Constitutive and treatment-induced CXCL8-signalling selectively modulates the efficacy of anti-metabolite therapeutics in metastatic prostate cancer.
ABSTRACT: BACKGROUND:The current study was undertaken to characterize the effect of anti-metabolites on inducing CXCL8 signaling and determining whether the constitutive and/or drug-induced CXCL8 signaling in metastatic prostate cancer (CaP) cells modulates their sensitivity to this class of agent. METHODS:The response of metastatic CaP cells to 5-Fluorouracil (5-FU), Pemetrexed or Tomudex was determined using cell count assays, flow cytometry and PARP cleavage analysis. Quantitative-PCR, ELISA and immunoblots were employed to determine effects of drugs or CXCL8 administration on target gene/protein expression. RESULTS:Administration of 5-FU but not pemetrexed potentiated CXCL8 secretion and increased CXCR1 and CXCR2 gene expression in metastatic PC3 cells. Consistent with this, the inhibition of CXCL8 signaling using a CXCR2 antagonist, AZ10397767, increased the cytotoxicity of 5-FU by 4-fold (P<0.001), and increased 5-FU-induced apoptosis in PC3 cells (P<0.01). In contrast, while administration of AZ10397767 had no effect on the sensitivity of pemetrexed, the CXCR2 antagonist exerted the greatest effect in increasing the sensitivity of PC3 cells to Tomudex, a directed thymidylate synthase (TS) inhibitor. Subsequent experiments confirmed that administration of recombinant human CXCL8 increased TS expression, a response mediated in part by the CXCR2 receptor. Moreover, siRNA-mediated knockdown of the CXCL8-target gene Bcl-2 increased the sensitivity of PC3 cells to 5-FU. CONCLUSIONS:CXCL8 signaling provides a selective resistance of metastatic prostate cancer cells to specific anti-metabolites by promoting a target-associated resistance, in addition to underpinning an evasion of treatment-induced apoptosis.
Project description:Impaired PTEN function is a genetic hallmark of aggressive prostate cancers (CaP) and is associated with increased CXCL8 expression and signaling. The current aim was to further characterize biological responses and mechanisms underpinning CXCL8-promoted progression of PTEN-depleted prostate cancer, focusing on characterizing the potential interplay between CXCL8 and other disease-promoting chemokines resident within the prostate tumor microenvironment. Autocrine CXCL8-stimulation (i) increased expression of CXCR1 and CXCR2 in PTEN-deficient CaP cells suggesting a self-potentiating signaling axis and (ii) induced expression of CXCR4 and CCR2 in PTEN-wild-type and PTEN-depleted CaP cells. In contrast, paracrine CXCL8 signaling induced expression and secretion of the chemokines CCL2 and CXCL12 from prostate stromal WPMY-1 fibroblasts and monocytic macrophage-like THP-1 cells. In vitro studies demonstrated functional co-operation of tumor-derived CXCL8 with stromal-derived chemokines. CXCL12-induced migration of PC3 cells and CCL2-induced proliferation of prostate cancer cells were dependent upon intrinsic CXCL8 signaling within the prostate cancer cells. For example, in co-culture experiments, CXCL12/CXCR4 signaling but not CCL2/CCR2 signaling supported fibroblast-mediated migration of PC3 cells while CXCL12/CXCR4 and CCL2/CCR2 signaling underpinned monocyte-enhanced migration of PC3 cells. Combined inhibition of both CXCL8 and CXCL12 signaling was more effective in inhibiting fibroblast-promoted cell motility while repression of CXCL8 attenuated CCL2-promoted proliferation of prostate cancer cells. We conclude that tumor-derived CXCL8 signaling from PTEN-deficient tumor cells increases the sensitivity and responsiveness of CaP cells to stromal chemokines by concurrently upregulating receptor expression in cancer cells and inducing stromal chemokine synthesis. Combined chemokine targeting may be required to inhibit their multi-faceted actions in promoting the invasion and proliferation of aggressive CaP.
