment response in NSCLC patients. Overall, these results may contribute to the identification of novel circulating biomarkers for rational immunotherapy approaches in patients affected by NSCLC.

Pancreatic cancer (PC) is one of the deadliest carcinomas and in most cases, which are diagnosed with locally advanced or metastatic disease, current therapeutic options are highly unsatisfactory. Based on the anti-proliferative effects shown by nitroxoline, an old urinary antibacterial agent, we explored a large library of newly synthesised derivatives to unravel the importance of the OH moiety and pyridine ring of the parent compound. The new derivatives showed a valuable anti-proliferative effect and some displayed a greater effect as compared to nitroxoline against three pancreatic cancer cell lines with different genetic profiles. In particular, in silico pharmacokinetic data, clonogenicity assays and selectivity indexes of the most promising compounds showed several advantages for such derivatives, as compared to nitroxoline. Moreover, some of these novel compounds had stronger effects on cell viability and/or clonogenic capacity in PC cells as compared to erlotinib, a targeted agent approved for PC treatment.

Pancreatic cancer (PC) is a lethal malignancy with rising incidence and limited therapeutic options. Obesity is a well-established risk factor for PC development. Moreover, it negatively affects outcome in PC patients. Excessive fat accumulation in obese, over- and normal-weight individuals induces metabolic and inflammatory changes of adipose tissue microenvironment leading to a dysfunctional adipose "organ". This may drive the association between abnormal fat accumulation and pancreatic cancer. In this review, we describe several molecular mechanisms that underpin this association at both local and systemic levels. We focus on the role of adipose tissue-derived circulating factors including adipokines, hormones and pro-inflammatory cytokines, as well as on the impact of the local adipose tissue in promoting PC. A discussion on potential therapeutic interventions, interfering with pro-tumorigenic effects of dysfunctional adipose tissue in PC, is included. Considering the raise of global obesity, research efforts to uncover the molecular basis of the relationship between pancreatic cancer and adipose tissue dysfunction may provide novel insights for the prevention of this deadly disease. In addition, these efforts may uncover novel targets for personalized interventional strategies aimed at improving the currently unsatisfactory PC therapeutic options.

An agonist-antagonist switching strategy was performed to discover novel PPARα antagonists. Phenyldiazenyl derivatives of fibrates were developed, bearing sulfonimide or amide functional groups. A second series of compounds was synthesized, replacing the phenyldiazenyl moiety with amide or urea portions. Final compounds were screened by transactivation assay, showing good PPARα antagonism and selectivity at submicromolar concentrations. When tested in cancer cell models expressing PPARα, selected derivatives induced marked effects on cell viability. Notably, 3c , 3d , and 10e displayed remarkable antiproliferative effects in two paraganglioma cell lines, with CC 50 lower than commercial PPARα antagonist GW6471 and a negligible toxicity on normal fibroblast cells. Docking studies were also performed to elucidate the binding mode of these compounds and to help interpretation of SAR data., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

We recently identified nitroxoline as a repurposed drug candidate in pancreatic cancer (PC) showing a dose-dependent antiproliferative activity in different PC cell lines. This antibiotic is effective in several in vitro and animal cancer models. To date, the mechanisms of nitroxoline anticancer action are largely unknown. Using shotgun proteomics we identified 363 proteins affected by nitroxoline treatment in AsPC-1 pancreatic cancer cells, including 81 consistently deregulated at both 24- and 48-hour treatment. These proteins previously unknown to be affected by nitroxoline were mostly downregulated and interconnected in a single highly-enriched network of protein-protein interactions. Integrative proteomic and functional analyses revealed nitroxoline-induced downregulation of Na/K-ATPase pump and β-catenin, which associated with drastic impairment in cell growth, migration, invasion, increased ROS production and induction of DNA damage response. Remarkably, nitroxoline induced a previously unknown deregulation of molecules with a critical role in cell bioenergetics, which resulted in mitochondrial depolarization. Our study also suggests that deregulation of cytosolic iron homeostasis and of co-translational targeting to membrane contribute to nitroxoline anticancer action. This study broadens our understanding of the mechanisms of nitroxoline action, showing that the drug modulates multiple proteins crucial in cancer biology and previously unknown to be affected by nitroxoline.

