Targeting NPRL2 to enhance the efficacy of Olaparib in castration-resistant prostate cancer

Castration-resistant prostate cancer (CRPC) lacks effective treatment, and studies have shown that PARPi inhibitors, such as Olaparib, are somewhat effective; however, the efficacy of Olaparib in CRPC still needs to be further improved. Nitrogen permease regulator-like 2 (NPRL2) is reported to be a tumor suppressor candidate gene and is closely related to the DNA repair pathway, which can affect the sensitivity of many chemotherapeutic drugs. However, there is no research on whether NPRL2 is associated with sensitivity to Olaparib. Hence, in the present study, we examined the NPRL2 expression levels in several PCa cell lines (LNCaP, PC3, and enzalutamide-resistant LNCaP, named LNPER) by Western blot. In addition, we investigated the role of NPRL2 expression and silencing in cell proliferation and in the regulation of ataxia telangiectasia mutated (ATM), which can mediate DNA repair and sensitivity to Olaparib. Furthermore, we performed in vitro and in vivo experiments to determine the mechanism of action of NPRL2 in adjusting Olaparib sensitivity. Our findings demonstrated that the NPRL2 expression level was upregulated in PCa cells, especially CRPC cells. NPRL2 overexpression promoted growth and resistance to Olaparib, and NPRL2 silencing inhibited proliferation, enhanced sensitivity to Olaparib, and increased CRRL2 expression in PCa cells. In addition, the Olaparib-induced growth delay in NPRL2-silenced PC3 tumors in mice correlated with ATM signaling downregulation, an apoptosis increase and migration/invasion suppression. Our results indicate that NPRL2 silencing enhances sensitivity to Olaparib treatment in CRPC and that NPRL2 may serve as a potential therapeutic target and predict resistance to Olaparib in CRPC.     

KIAA1529 regulates RAD51 expression to confer PARP inhibitors resistance in ovarian cancer

PARP inhibitors (PARPi) are currently used as first-line therapy for advanced and recurrent ovarian cancer, but the clinical efficacy is limited by drug resistance. We aimed to investigate the role of KIAA1529 in PARPi resistance in ovarian cancer. The expression of KIAA1529 was determined in ovarian cancer cells using qRT‒PCR and western blotting. Immunohistochemistry was used to examine the expression of KIAA1529 in primary ovarian cancer and recurrent ovarian cancer tissues. The effects of KIAA1529 on PARPi resistance were evaluated by knocking down KIAA1529 expression in ovarian cancer cells and assessing cell viability by CCK8 assays, apoptosis by flow cytometry, and homologous recombination (HR) repair by immunofluorescence analysis. The interaction between KIAA1529 and RAD51 was examined by western blotting. KIAA1529 was confirmed to be expressed in all ovarian cancer cell lines, and high expression of KIAA1529 was observed in recurrent ovarian cancer tissues. Inhibiting KIAA1529 expression increased the sensitivity of ovarian cancer cells to PARPi treatment. Furthermore, KIAA1529 increased the expression of the downstream effector RAD51 via Aurora-A, and HR was restored in ovarian cancer cells. This study demonstrates that KIAA1529 regulates RAD51 expression through Aurora-A to restore HR, which confers resistance to PARPi in ovarian cancer cells. These findings could provide a novel therapeutic target to overcome PARPi resistance in ovarian cancer.     

