Deubiquitinases in Ovarian Cancer: Role in Drug Resistance and Tumor Aggressiveness.

Ovarian cancer is a lethal disease due to late diagnosis and occurrence of drug resistance that limits the efficacy of platinum-based therapy. Drug resistance mechanisms include both tumor intrinsic and tumor microenvironment-related factors. A role for deubiquitinases (DUBs) is starting to emerge in ovarian cancer. DUBs are a large family of enzymes that remove ubiquitin from target proteins and participate in processes affecting drug resistance such as DNA damage repair and apoptosis. Besides, DUBs modulate the functions of T cell populations favoring an immune suppressed microenvironment. Three DUBs are proteasome-associated, whereas the large majority are not. Among the former DUBs, USP14 has been proposed to modulate transcription factors such as Bcl6 and BACH1. In addition, RPN11/PSMD14 interferes with various processes including epithelial mesenchymal transition, also favored by non-proteasomal DUBs such as USP1 by acting on Snail. Besides, USP8 by stabilizing HER family receptors can confer drug resistance. Overall, DUBs appear to be druggable, with several inhibitors under development. Based on DUBs biological role, DUBs targeting appears promising in view of combination strategies involving different therapeutic approaches. Here, we summarize the relevance of DUBs in ovarian carcinoma and provide insights into future challenges for the treatment of this disease.

KiSS-1 Modulation by Epigenetic Agents Improves the Cisplatin Sensitivity of Lung Cancer Cells.

Epigenetic alterations my play a role in the aggressive behavior of Non-Small Cell Lung Cancer (NSCLC). Treatment with the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA, vorinostat) has been reported to interfere with the proliferative and invasive potential of NSCLC cells. In addition, the DNA methyltransferase inhibitor azacytidine (AZA, vidaza) can modulate the levels of the metastasis suppressor KiSS-1. Thus, since cisplatin is still clinically available for NSCLC therapy, the aim of this study was to evaluate drug combinations between cisplatin and SAHA as well as AZA using cisplatin-sensitive H460 and -resistant H460/Pt NSCLC cells in relation to KiSS-1 modulation. An analysis of drug interaction according to the Combination-Index values indicated a more marked synergistic effect when the exposure to SAHA or AZA preceded cisplatin treatment with respect to a simultaneous schedule. A modulation of proteins involved in apoptosis (p53, Bax) was found in both sensitive and resistant cells, and compared to the treatment with epigenetic agents alone, the combination of cisplatin and SAHA or AZA increased apoptosis induction. The epigenetic treatments, both as single agents and in combination, increased the release of KiSS-1. Finally, the exposure of cisplatin-sensitive and -resistant cells to the kisspeptin KP10 enhanced cisplatin induced cell death. The efficacy of the combination of SAHA and cisplatin was tested in vivo after subcutaneous inoculum of parental and resistant cells in immunodeficient mice. A significant tumor volume inhibition was found when mice bearing advanced tumors were treated with the combination of SAHA and cisplatin according to the best schedule identified in cellular studies. These results, together with the available literature, support that epigenetic drugs are amenable for the combination treatment of NSCLC, including patients bearing cisplatin-resistant tumors.

Therapeutic Strategies to Improve the Treatment of Pleural Mesothelioma.

Pleural mesothelioma is a rare neoplastic disease with aggressive features. Patient survival is poor due to the lack of early symptoms and the absence of effective therapeutic strategies. The development of pleural mesothelioma is mainly associated with asbestos exposure and related chronic inflammation. From a molecular-based perspective, this disease is a heterogeneous tumor lacking actionable alterations. The median overall survival of patients affected by this tumor does not exceed 16 months from diagnosis. Molecular and biochemical approaches have shown that this disease is characterized by resistance to drug-induced apoptosis associated with the activation of cell survival pathways and expression of anti-apoptotic proteins. Thus, there is an urgent need to develop efficient and safe therapeutic strategies. Here, we review the pharmacological options available for the treatment of this disease with specific reference to the antitumor agents used in systemic therapies. In addition, novel pharmacological approaches, such as drug delivery tools, to improve pleural mesothelioma treatment are discussed.

Alterations in Plasma Lipid Profiles Associated with Melanoma and Therapy Resistance.