Pancreatic cancer (PC) is one of the most lethal, chemoresistant malignancies and it is of paramount importance to find more effective therapeutic agents. Repurposing of non-anticancer drugs may expand the repertoire of effective molecules. Studies on repurposing of benzimidazole-based anthelmintics in PC and on their interaction with agents approved for PC therapy are lacking. We analyzed the effects of four Food and Drug Administration (FDA)-approved benzimidazoles on AsPC-1 and Capan-2 pancreatic cancer cell line viability. Notably, parbendazole was the most potent benzimidazole affecting PC cell viability, with half maximal inhibitory concentration (IC 50 ) values in the nanomolar range. The drug markedly inhibited proliferation, clonogenicity and migration of PC cell lines through mechanisms involving alteration of microtubule organization and formation of irregular mitotic spindles. Moreover, parbendazole interfered with cell cycle progression promoting G2/M arrest, followed by the emergence of enlarged, polyploid cells. These abnormalities, suggesting a mitotic catastrophe, culminated in PC cell apoptosis, are also associated with DNA damage in PC cell lines. Remarkably, combinations of parbendazole with gemcitabine, a drug employed as first-line treatment in PC, synergistically decreased PC cell viability. In conclusion, this is the first study providing evidence that parbendazole as a single agent, or in combination with gemcitabine, is a repurposing candidate in the currently dismal PC therapy.

The reduced activation of PPARs has a positive impact on cancer cell growth and viability in multiple preclinical tumor models, suggesting a new therapeutic potential for PPAR antagonists. In the present study, the benzothiazole amides 2a-g were synthesized and their activities on PPARs were investigated. Transactivation assay showed a moderate activity of the novel compounds as PPARα antagonists. Notably, in cellular assays they exhibited cytotoxicity in pancreatic, colorectal and paraganglioma cancer cells overexpressing PPARα. In particular, compound 2b showed the most remarkable inhibition of viability (greater than 90%) in two paraganglioma cell lines, with IC 50 values in the low micromolar range. In addition, 2b markedly impaired colony formation capacity in the same cells. Taken together, these results show a relevant anti-proliferative potential of compound 2b, which appears particularly effective in paraganglioma, a rare tumor poorly responsive to chemotherapy., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Pancreatic cancer (PC) is the fourth most common cause of cancer death. Combination therapies with classical chemotherapeutic agents improved treatment of advanced PC at the cost of a relevant toxicity, but the 5-year survival rate remains below 5%. Consequently, new therapeutic options for this disease are urgently needed. In this study, we explored the effect of two repurposed drug candidates nelfinavir and nitroxoline, approved for non-anticancer human use, in PC cell lines. Nelfinavir and nitroxoline were tested as single agents, or in combinations with or without erlotinib, a targeted drug approved for PC treatment., Methods: The effects of the drugs on the viability of AsPC-1, Capan-2 and BxPC-3 PC cell lines were assessed by MTT. The impact of the treatments on cell cycle distribution and apoptosis was analyzed by flow cytometry. The effects of treatments on proteins relevant in cell cycle regulation and apoptosis were evaluated by western blot. Self-renewal capacity of PC cell lines after drug treatments was assessed using a clonogenic assay., Results: When used as single agents, nelfinavir and nitroxoline decreased viability, affected cell cycle and reduced the expression of relevant cell cycle proteins. The effects on apoptosis were variable among PC cell lines. Moreover, these agents drastically impaired clonogenic activity of the three PC cell lines. Combinations of nelfinavir and nitroxoline, with or without erlotinib, resulted in dose- and cell-dependent synergistic effects on cell viability. These effects were paralleled by cell cycle alterations and more consistent apoptosis induction as compared to single agents. Treatments with drug combinations induced drastic impairment of clonogenic activity in the three cell lines., Conclusions: This study shows that two non-antitumor drugs, nelfinavir and nitroxoline, as single agents or in combination have antitumor effects that appear comparable, or in some case more pronounced than those of erlotinib in three PC cell lines. Our results support repurposing of these approved drugs as single agents or in combination for PC treatment.