A2B adenosine receptor blockade inhibits growth of prostate cancer cells

The role of the A_2B adenosine receptor (AR) in prostate cell death and growth was studied. The A_2B AR gene expression quantified by real-time quantitative RT-PCR and Western blot analysis was the highest among four AR subtypes (A₁, A_2A, A_2B, and A₃) in all three commonly used prostate cancer cell lines, PC-3, DU145, and LNCaP. We explored the function of the A_2B AR using PC-3 cells as a model. The A_2B AR was visualized in PC-3 cells by laser confocal microscopy. The nonselective A_2B AR agonist NECA and the selective A_2B AR agonist BAY60-6583, but not the A_2A AR agonist CGS21680, concentration-dependently induced adenosine 3′,5′-cyclic monophosphate (cyclic AMP) accumulation. NECA diminished lactate dehydrogenase (LDH) release, TNF-α-induced increase of caspase-3 activity, and cycloheximide (CHX)-induced morphological changes typical of apoptosis in PC-3 cells, which were blocked by a selective A_2B AR antagonist PSB603. NECA-induced proliferation of PC-3 cells was diminished by siRNA specific for the A_2B AR. The selective A_2B AR antagonist PSB603 was shown to inhibit cell growth in all three cell lines. Thus, A_2B AR blockade inhibits growth of prostate cancer cells, suggesting selective A_2B AR antagonists as potential novel therapeutics.     

HMGB3 promotes PARP inhibitor resistance through interacting with PARP1 in ovarian cancer

Poly (ADP-ribose) polymerase (PARP) inhibitor (PARPi) resistance remains a therapeutic challenge in ovarian cancer. High-mobility group box 3 (HMGB3) plays significant roles in the development of drug resistance of many cancers. However, the function of HMGB3 in PARPi resistance is poorly understood. In the current study, we clarified that HMGB3 was aberrantly overexpressed in high-grade serous ovarian carcinoma (HGSOC) tissues, and high HMGB3 levels indicated shorter overall survival and drug resistance in HGSOC. The overexpression of HMGB3 increased the insensitivity of ovarian cancer to PARPi, whereas HMGB3 knockdown reduced PARPi resistance. Mechanistically, PARP1 was identified as a novel interaction partner of HMGB3, which could be blocked using olaparib and was enhanced upon DNA damage conditions. We further showed that loss of HMGB3 induced PARP1 trapping at DNA lesions and inhibited the PARylation activity of PARP1, resulting in an increased DNA damage response and cell apoptosis. The PARPi-resistant role of HMGB3 was also verified in a xenograft mouse model. In conclusion, HMGB3 promoted PARPi resistance via interacting with PARP1, and the targeted inhibition of HMGB3 might overcome PARPi resistance in ovarian cancer therapy.Subject terms: Chemotherapy, Ovarian cancer, Ovarian cancer, Cancer therapeutic resistance     

Adenosine receptors regulate exosome production

Exosomes, small-sized extracellular vesicles, carry components of the purinergic pathway. The production by cells of exosomes carrying this pathway remains poorly understood. Here, we asked whether type 1, 2A, or 2B adenosine receptors (A₁Rs, A_2ARs, and A_2BRs, respectively) expressed by producer cells are involved in regulating exosome production. Preglomerular vascular smooth muscle cells (PGVSMCs) were isolated from wildtype, A₁R^?/?, A_2AR^?/?, and A_2BR^?/? rats, and exosome production was quantified under normal or metabolic stress conditions. Exosome production was also measured in various cancer cells treated with pharmacologic agonists/antagonists of A₁Rs, A_2ARs, and A_2BRs in the presence or absence of metabolic stress or cisplatin. Functional activity of exosomes was determined in Jurkat cell apoptosis assays. In PGVSMCs, A₁R and A_2AR constrained exosome production under normal conditions (p?=?0.0297; p?=?0.0409, respectively), and A₁R, A_2AR, and A_2BR constrained exosome production under metabolic stress conditions. Exosome production from cancer cells was reduced (p?=?0.0028) by the selective A_2AR agonist CGS 21680. These exosomes induced higher levels of Jurkat apoptosis than exosomes from untreated cells or cells treated with A₁R and A_2BR agonists (p?=?0.0474). The selective A_2AR antagonist SCH 442416 stimulated exosome production under metabolic stress or cisplatin treatment, whereas the selective A_2BR antagonist MRS 1754 reduced exosome production. Our findings indicate that A_2ARs suppress exosome release in all cell types examined, whereas effects of A₁Rs and A_2BRs are dependent on cell type and conditions. Pharmacologic targeting of cancer with A_2AR antagonists may inadvertently increase exosome production from tumor cells.