Project description:Suppression of the host's immune system plays a major role in cancer progression. Tumor signaling of programmed death 1 (PD1) on T cells and expansion of myeloid-derived suppressor cells (MDSCs) are major mechanisms of tumor immune escape. We sought to target these pathways in rhabdomyosarcoma (RMS), the most common soft tissue sarcoma of childhood. Murine RMS showed high surface expression of PD-L1, and anti-PD1 prevented tumor growth if initiated early after tumor inoculation; however, delayed anti-PD1 had limited benefit. RMS induced robust expansion of CXCR2(+)CD11b(+)Ly6G(hi) MDSCs, and CXCR2 deficiency prevented CD11b(+)Ly6G(hi) MDSC trafficking to the tumor. When tumor trafficking of MDSCs was inhibited by CXCR2 deficiency, or after anti-CXCR2 monoclonal antibody therapy, delayed anti-PD1 treatment induced significant antitumor effects. Thus, CXCR2(+)CD11b(+)Ly6G(hi) MDSCs mediate local immunosuppression, which limits the efficacy of checkpoint blockade in murine RMS. Human pediatric sarcomas also produce CXCR2 ligands, including CXCL8. Patients with metastatic pediatric sarcomas display elevated serum CXCR2 ligands, and elevated CXCL8 is associated with diminished survival in this population. We conclude that accumulation of MDSCs in the tumor bed limits the efficacy of checkpoint blockade in cancer. We also identify CXCR2 as a novel target for modulating tumor immune escape and present evidence that CXCR2(+)CD11b(+)Ly6G(hi) MDSCs are an important suppressive myeloid subset in pediatric sarcomas. These findings present a translatable strategy to improve the efficacy of checkpoint blockade by preventing trafficking of MDSCs to the tumor site.
Project description:The CXCR2 chemokine receptor is a G-protein-coupled receptor that undergoes clathrin-mediated endocytosis upon ligand binding. The trafficking of CXCR2 is crucial for cells to maintain a proper chemotactic response. The mechanisms that regulate the recycling/degradation sorting decision are unknown. In this study, we used dominant-negative (T19N) and GTPase-deficient activated (Q63L) RhoB mutants, as well as RhoB small interfering RNA (siRNA) to investigate the role of RhoB in CXCR2 trafficking. Expression of either of the RhoB mutants or transfection of RhoB siRNA impaired CXCR2-mediated chemotaxis. Expression of RhoB T19N and transfection of RhoB siRNA impaired sorting of CXCR2 to the lysosome after 3 hours of CXCL8 stimulation and impaired CXCL8-induced CXCR2 degradation. In cells expressing the RhoB Q63L mutant, CXCR2 recycling through the Rab11a recycling compartment was impaired after 30 minutes of CXCL8 stimulation as was CXCL8-induced CXCR2 degradation. For cells expressing activated RhoB, CXCR2 colocalized with Rab4, a marker for the rapid recycling pathway, and with the mannose-6-phosphate receptor, which traffics between the trans-Golgi network and endosomes. These data suggest that CXCR2 recycles through alternative pathways. We conclude that oscillation of RhoB GTPase activity is essential for appropriate sorting decisions, and for directing CXCR2 degradation and recycling--events that are required for optimal chemotaxis.