Paragangliomas (PGLs) are infiltrating autonomic nervous system tumors that cause important morbidity. At present, surgery is the only effective therapeutic option for this rare tumor. Thus, new agents for PGL treatment should be identified. Using unique PGL cell models established in our laboratory, we evaluated the effect of dichloroacetate (DCA) as single agent or in a novel combination with other metabolic drugs, including GW6471 and metformin. DCA and metformin had not been tested before in PGL. DCA reduced PGL cell viability and growth through mechanisms involving reactivation of PDH complex leading to promotion of oxidative metabolism, with lowering of lactate and enhanced ROS production. This resulted in cell cycle inhibition and induction of apoptosis in PGL cells, as shown by flow cytometry and immunoblot analyses. Moreover, DCA drastically impaired clonogenic activity and migration of PGL cells. Also metformin reduced PGL cell viability as single agent and the combinations of DCA, GW6471 and metformin had strong effects on cell viability. Furthermore, combined treatments had drastic and synergistic effects on clonogenic ability. In conclusion, DCA, GW6471 and metformin as single agents and in combination appear to have promising antitumor effects in unique cell models of PGL.

The effects on cancer-cell proliferation and differentiation mediated by peroxisome proliferator-activated receptors (PPARs) have been widely studied, and pleiotropic outcomes in different cancer models and under different experimental conditions have been obtained. Interestingly, few studies report and little preclinical evidence supports the potential antitumor activity of PPAR antagonists. This review focuses on recent findings on the antitumor in vitro and in vivo effects observed for compounds able to inhibit the three PPAR subtypes in different tumor models, providing a rationale for the use of PPAR antagonists in the treatment of tumors expressing the corresponding receptors., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Head and neck paragangliomas (HNPGLs) are rare tumors that may cause important morbidity, because of their tendency to infiltrate the skull base. At present, surgery is the only therapeutic option, but radical removal may be difficult or impossible. Thus, effective targets and molecules for HNPGL treatment need to be identified. However, the lack of cellular models for this rare tumor hampers this task. PPARα receptor activation was reported in several tumors and this receptor appears to be a promising therapeutic target in different malignancies. Considering that the role of PPARα in HNPGLs was never studied before, we analyzed the potential of modulating PPARα in a unique model of HNPGL cells. We observed an intense immunoreactivity for PPARα in HNPGL tumors, suggesting that this receptor has an important role in HNPGL. A pronounced nuclear expression of PPARα was also confirmed in HNPGL-derived cells. The specific PPARα agonist WY14643 had no effect on HNPGL cell viability, whereas the specific PPARα antagonist GW6471 reduced HNPGL cell viability and growth by inducing cell cycle arrest and caspase-dependent apoptosis. GW6471 treatment was associated with a marked decrease of CDK4, cyclin D3 and cyclin B1 protein expression, along with an increased expression of p21 in HNPGL cells. Moreover, GW6471 drastically impaired clonogenic activity of HNPGL cells, with a less marked effect on cell migration. Notably, the effects of GW6471 on HNPGL cells were associated with the inhibition of the PI3K/GSK3β/β-catenin signaling pathway. In conclusion, the PPARα antagonist GW6471 reduces HNPGL cell viability, interfering with cell cycle and inducing apoptosis. The mechanisms affecting HNPGL cell viability involve repression of the PI3K/GSK3β/β-catenin pathway. Therefore, PPARα could represent a novel therapeutic target for HNPGL.

The present study aimed to investigate the expression of human epidermal growth factor receptors (HERs) (HER1/HER2/HER3/HER4) and their phosphorylated forms (p-HER1/p-HER2/p-HER3/p-HER4) in pulmonary carcinoids (PCs). HER and p-HER protein expression was assessed by immunohistochemistry on tissue microarrays in 37 specimens of sporadic PCs, 29 typical carcinoids (TCs) and 8 atypical carcinoids (ACs). When compared with the ACs, the TCs did not exhibit any differences in terms of HER/p-HER expression. The tumors of this study have previously been characterized for the expression of menin and the mutational status of menin 1 ( MEN1 ), a gene strongly implicated in the pathogenesis of PCs. In the present study, it was found that the cytoplasmic ('disarrayed'), but not nuclear ('arrayed') expression of menin was positively correlated with HER3 (P=0.004), HER4 (P=0.015), p-HER1 (P=0.005), p-HER3 (P<0.001), and p-HER4 (P=0.001) expression. Moreover, HER3 and p-HER3 were found to be significantly more expressed in PCs with MEN1 variants, than in tumors with MEN1 wild-type (P=0.000 and P=0.025, respectively). These findings suggest the potential clinical use of HER inhibitors in the treatment of patients with PCs, particularly for individuals with p-HER3-positive PCs harboring MEN1 gene variants.