Dysfunctions of lipid metabolism are associated with tumor progression and treatment resistance of cutaneous melanoma. BRAF/MEK inhibitor resistance is linked to alterations of melanoma lipid pathways. We evaluated whether a specific lipid pattern characterizes plasma from melanoma patients and their response to therapy. Plasma samples from patients and controls were analyzed for FASN and DHCR24 levels and lipidomic profiles. FASN and DHCR24 expression resulted in association with disease condition and related to plasma cholesterol and triglycerides in patients at different disease stages (n = 144) as compared to controls (n = 115). Untargeted lipidomics in plasma (n = 40) from advanced disease patients and controls revealed altered levels of different lipids, including fatty acid derivatives and sphingolipids. Targeted lipidomics identified higher levels of dihydroceramides, ceramides, sphingomyelins, ganglioside GM3, sphingosine, sphingosine-1-phosphate, and dihydrosphingosine, saturated and unsaturated fatty acids. When melanoma patients were stratified based on a long/short-term clinical response to kinase inhibitors, differences in plasma levels were shown for saturated fatty acids (FA 16:0, FA18:0) and oleic acid (FA18:1). Our results associated altered levels of selected lipid species in plasma of melanoma patients with a more favorable prognosis. Although obtained in a small cohort, these results pave the way to lipidomic profiling for melanoma patient stratification.

Kisspeptin-mediated improvement of sensitivity to BRAF inhibitors in vemurafenib-resistant melanoma cells.

Metastatic dissemination is still one of the major causes of death of melanoma's patients. KiSS1 is a metastasis suppressor originally identified in melanoma cells, known to play an important physiological role in mammals' development and puberty. It has been previously shown that expression of KiSS1 could be increased in lung cancer cells using epigenetic agents, and that KiSS1 could have a pro-apoptotic action in combination with cisplatin. Thus, the aim of the present study was to examine in human melanoma vemurafenib sensitive- and -resistant BRAF mutant cells characterized by different mutational profiles and KiSS1, KiSS1 receptor and KiSS1 drug-induced release, if peptides derived from KiSS1 cleavage, i.e., kisspeptin 54, could increase the sensitivity to vemurafenib of human melanoma, using cellular, molecular and biochemical approaches. We found that kisspeptin 54 increases vemurafenib pro-apoptotic activity in a statistically significant manner, also in drug resistant cellular models. The efficacy of the combination appears to reflect the intrinsic susceptibility of each cell line to PLX4032-induced apoptosis, together with the different mutational profile as well as perturbation of proteins regulating the apoptotic pathway, The results presented here highlight the possibility to exploit KiSS1 to modulate the apoptotic response to therapeutically relevant agents, suggesting a multitasking function of this metastasis suppressor.

Synthesis of N-oxyamide analogues of protein kinase B (Akt) targeting anionic glycoglycerolipids and their antiproliferative activity on human ovarian carcinoma cells.

N-Oxyamides of bioactive anionic glycoglycerolipids based on 2-O-ß-D-glucosylglycerol were efficiently prepared. However, the oxidation step of the primary hydroxyl group of the glucose moiety in the presence of the N-oxyamide function appeared to be a difficult task that was nevertheless conveniently achieved for the first time by employing a chemoenzymatic laccase/TEMPO procedure. The obtained N-oxyamides exhibited a higher inhibition of proliferation of ovarian carcinoma IGROV-1 cells in serum-free medium than in complete medium, similarly to the corresponding bioactive esters. Stability and serum binding studies indicated that the observed reduced activity of the compounds in complete medium could be mainly due to a binding effect of serum proteins rather than the hydrolytic degradation of glycoglycerolipid acyl chains. Furthermore, the results of the cellular studies under serum-free conditions suggested that the N-oxyamide group could increase the antiproliferative activity of a glycoglycerolipid independently of the presence of the anionic carboxylic group. Cellular studies in other cell lines besides IGROV-1 also support a certain degree of selectivity of this series of compounds for tumor cells with Akt hyperactivation.

The deubiquitinase USP8 regulates ovarian cancer cell response to cisplatin by suppressing apoptosis.