Project description:The IL-8 (CXCL8) receptors CXCR1 and CXCR2 couple to G?i to induce leukocyte recruitment and activation at sites of inflammation. We recently showed that CXCR1 couples predominantly to the G protein-coupled receptor kinase (GRK)2, whereas CXCR2 interacts with GRK6 to regulate cellular responses. In addition to G protein-coupled receptors, GRKs displayed a more diverse protein/protein interaction in cells. In this study, we sought to identify GRK6 binding partner(s) that may influence CXCL8 activities, using RBL-2H3 cells stably expressing CXCR1 (RBL-CXCR1) or CXCR2 (RBL-CXCR2), as well as human and murine neutrophils. Our data demonstrated that, upon CXCR2 activation, GRK6 interacts with activator of G protein signaling (AGS)3 and G?i2 to form a GRK6/AGS3/G?i2 complex. This complex is time dependent and peaked at 2-3 min postactivation. GTP?S pretreatment blocked GRK6/AGS3/G?i2 formation, suggesting that this assembly depends on G protein activation. Surprisingly, CXCR2 activation induced AGS3 phosphorylation in a PKC-dependent, but GRK6-independent, fashion. Overexpression of AGS3 in RBL-CXCR2 significantly inhibited CXCL8-induced Ca(2+) mobilization, phosphoinositide hydrolysis, and chemotaxis. In contrast, short hairpin RNA inhibition of AGS3 enhanced CXCL8-induced Ca(2+) mobilization, receptor resistance to desensitization, and recycling to the cell surface, with no effect on receptor internalization. Interestingly, RBL-CXCR2-AGS3(-/-) cells displayed a significant increase in CXCR2 expression on the cell surface but decreased ERK1/2 and P38 MAPK activation. Taken together, these results indicate that GRK6 complexes with AGS3-G?i2 to regulate CXCR2-mediated leukocyte functions at different levels, including downstream effector activation, receptor trafficking, and expression at the cell membrane.
Project description:Pro-inflammatory mediators, like prostaglandin (PG) and chemokines, promote tumourigenesis by enhancing cell proliferation, migration of immune cells and recruitment of blood vessels. Recently we showed elevated expression of the chemokine (C-X-C motif) receptor 2 (CXCR2) in endometrial adenocarcinomas localized to neutrophils and neoplastic epithelial and vascular cells. Furthermore we found that PGF(2alpha)-F-prostanoid (FP) receptor regulates the expression of the CXCR2 ligand CXCL1, to promote neutrophil chemotaxis in endometrial adenocarcinomas. In the present study we identified another CXCR2 ligand, CXCL8 as a target for PGF(2alpha)-FP receptor signalling which enhances epithelial cell proliferation in endometrial adenocarcinoma cells in vitro and in nude mice in vivo. We found that PGF(2alpha)-FP receptor interaction induces CXCL8 expression in endometrial adenocarcinoma cells via the protein kinase C-calcium-calcineurin-NFAT signaling pathway. Promoter analysis revealed that CXCL8 transcriptional activation by PGF(2alpha) signaling is mediated by cooperative interactions between the AP1 and NFAT binding sites. Furthermore, PGF(2alpha) via the FP receptor induced the expression of the regulator of calcineurin 1 isoform 4 (RCAN1-4) via the calcineurin/NFAT pathway in a reciprocal manner to CXCL8. Using an adenovirus to overexpress RCAN1-4, we found that RCAN1-4 is a negative regulator of CXCL8 expression in endometrial adenocarcinoma cells. Taken together our data have elucidated the molecular and cellular mechanism whereby PGF(2alpha) regulates CXCL8 expression via the FP receptor in endometrial adenocarcinomas and have highlighted RCAN1-4 as a negative regulator of CXCL8 expression which may be exploited therapeutically to inhibit CXCL8-mediated tumour development.
Project description:Tumor resistance to apoptosis is one the main causes of anticancer treatment failure. Previous studies showed that LMW-PTP overexpression enhances resistance of cancer cells to traditional anticancer drugs. Today, the role of LMW-PTP in inducing resistance to apoptosis in melanoma cells remains to be elucidated. Experimental setting include MTT assay, Annexin V/Pi method, and colony assay to assess whether silencing of LMW-PTP improves the sensitivity of A375 to dacarbazine, 5-FU, and radiotherapy. Pharmacological targeting of LMW-PTP was obtained using Morin, a LMW-PTP inhibitor. The ability of Morin to improve the effectiveness of anticancer drugs and radiotherapy was also studied. Moreover, PC3 cells were used as an alternative cellular model to confirm the data obtained with melanoma cells. We found that LMW-PTP silencing improves the effectiveness of dacarbazine, 5-FU, and radiotherapy. Identical results were obtained in vivo when Morin was used to target LMW-PTP. We demonstrated that Morin synergizes with dacarbazine, improving its cytotoxic activity. However, we showed that the combined treatment, Morin-anticancer drug, does not affect the viability of noncancerous cells. Knockdown of LMW-PTP sensitizes also PC3 cells to docetaxel and radiotherapy. In conclusion, we showed that LMW-PTP targeting improves effectiveness of anticancer drugs used for treatment of melanoma. Moreover, our results suggest that Morin could be used as adjuvant to improve the outcome of patients affected by metastatic melanoma.