     

Inhibition of PKM2 Enhances Sensitivity of Olaparib to Ovarian Cancer Cells and Induces DNA Damage

Poly (ADP-ribose) polymerase inhibitors (PARPi) have showed clinical benefit as maintenance therapy in advanced ovarian cancer by impairing the homologous recombination (HR) pathway. Pyruvate kinase M2 (PKM2), the significant cancer metabolic biomarker, integrates with DNA damage to directly promote HR. We aimed to investigate the role and molecular mechanism of PKM2 downregulation on sensitization of ovarian cancer cells to PARPi. Inhibitory effects in vitro were assessed by cell viability, clone formation, transwell assay, and flow cytometry. Downregulation of PKM2 by siRNA or small molecular inhibitor shikonin (Sk) enhanced anti-tumour activity of olaparib (Ola) in ovarian cancer cells. Silencing PKM2 or Sk synergized with Ola and reduced cell growth, colony formation and migration, and induced apoptosis. Western blot and immunofluorescence demonstrated that inhibition of PKM2 amplified Ola-induced γH2AX and phospho-ATM (p-ATM) activation and interfered with BRCA1 accumulation in the nucleus. A xenograft animal model demonstrated in vivo antitumor combination effect of Sk and Ola. Furthermore, Western blot and immunofluorenscent analyses of tissue samples revealed that treatment of Sk increased DNA damage, reduced expression of BRCA1 and PKM2. Therefore, this study identified that PKM2 downregulation is a novel therapeutic strategy to enhance Ola effectiveness in treating ovarian cancer.     

Caveolae Contribute to the Apoptosis Resistance Induced by the α1A-Adrenoceptor in Androgen-Independent Prostate Cancer Cells

Background

During androgen ablation prostate cancer cells'' growth and survival become independent of normal regulatory mechanisms. These androgen-independent cells acquire the remarkable ability to adapt to the surrounding microenvironment whose factors, such as neurotransmitters, influence their survival. Although findings are becoming evident about the expression of α_1A-adrenoceptors in prostate cancer epithelial cells, their exact functional role in androgen-independent cells has yet to be established. Previous work has demonstrated that membrane lipid rafts associated with key signalling proteins mediate growth and survival signalling pathways in prostate cancer cells.

Methodology/Principal Findings

In order to analyze the membrane topology of the α_1A-adrenoceptor we explored its presence by a biochemical approach in purified detergent resistant membrane fractions of the androgen-independent prostate cancer cell line DU145. Electron microscopy observations demonstrated the colocalisation of the α_1A-adrenoceptor with caveolin-1, the major protein component of caveolae. In addition, we showed that agonist stimulation of the α_1A-adrenoceptor induced resistance to thapsigargin-induced apoptosis and that caveolin-1 was necessary for this process. Further, immunohistofluorescence revealed the relation between high levels of α_1A-adrenoceptor and caveolin-1 expression with advanced stage prostate cancer. We also show by immunoblotting that the TG-induced apoptosis resistance described in DU145 cells is mediated by extracellular signal-regulated kinases (ERK).

Conclusions/Significance

In conclusion, we propose that α_1A-adrenoceptor stimulation in androgen-independent prostate cancer cells via caveolae constitutes one of the mechanisms contributing to their protection from TG-induced apoptosis.     