The identification of therapeutic approaches to improve response to platinum-based therapies is an urgent need for ovarian carcinoma. Deubiquitinases are a large family of ubiquitin proteases implicated in a variety of cellular functions and may contribute to tumor aggressive features through regulation of processes such as proliferation and cell death. Among the subfamily of ubiquitin-specific peptidases, USP8 appears to be involved in modulation of cancer cell survival by still poorly understood mechanisms. Thus, we used ovarian carcinoma cells of different histotypes, including cisplatin-resistant variants with increased survival features to evaluate the efficacy of molecular targeting of USP8 as a strategy to overcome drug resistance/modulate cisplatin response. We performed biochemical analysis of USP8 activity in pairs of cisplatin-sensitive and -resistant cells and found increased USP8 activity in resistant cells. Silencing of USP8 resulted in decreased activation of receptor tyrosine kinases and increased sensitivity to cisplatin in IGROV-1/Pt1 resistant cells as shown by colony forming assay. Increased cisplatin sensitivity was associated with enhanced cisplatin-induced caspase 3/7 activation and apoptosis, a phenotype also observed in cisplatin sensitive cells. Increased apoptosis was linked to FLIPL decrease and cisplatin induction of caspase 3 in IGROV-1/Pt1 cells, cisplatin-induced claspin and survivin down-regulation in IGROV-1 cells, thereby showing a decrease of anti-apoptotic proteins. Immunohistochemical staining on 65 clinical specimens from advanced stage ovarian carcinoma indicated that 40% of tumors were USP8 positive suggesting that USP8 is an independent prognostic factor for adverse outcome when considering progression free survival as a clinical end-point. Taken together, our results support that USP8 may be of diagnostic value and may provide a therapeutic target to improve the efficacy of platinum-based therapy in ovarian carcinoma.

Increased serum levels of KiSS1-derived peptides in non-small cell lung cancer patient liquid biopsies and biological relevance.

Background: The secreted products of the metastasis suppressor gene KiSS1 may represent useful biomarkers in non-small cell lung cancer (NSCLC) but their levels in patients have remained poorly investigated. We previously found that forced expression of KiSS1 decreased the invasive capability of NSCLC drug-resistant cells and a pro-apoptotic role for KiSS1 has been proposed in head and neck cancer. Thus, we designed a translational investigation including a pilot study to analyze KiSS1 levels in liquid biopsies, and in vitro experiments to explore the biological relevance of KiSS1 modulation. Methods: KiSS1-derived peptide levels in liquid biopsies from 60 NSCLC patients were assayed by ELISA. Preclinical experiments were carried out using quantitative real time polymerase chain reaction (qRT-PCR), ELISA, annexin V-binding and caspase activation assays. Results: We compared KiSS1 release in 3 different matrices (serum, plasma and urine) and the highest levels were detectable in serum (range, 0-4.5 ng/mL). We observed increased levels of seric KiSS1 in NSCLC patients as compared to healthy donors. KiSS1 serum concentrations, after surgical procedure and/or adjuvant therapy. We observed differences among disease stages in urine samples. In preclinical models, KiSS1 mRNA levels were increased by short term exposure to azacytidine, enhanced KiSS1 release was induced by the combination of azacytidine and cisplatin and KiSS1-derived peptides enhanced cisplatin-induced apoptosis. KiSS1 increase was observed upon exposure neurons-enriched cultures to tumor cell conditioned medium. Conclusions: Our results showing a peculiar modulation of KiSS1 levels in liquid biopsies of NSCLC patients and a regulation of cisplatin-induced apoptosis by KiSS1-derived peptides support an involvement of KiSS1 in cell response to treatment and highlight its promising features as a potential biomarker in NSCLC.

Targeting of the Lipid Metabolism Impairs Resistance to BRAF Kinase Inhibitor in Melanoma.