Project description:The chemokine system has been reported to be utilized and manipulated by tumor cells in order to promote local tumor growth and distant dissemination. The present study aimed to investigate the expression of three chemokine ligand-receptor axes in lung carcinoma tissues. Tumor and healthy normal tissue samples were obtained from 120 lung carcinoma patients following surgical resection. Immunohistochemistry and reverse transcription quantitative polymerase chain reaction were used in order to identify the protein and messenger (m)RNA expression of chemokines, including chemokine (C-X-C motif) ligand (CXCL)12/stromal cell-derived factor (SDF)-1, CXCL8/interleukin (IL)-8, chemokine (C-C motif) ligand (CCL)19 and CCL21, and the corresponding chemokine receptors, chemokine (C-X-C motif) receptor (CXCR)4, CXCR1, CXCR2 and chemokine (C-C motif) receptor (CCR)7, respectively. The results revealed that compared with the normal lung tissues, lung carcinoma tissues expressed significantly higher mRNA levels of CXCL12/SDF-1, CXCR4, CXCL8/IL-8, CXCR2, CCL19 and CCR7 (P<0.01). In four histological subtypes, adenocarcinoma presented dominant expression of CXCR4, CXCR2, CXCL8/IL-8 and CCL19 (P<0.05). In addition, it was demonstrated that tumor staging was inversely correlated with chemokine receptor CCR7 and CXCR2 mRNA expression as well as positively correlated with CXCL12/SDF-1, CXCL8/IL-8 and CCL19 mRNA levels (P<0.05). Lymph node metastasis presented a positive correlation with CXCR4, CXCR2 and CXCL8/IL-8 expression and a negative correlation with CCL19 and CCR7 expression (P<0.05). Furthermore, vascular invasion was more prevalent in patients with higher expression levels of CXCR4, CCR7 or CCL19 (P<0.01). In conclusion, these data suggested that the ligand-receptor interaction of CXCL8-CXCR2, CXCL12-CXCR4 and CCL19-CCR7 may be involved in the tumorigenesis of lung carcinoma. Higher expression levels of chemokines and lower expression of chemokine receptors indicated poor tumor staging. The CXC chemokine receptors, CXCR4 and CXCR2, promoted lymphatic metastasis through the activation of their specific ligands, while CCL19 and its receptor CCR7 had an essential role in hematogenous metastasis of lung carcinoma.
Project description:Since the CXC chemokine receptor CXCR2 and its cognate ligand CXCL8 (IL-8) critically regulate neutrophil trafficking during inflammation, they have been implicated in a number of inflammatory lung diseases. Several CXCR2 antagonists have been described and the blockade of CXCR2 has shown promise in pre-clinical disease models and early clinical trials. However, given its potential, there are fewer distinct classes of antagonists of CXCR2 than of other clinically relevant molecular targets. Thus, we sought to identify additional classes of compounds that alter CXCR2 function.We used the CXCR2 Tango(TM) assay to screen an in-house library of highly diverse chemical compounds. CX4338 [2-(benzylamino)-4,4-dimethyl-6-oxo-N-phenylcyclohex-1-enecarbothioamide] was identified from our screen and additional studies to characterize the compound were performed. Receptor internalization and second-messenger assays were used to assess the effects of CX4338 on CXCR2-mediated signalling. Wound healing, transwell cell migration and LPS-induced lung inflammation in mice were used to determine the in vitro and in vivo effects of CX4338.CX4338 selectively inhibited CXCR2-mediated recruitment of ?-arrestin-2 and receptor internalization, while enhancing CXCR2-mediated MAPK activation. Additionally, CX4338 inhibited CXCL8-induced chemotaxis in CXCR2-overexpressing cells and human neutrophils. In vivo, CX4338 significantly reduced neutrophils in bronchoalveolar lavage induced by LPS in mice.A novel compound CX4338 inhibited CXCR2-mediated cell migration with a mechanism of action not previously reported. Also, selective inhibition of CXCR2-mediated ?-arrestin-2 activation is sufficient to inhibit CXCL8-mediated chemotaxis.