A<Subscript>2B</Subscript>adenosine receptor activity is reduced in neutrophils from patients with systemic sclerosis

We conducted the present study to investigate protein expression and functioning of A_2A and A_2B adenosine receptors (ARs) in neutrophils of patients affected by systemic sclerosis (SSc). The presence of A_2A and A_2B ARs was assessed by immunoblotting using specific antibodies. Equilibrium A_2A and A_2B ARs binding parameters were evaluated by radioligand binding assay. Functional studies were conducted to investigate coupling of the A_2B AR to the adenylyl cyclase pathway. This is the first report of the use of Western blot analysis to confirm the presence of A_2A and A_2B ARs in human neutrophils. No significant changes in A_2A AR binding parameters or expression levels were detected between SSc patients and healthy control individuals. A significant decrease (65%) in the maximum density of A_2B AR binding sites occurred in SSc neutrophils, whereas no changes in the affinity constant values were found. Moreover, a decrease in A_2B AR mediated adenylyl cyclase activity was observed in patients with SSc. Our findings demonstrate the occurrence of selective alterations in A_2B AR density and signalling in SSc.     

Prognostic role of Wnt and Fzd gene families in acute myeloid leukaemia

Wnt-Fzd signalling pathway plays a critical role in acute myeloid leukaemia (AML) progression and oncogenicity. There is no study to investigate the prognostic value of Wnt and Fzd gene families in AML. Our study screened 84 AML patients receiving chemotherapy only and 71 also undergoing allogeneic haematopoietic stem cell transplantation (allo-HSCT) from the Cancer Genome Atlas (TCGA) database. We found that some Wnt and Fzd genes had significant positive correlations. The expression levels of Fzd gene family were independent of survival in AML patients. In the chemotherapy group, AML patients with high Wnt2B or Wnt11 expression had significantly shorter event-free survival (EFS) and overall survival (OS); high Wnt10A expressers had significantly longer OS than the low expressers (all P < .05), whereas, in the allo-HSCT group, the expression levels of Wnt gene family were independent of survival. We further found that high expression of Wnt10A and Wnt11 had independent prognostic value, and the patients with high Wnt10A and low Wnt11 expression had the longest EFS and OS in the chemotherapy group. Pathway enrichment analysis showed that genes related to Wnt10A, Wnt11 and Wnt 2B were mainly enriched in ‘cell morphogenesis involved in differentiation’, ‘haematopoietic cell lineage’, ‘platelet activation, signalling and aggregation’ and ‘mitochondrial RNA metabolic process’ signalling pathways. Our results indicate that high Wnt2B and Wnt11 expression predict poor prognosis, and high Wnt10A expression predicts favourable prognosis in AML, but their prognostic effects could be neutralized by allo-HSCT. Combined Wnt10A and Wnt11 may be a novel prognostic marker in AML.     

PARP inhibitor Olaparib increases the sensitization to radiotherapy in FaDu cells

Radioresistance causes a major problem for improvement of outcomes of patients treated with radiation. Targeting for DNA repair deficient mechanisms is a hallmark of sensitization to resistance. We tested whether Olaparib, a (poly) ADP‐ribose polymerase (PARP) inhibitor, can sensitize the radioresistant FaDu cells to radiotherapy. Radioresistant FaDu cells, called FaDu‐RR cells, were used as the radioresistant hypopharyngeal cancer models. The expression of PARP1 was detected in both FaDu and FaDu‐RR cells. The role of Olaparib in radiosensitization was analysed with several assays including clonogenic cell survival, cell proliferation and cell cycle, and radioresistant xenograft. High expression of PARP1 had a significant effect on enhancing radioresistance in FaDu‐RR cells compared with FaDu cells. After treatment of Olaparib, FaDu‐RR cells showed significantly less and smaller surviving colonies, lower proliferation ability and G2/M arrest than those in the group without treatment. Moreover, Olaparib significantly reduced growth of tumours in FaDu‐RR cell xenografts treated with ionizing radiation. Olaparib can significantly inhibit PARP1 expression and consequently has significant effects on radiosensitization in FaDu‐RR cells. These results indicate that Olaparib may help individualize treatment and improve their outcomes of hypopharyngeal cancer patients treated with radiation.     