Drug resistance limits the achievement of persistent cures for the treatment of melanoma, in spite of the efficacy of the available drugs. The aim of the present study was to explore the involvement of lipid metabolism in melanoma resistance and assess the effects of its targeting in cellular models of melanoma with acquired resistance to the BRAF-inhibitor PLX4032/Vemurafenib. Since transcriptional profiles pointed to decreased cholesterol and fatty acids synthesis in resistant cells as compared to their parental counterparts, we examined lipid composition profiles of resistant cells, studied cell growth dependence on extracellular lipids, assessed the modulation of enzymes controlling the main nodes in lipid biosynthesis, and evaluated the effects of targeting Acetyl-CoA Acetyltransferase 2 (ACAT2), the first enzyme in the cholesterol synthesis pathway, and Acyl-CoA Cholesterol Acyl Transferase (ACAT/SOAT), which catalyzes the intracellular esterification of cholesterol and the formation of cholesteryl esters. We found a different lipid composition in the resistant cells, which displayed reduced saturated fatty acids (SFA), increased monounsaturated (MUFA) and polyunsaturated (PUFA), and reduced cholesteryl esters (CE) and triglycerides (TG), along with modulated expression of enzymes regulating biosynthetic nodes of the lipid metabolism. The effect of tackling lipid metabolism pathways in resistant cells was evidenced by lipid starvation, which reduced cell growth, increased sensitivity to the BRAF-inhibitor PLX4032, and induced the expression of enzymes involved in fatty acid and cholesterol metabolism. Molecular targeting of ACAT2 or pharmacological inhibition of SOAT by avasimibe showed antiproliferative effects in melanoma cell lines and a synergistic drug interaction with PLX4032, an effect associated to increased ferroptosis. Overall, our findings reveal that lipid metabolism affects melanoma sensitivity to BRAF inhibitors and that extracellular lipid availability may influence tumor cell response to treatment, a relevant finding in the frame of personalized therapy. In addition, our results indicate new candidate targets for drug combination treatments.

Curcumin and Related Compounds in Cancer Cells: New Avenues for Old Molecules.

Curcumin and related compounds are known for the large spectrum of activities. The chemical features of these compounds are important for their biological effects with a key role for the thiol-reactive α-ß unsaturated carbonyl groups. Curcumin derivatives may overcome the limitation of the bioavailability of the parent compound, while maintaining the key chemical features responsible for biological activities. Curcumin and related compounds show anti-viral, anti-fungal, anti-microbial and anti-tumor activities. The therapeutic effects of curcumin, used as a supplement in cancer therapy, have been documented in various cancer types, in which inhibition of cell growth and survival pathways, induction of apoptosis and other cell death pathways have been reported. Curcumin-induced apoptosis has been linked both to the intrinsic and extrinsic apoptotic pathways. Necroptosis has also been involved in curcumin-induced toxicity. Among curcumin-induced effects, ferroptosis has also been described. The mechanism of curcumin toxicity can be triggered by reactive oxygen species-mediated endoplasmic reticulum stress. Curcumin targets have been identified in the context of the ubiquitin-proteasome system with evidence of inhibition of the proteasome proteolytic activities and cellular deubiquitinases. Curcumin has recently been shown to act on the tumor microenvironment with effects on cancer-associated fibroblasts and immune cells. The related product caffeic acid phenethyl ester has shown promising preclinical results with an effect on the inflammatory microenvironment. Here, we review the mechanisms underlying curcumin and derivatives toxicity towards cancer cells with particular emphasis on cell death pathways and the ubiquitin-proteasome system.

Synthesis and Investigation of the G-Quadruplex Binding Properties of Kynurenic Acid Derivatives with a Dihydroimidazoquinoline-3,5-dione Core.

G-quadruplexes are secondary structures originating from nucleic acid regions rich in guanines, which are well known for their involvement in gene transcription and regulation and DNA damage repair. In recent studies from our group, kynurenic acid (KYNA) derivative 1 was synthesized and found to share the structural features typical of G-quadruplex binders. Herein, structural modifications were conducted on this scaffold in order to assist the binding with a G-quadruplex, by introducing charged hydrophilic groups. The antiproliferative activity of the new analogues was evaluated on an IGROV-1 human ovarian cancer cell line, and the most active compound, compound 9, was analyzed with NMR spectrometry in order to investigate its binding mode with DNA. The results indicated that a weak, non-specific interaction was set with duplex nucleotides; on the other hand, titration in the presence of a G-quadruplex from human telomere d(TTAGGGT)4 showed a stable, although not strong, interaction at the 3'-end of the nucleotidic sequence, efficiently assisted by salt bridges between the quaternary nitrogen and the external phosphate groups. Overall, this work can be considered a platform for the development of a new class of potential G-quadruplex stabilizing molecules, confirming the crucial role of a planar system and the ability of charged nitrogen-containing groups to facilitate the binding to G-quadruplex grooves and loops.