Project description:The reduced folate carrier (RFC) and the proton-coupled folate transporter (PCFT) are ubiquitously expressed in normal and malignant mammalian tissues and in human solid tumor cell lines. This article addresses the extent to which PCFT contributes to transport of pemetrexed and to the activities of this and other antifolates relative to RFC at physiological pH. Either RFC or PCFT cDNA was stably transfected into a transporter-null HeLa cell variant to achieve activities similar to their endogenous function in wild-type HeLa cells. PCFT and RFC produced comparable increases in pemetrexed activity in growth medium with 5-formyltetrahydrofolate. However, PCFT had little or no effect on the activities of methotrexate, N-(5-[N-(3,4-dihydro-2-methyl-4-oxyquinazolin-6-ylmethyl)-N-methyl-amino]-2-thenoyl)-l-glutamic acid (raltitrexed, Tomudex; ZD1694), or N(alpha)-(4-amino-4-deoxypteroyl)-N(delta)-hemiphthaloyl-l-ornithine (PT523) in comparison with RFC irrespective of the folate growth source. PCFT, expressed at high levels in Xenopus laevis oocytes and in transporter-competent HepG2 cells, exhibited a high affinity for pemetrexed, with an influx K(m) value of 0.2 to 0.8 muM at pH 5.5. PCFT increased the growth inhibitory activity of pemetrexed, but not that of the other antifolates in HepG2 cells grown with 5-formyltetrahydrofolate at physiological pH. These findings illustrate the unique role that PCFT plays in the transport and pharmacological activity of pemetrexed. Because of the ubiquitous expression of PCFT in human tumors, and the ability of PCFT to sustain pemetrexed activity even in the absence of RFC, tumor cells are unlikely to become resistant to pemetrexed as a result of impaired transport because of the redundancy of these genetically distinct routes.
Project description:Neutrophils serve as critical players in the pathogenesis of liver diseases. Chemokine receptors CXCR1 and CXCR2 are required for neutrophil chemotaxis to the site of inflammation/injury and are crucial in hepatic inflammatory response. However, key mechanism of neutrophil-mediated liver injury in acute-on-chronic liver failure (ACLF) remains highly elusive; which could be targeted for the development of new therapeutic interventions.To demonstrate the role of CXCR1/CXCR2-expressing neutrophils in hepatic injury, we investigated CXCR1/CXCR2 receptor expression in 17 hepatitis B virus-related ACLF patients in comparison to 42 chronic hepatitis B and 18 healthy controls. Mechanism of neutrophil-mediated cell death was analyzed by in vitro coculture assays and correlated with the patient data. In addition, to find out any etiological-based variations in ACLF, 19 alcohol-related ACLF patients were also included.In ACLF, neutrophils have high expression of CXCR1/CXCR2 receptors, which potentially participate in hepatocyte death through early apoptosis and necrosis in contact-dependent and -independent mechanisms. Importantly, blockade of CXCR1/CXCR2 with SCH 527123 antagonist significantly reduced cell death by targeting both the mechanisms. No etiology-based differences were seen between ACLF groups. Importantly, absolute neutrophil count was particularly higher in clinically severe ACLF patients and non-survivors (p?<?0.0001). Multivariate analysis demonstrated ANC and CXCL8/IL-8 as a predictor of mortality. Further, receiver operating characteristics curve confirmed the cutoff of ANC >73.5% (sensitivity: 76.5% and specificity: 76.5%) and CXCL8/IL-8 >27% (sensitivity: 70% and specificity: 73%) in prediction of mortality.Blockade of CXCR1/CXCR2 diminished the production of inflammatory mediators and reduced cell death; therefore, pharmacological neutralization of CXCR1/CXCR2 could provide novel therapeutic target in the management of ACLF.