Background: To assess whether previously identified obesity-susceptibility loci were associated with overweight/obesity risk in a homogeneous population of Caucasian schoolchildren and whether these associations varied with age., Methods: Seven hundred and forty-five schoolchildren (353 boys, mean age: 8.3 ± 1.4 years) underwent anthropometric assessments. A saliva sample was collected for DNA extraction and assessment of 19 single-nucleotide polymorphisms previously associated with obesity., Results: Only the rs12970134 in the MC4R gene was significantly associated with overweight/obesity risk, with a higher frequency of the AA risk genotype in children with a BMI >85th (8.3%) than in those with a BMI <85th percentile (3.0%), p = 0.001; odds ratio (95% CI) of 1.544 (1.192-1.998), p = 0.001, after adjusting for age, sex and pubertal stage. BMI standard deviation scores (SDS) and waist-to-height ratio (W/Hr) progressively increased across the rs12970134 genotypes (GG vs. AG vs. AA): BMI SDS, p = 0.004; W/Hr, p = 0.009. When dividing the study population into two groups based on the median age of participants (8.3 years), the differences in BMI SDS and W/Hr across the MC4R genotypes persisted only in children older than 8.3 years., Conclusions: In a population of Caucasian schoolchildren, the rs12970134 MC4R variant was significantly associated with excess body weight, particularly in children older than 8 years of age., (© 2014 S. Karger AG, Basel.)

The identification of germline variants predisposing to hereditary nonpolyposis colorectal cancer (HNPCC) is crucial for clinical management of carriers, but several probands remain negative for such variants or bear variants of uncertain significance (VUS). Here we describe the results of integrative molecular analyses in 132 HNPCC patients providing evidences for improved genetic testing of HNPCC with traditional or next generation methods. Patients were screened for: germline allele-specific expression (ASE), nucleotide variants, rearrangements and promoter methylation of mismatch repair (MMR) genes; germline EPCAM rearrangements; tumor microsatellite instability (MSI) and immunohistochemical (IHC) MMR protein expression. Probands negative for pathogenic variants of MMR genes were screened for germline APC and MUTYH sequence variants. Most germline defects identified were sequence variants and rearrangements of MMR genes. Remarkably, altered germline ASE of MMR genes was detected in 8/22 (36.5%) probands analyzed, including 3 cases negative at other screenings. Moreover, ASE provided evidence for the pathogenic role and guided the characterization of a VUS shared by 2 additional probands. No germline MMR gene promoter methylation was observed and only one EPCAM rearrangement was detected. In several cases, tumor IHC and MSI diverged from germline screening results. Notably, APC or biallelic MUTYH germline defects were identified in 2/19 probands negative for pathogenic variants of MMR genes. Our results show that ASE complements gDNA-based analyses in the identification of MMR defects and in the characterization of VUS affecting gene expression, increasing the number of germline alterations detected. An appreciable fraction of probands negative for MMR gene variants harbors APC or MUTYH variants. These results indicate that germline ASE analysis and screening for APC and MUTYH defects should be included in HNPCC diagnostic algorithms.

The insulin/insulin-like growth factor pathway is involved in breast and colorectal cancer (CRC) development. In the present study, we analyzed the coding region and short intron-exon borders of the insulin receptor substrate 1 and 2 (IRS‑1 and IRS‑2) genes in 12 cell lines derived from breast cancer (BC), 14 cell lines derived from CRC and 33 primary CRCs. The nucleotide variants identified in BC were 3 in IRS‑1, 1 of which (p.Arg267Cys) was novel and with a pathogenic potential as predicted by in silico analysis and 6 in IRS‑2. Twenty‑one variants in IRS‑1 and 18 in IRS‑2 were identified in the CRC samples. These included 11 novel IRS‑1 variants detected exclusively in CRCs, which included 5 missense (p.Pro559Leu, p.Gln655His, p.Asp1014Gly, p.Asp1181His and pPro1203Ser) with a pathogenic potential as predicted by in silico analysis, 2 frameshifts predicted to generate a truncated protein, 1 splice-site mutation and 3 silent variants. In the CRC samples we also identified 7 novel IRS‑2 variants, including 4 missense variants, which included 2 (p.Asp782Asn and p.Gly1230Ser) with a pathogenic potential as predicted by in silico analysis, 2 frame insertion mutations and 1 silent variant. Most of the novel IRS‑1 and IRS‑2 variants may be involved in the modulation of IRS-1 or IRS‑2 functions and could be relevant to breast and colorectal tumorigenesis.