Adenosine A2B receptor antagonist suppresses differentiation to regulatory T cells without suppressing activation of T cells

Extracellular adenosine activates P1 receptors (A₁, A_2A, A_2B, A₃) on cellular membranes. Here, we investigated the involvement of P1 receptor-mediated signaling in differentiation to regulatory T cells (Treg). Treg were induced in vitro by incubating isolated CD4⁺CD62L⁺ naïve murine T cells under Treg-skewing conditions. Antagonists of A₁ and A_2B receptors suppressed the expression of Foxp3, a specific marker of Treg, and the production of IL-10, suggesting the involvement of A₁ and A_2B receptors in differentiation to Treg. We also investigated the effect of these antagonists on T cell activation, which is essential for differentiation to Treg, and found that A₁ antagonist, but not A_2B antagonist, suppressed T cell activation. We conclude that A₁ and A_2B receptors are both involved in differentiation to Treg, but through different mechanisms. Since A_2B antagonist blocked differentiation to Treg without suppressing T cell activation, it is possible that blockade of A_2B receptor would facilitate tumor immunity.     

Human omental adipose-derived mesenchymal stem cells enhance autophagy in ovarian carcinoma cells through the STAT3 signalling pathway

BackgroundOur previous study showed that human omental adipose-derived stem cells (ADSCs) promote ovarian cancer growth and metastasis. In this study, the role of autophagy in the ovarian cancer-promoting effects of omental ADSCs was further determined.MethodsThe growth and invasion of ovarian cancer cells were detected by CCK-8 and Transwell assays, respectively. The autophagy of ovarian cancer cells transfected with MRFP-GFP-LC3 adenoviral vectors was evaluated by confocal microscopy and western blot assay. Transfection of STAT3 siRNA was used to inhibit the expression of STAT3.ResultsOur results show that autophagy plays a vital role in ovarian cancer and is promoted by ADSCs. Specifically, we show that proliferation and invasion are correlated with autophagy induction by ADSCs in two ovarian cancer cell lines under hypoxic conditions. Mechanistically, ADSCs activate the STAT3 signalling pathway, thereby promoting autophagy. Knockdown of STAT3 expression using siRNA decreased hypoxia-induced autophagy and decreased the proliferation and metastasis of ovarian cancer cells.ConclusionTaken together, our data indicate that STAT3-mediated autophagy induced by ADSCs promotes ovarian cancer growth and metastasis.     

Fall in oxygen tension of culture medium stimulates the adenosinergic signalling of a human T cell line

We examined the short-course expression of various parameters involved in the adenosinergic signalling of a human T cell line during in vitro decrease of the medium culture oxygen tension mimicking in vivo hypoxia. Fall of 92 mmHg in oxygen tension of culture medium induced in CEM, a CD4+ human T cell line, a continuous production of hypoxia-inducing factor-1α with a plateau value at 9 h, a rapid increase in adenosine production peaking at 3 h and a decrease in adenosine deaminase peaking at 6 h. The adenosine A_2A receptor (A_2AR) protein level of CEM cells was enhanced with a peak at 6 h. Intracellular 3′,5′-cyclic adenosine monophosphate accumulated in CEM cells with a maximal level at 9 h. These results show that a human-cultured T cells line can upregulate its own adenosine production and A_2AR expression during exposure to acute hypoxia. Hypoxia-increased stimulation of the adenosinergic signalling of T cells may have immunosuppressive properties and, consequently, A_2AR agonists may have therapeutic relevance.     

Clinical and prognostic effects of CDKN2A,CDKN2B and CDH13 promoter methylation in ovarian cancer: a study using meta-analysis and TCGA data

Abstract

Background: Promoter methylation of tumour suppressor genes (TSGs) CDKN2A, CDKN2B and CDH13 has been reported in ovarian cancer. However, the clinicopathological characteristics and prognostic role of CDKN2A, CDKN2B and CDH13 promoter methylation in ovarian carcinoma remained unclear.

Methods: The pooled odds ratio (OR) or hazard ratios (HRs) with their 95% confidence intervals (95% CIs) were calculated in this meta-analysis. The Cancer Genome Atlas data were obtained to confirm the role of CDKN2A, CDKN2B and CDH13 methylation in ovarian cancer.