P301L tau mutation leads to alterations of cell cycle, DNA damage response and apoptosis: Evidence for a role of tau in cancer.

Tau is a microtubule-associated protein, coded by the MAPT gene, which regulates microtubule (MT) polymerization and dynamics. Due to its key role in neurons, it is a major player in neurodegenerative diseases known as "tauopathies". Since tau has emerged as a multitasking protein with a role in genome stability, it may act both in neurodegeneration and cancer. After demonstrating that tau can be considered as a risk factor for cancer, here we explored the mechanisms linking mutated tau to dysregulation of cancer-relevant processes, by employing lymphoblastoid cell lines (LCL) from patients affected by genetic tauopathy carrying the MAPT P301L mutation and healthy controls (wild-type, wt). In mutated LCL, we found reduced sensitivity to MT perturbation, along with decreased G2/M accumulation and cyclin B1 levels. Furthermore, mutated LCL displayed lower levels of phospho-Chk1 and phospho-Chk2 following hydrogen peroxide-induced oxidative stress, indicating a poorly effective DNA damage checkpoint, as well as reduced basal levels of p53. Such cells also exhibited lower levels of Bax and cleaved caspase-3, and increased levels of Cdc25A, upon oxidative stress, accounting for diminished apoptosis. Overall, these findings point to tau as a key player in biological pathways relevant for cancer, as evidenced by the differential response of mutated and wt cells to MT and DNA perturbation. The modulation of p53 is intriguing given its function as guardian of the genome.

The mTOR/4E-BP1/eIF4E Signalling Pathway as a Source of Cancer Drug Targets.

The mechanistic/mammalian target of rapamycin (mTOR) is the crucial hub of signalling pathways that regulate essential steps in the cell life cycle. Once incorporated in the mTORC1 complex, mTOR phosphorylates the eukaryotic initiation factor 4E (eIF4E)- binding protein 1 (4E-BP1), which then releases eIF4E. When not bound to 4EBPs, eIF4E recognizes the mRNA 5'-cap structure and, together with eIF4A and eIF4G, it forms the eIF4F complex that recruits the ribosome on the mRNA. Under normal conditions, the cellular concentration of eIF4E is very low, making eIF4E the limiting factor in the initiation of protein synthesis. The vast majority of cancer types are characterized by the simultaneous deregulation of the mTOR/4E-BP1 signalling pathway and upregulation of eIF4E, which lead to an increased expression of cancer-promoting genes and deregulated cellular growth. Over the last decades, a growing number of selective inhibitors of the mTOR/4E-BP1/eIF4E pathway have been discovered or designed. Several inhibitors with encouraging preclinical results have been tested in clinical trials. This review summarizes the most recent research on drug development against mTOR, 4E-BP1, and eIF4E, describing the design rationale and the available structural and functional data on the most promising compounds.

Caffeic acid phenethyl ester targets ubiquitin-specific protease 8 and synergizes with cisplatin in endometrioid ovarian carcinoma cells.

Deubiquitinases (DUBs) mediate the removal of ubiquitin from diverse proteins that participate in the regulation of cell survival, DNA damage repair, apoptosis and drug resistance. Previous studies have shown an association between activation of cell survival pathways and platinum-drug resistance in ovarian carcinoma cell lines. Among the strategies available to inhibit DUBs, curcumin derivatives appear promising, thus we hypothesized their use to enhance the efficacy of cisplatin in ovarian carcinoma preclinical models. The caffeic acid phenethyl ester (CAPE), inhibited ubiquitin-specific protease 8 (USP8), but not proteasomal DUBs in cell-free assays. When CAPE was combined with cisplatin in nine cell lines representative of various histotypes a synergistic effect was observed in TOV112D cells and in the cisplatin-resistant IGROV-1/Pt1 variant, both of endometrioid type and carrying mutant TP53. In the latter cells, persistent G1 accumulation upon combined treatment associated with p27kip1 protein levels was observed. The synergy was not dependent on apoptosis induction, and appeared to occur in cells with higher USP8 levels. In vivo antitumor activity studies supported the advantage of the combination of CAPE and cisplatin in the subcutaneous model of cisplatin-resistant IGROV-1/Pt1 ovarian carcinoma as well as CAPE activity on intraperitoneal disease. This study reveals the therapeutic potential of CAPE in cisplatin-resistant ovarian tumors as well as in tumors expressing USP8.

Recent Progresses in Conjugation with Bioactive Ligands to Improve the Anticancer Activity of Platinum Compounds.

Platinum (Pt) drugs, including cisplatin, are widely used for the treatment of solid tumors. Despite the clinical success, side effects and occurrence of resistance represent major limitations to the use of clinically available Pt drugs. To overcome these problems, a variety of derivatives have been designed and synthetized. Here, we summarize the recent progress in the development of Pt(II) and Pt(IV) complexes with bioactive ligands. The development of Pt(II) and Pt(IV) complexes with targeting molecules, clinically available agents, and other bioactive molecules is an active field of research. Even if none of the reported Pt derivatives has been yet approved for clinical use, many of these compounds exhibit promising anticancer activities with an improved pharmacological profile. Thus, planning hybrid compounds can be considered as a promising approach to improve the available Pt-based anticancer agents and to obtain new molecular tools to deepen the knowledge of cancer progression and drug resistance mechanisms.

A trifunctional self-immolative spacer enables drug release with two non-sequential enzymatic cleavages.

Cyclative cleavage of an amine-carbamate self-immolative spacer to deliver a hydroxyl cargo was inhibited by spacer derivatisation with a phosphate monoester handle. This trifunctional spacer was installed in a model anticancer prodrug that showed fast drug release only when incubated with both a protease and a phosphatase enzyme.

Synergistic Effect of Perampanel and Temozolomide in Human Glioma Cell Lines.

Glioblastoma is characterized by a high proliferative rate and drug resistance. The standard of care includes maximal safe surgery, followed by radiotherapy and temozolomide chemotherapy. The expression of glutamate receptors has been previously reported in human glioma cell lines. The aim of this study was to examine the cellular effects of perampanel, a broad-spectrum antiepileptic drug acting as an α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA) glutamate receptor antagonist, alone or in combination with temozolomide. Four human glioma cell lines were exposed to different concentrations of perampanel and temozolomide, alone or in combination. The type of drug interaction was assessed using the Chou-Talalay method. Apoptosis, cell cycle perturbation, and glutamate receptors (GluRs) subunit expression were assessed by flow cytometry. Perampanel significantly inhibited the growth, inducing high levels of apoptosis. A strong synergistic effect of the combination of perampanel with temozolomide was detected in U87 and A172, but not in U138. Treatment with perampanel resulted in an increased GluR2/3 subunit expression in U87 and U138. Perampanel displays a pro-apoptotic effect on human glioblastoma cell lines when used alone, possibly due to increased GluR2/3 expression. The observed synergistic effect of the combination of temozolomide with perampanel suggests further investigation on the impact of this combination on oncologic outcomes in glioblastoma.

Deregulated FASN Expression in BRAF Inhibitor-Resistant Melanoma Cells Unveils New Targets for Drug Combinations.

Metabolic changes promoting cell survival are involved in metastatic melanoma progression and in the development of drug resistance. In BRAF-inhibitor resistant melanoma cells, we explored the role of FASN, an enzyme involved in lipogenesis overexpressed in metastatic melanoma. Resistant melanoma cells displaying enhanced migratory and pro-invasive abilities increased sensitivity to the BRAF inhibitor PLX4032 upon the molecular targeting of FASN and upon treatment with the FASN inhibitor orlistat. This behavior was associated with a marked apoptosis and caspase 3/7 activation observed for the drug combination. The expression of FASN was found to be inversely associated with drug resistance in BRAF-mutant cell lines, both in a set of six resistant/sensitive matched lines and in the Cancer Cell Line Encyclopedia. A favorable drug interaction in resistant cells was also observed with U18666 A inhibiting DHCR24, which increased upon FASN targeting. The simultaneous combination of the two inhibitors showed a synergistic interaction with PLX4032 in resistant cells. In conclusion, FASN plays a role in BRAF-mutated melanoma progression, thereby creating novel therapeutic opportunities for the treatment of melanoma.