Results: CDKN2A, CDKN2B and CDH13 promoter methylation was higher in ovarian cancer than in normal ovarian tissues. CDH13 promoter methylation was correlated with tumour histology (serous vs. non-serous type: OR?=?0.33, p?=?0.031). CDKN2A promoter methylation was not linked to overall survival (OS), but it was correlated with a poor prognosis in progression-free survival (HR?=?1.55, p?=?0.004). TCGA data showed no correlation between CDKN2A, CDKN2B and CDH13 methylation and OS as well as disease-free survival (DFS).

Conclusions: CDKN2A, CDKN2B and CDH13 promoter methylation may correlate with the increased risk of ovarian cancer. CDKN2A promoter methylation may be an independent prognostic biomarker for predicting progression-free survival.     

Osteoblast differentiation and survival: A role for A2B adenosine receptor allosteric modulators

The A_2B adenosine receptor (A_2B AR), activated in response to high levels of endogenous adenosine, is the major AR subtype involved in mesenchymal stem cell (MSC) differentiation to osteoblasts and bone formation. For this reason, targeting of A_2B AR with selective allosteric modulators may represent a promising pharmacological approach to the treatment of bone diseases.Herein, we report the characterization of a 3-keto-indole derivative, 2-(1-benzyl-1H-indol-3-yl)-2-oxo-N-phenylacetamide (KI-7), as A_2B AR positive allosteric modulator in MSCs, demonstrating that this compound is able to potentiate the effects of either adenosine and synthetic orthosteric A_2B AR agonists in mediating osteoblast differentiation in vitro. In detail, we observed that MSC treatment with KI-7 determined an increase in the expression of osteoblast-related genes (Runx2 and osterix) and osteoblast marker proteins (phosphatase alkaline and osteocalcin), associated with a stimulation of osteoblast mineralization.In the early phase of differentiation programme, KI-7 significantly potentiated physiological and A_2B AR agonist-mediated down-regulation of IL-6 release. Conversely, during the late stage of differentiation, when most of the cells have an osteoblast phenotype, KI-7 caused a sustained raise in IL-6 levels and an improvement in osteoblast viability. These data suggest that a positive allosteric modulation of A_2B AR not only favours MSC commitment to osteoblasts, but also ensures a greater survival of mature osteoblasts. Our study paves the way for a therapeutic use of selective positive allosteric modulators of A_2B AR in the control of osteoblast differentiation, bone formation and fracture repair.     

MARCH5 RNA promotes autophagy,migration, and invasion of ovarian cancer cells

MARCH5 is a crucial regulator of mitochondrial fission. However, the expression and function of MARCH5 in ovarian cancer have not been determined. This study investigated the expression and function of MARCH5 in ovarian cancer with respect to its potential role in the tumorigenesis of the disease as well as its usefulness as an early diagnostic marker. We found that the expression of MARCH5 was substantially upregulated in ovarian cancer tissue in comparison with the normal control. Silencing MARCH5 in SKOV3 cells decreased TGFB1-induced cell macroautophagy/autophagy, migration, and invasion in vitro and in vivo, whereas the ectopic expression of MARCH5 in A2780 cells had the opposite effect. Mechanistic investigations revealed that MARCH5 RNA may function as a competing endogenous RNA (ceRNA) to regulate the expression of SMAD2 and ATG5 by competing for MIR30A. Knocking down SMAD2 or ATG5 can block the effect of MARCH5 in A2780 cells. Also, silencing the expression of MARCH5 in SKOV3 cells can inhibit the TGFB1-SMAD2/3 pathway. In contrast, the ectopic expression of MARCH5 in A2780 cells can activate the TGFB1-SMAD2/3 pathway. In turn, the TGFB1-SMAD2/3 pathway can regulate MARCH5 and ATG5 through MIR30A. Overall, the results of this study identified MARCH5 as a candidate oncogene in ovarian cancer and a potential target for ovarian cancer therapy.