Tumor-derived CXCL8 signaling augments stroma-derived CCL2-promoted proliferation and CXCL12-mediated invasion of PTEN-deficient prostate cancer cells

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.</p

Cabozantinib attenuates CXCL8-driven macrophage dependent migration and invasion of PTEN-deficient prostate cancer cells

Abstract Background: The interaction between a tumor and the surrounding non-malignant stroma impacts on nearly every aspect of malignant progression. The heterogeneity of the tumor microenvironment (TME), however, makes mapping this interaction difficult. Each stromal population contributes to various functions and each tumor type has its own distinct pathway of interaction with different stromal populations. The majority of these interactions are mediated by soluble factors secreted into the TME. The loss of the tumor suppressor PTEN, an early event in prostate tumorigenesis, results in a subsequent increase in secretion of the pro-inflammatory CXCL8 chemokine into the TME (Maxwell et al 2013). The specific role of CXCL8 signaling within the prostate TME has not been fully classified and warrants further investigation. Methods: PTENnull PC3 cells were cultured in the absence and presence of human macrophage-representative THP-1 monocytes. Tumor-derived CXCL8 signaling was attenuated using (i) CXCL8 neutralizing antibodies, (ii) CXCR1 and CXCR2 receptor-targeting small peptide inhibitors (PEPducins), and (iii) a CXCL8-targeted shRNA method to generate stable PC3-120p cells with reduced CXCL8 secretion – a control line termed PC3-NTp was co-developed. PC3 cell motility and invasion was assessed by real-time transwell invasion through MatrigelTM and wound closure assays. Phospho-receptor tyrosine kinase (RTK) arrays were used to identify signaling pathways downstream of CXCL8 signaling which are relevant to prostate tumor cross-talk with infiltrating monocytes. Results: The presence of THP-1 cells more than doubled the rate of PC3 invasion through MatrigelTM (2.4-fold, p=0.0098) and increased wound closure after 6 h (50.63% v 25.65% for PC3 cells alone; p=0.0108). Macrophage-dependent enhancement of invasion and motility was attenuated following inhibition of CXCL8 signaling (i) by neutralizing antibody (90% decrease in normalized invasion and 43% decrease in wound closure, p&amp;lt;0.001 and p=0.03 respectively relative to IgG) and (ii) PEPducin inhibition of CXCR1 and CXCR2 (38% reduction in wound closure, p=0.0002) compared to non-targeting peptides. Low CXCL8-secreting PC3-120p cells were unable to initiate THP-1-potentiated motility and invasion, however, this response was rescued by addition of exogenous human CXCL8. Exogenous CXCL8 was shown to up-regulate secretion of RTK-activating cytokines and growth factors from THP-1 cells. Addition of the RTK inhibitor cabozantinib (XL184) but not crizotinib abrogated THP-1-dependent invasion and wound healing response of PTEN-deficient PC3 cells. Differential analysis of RTKs regulated by both cabozantinib and CXCL8 neutralizing antibody in co-culture identify c-Kit, RET, VEGFR1 and VEGFR2 as potential downstream regulators of CXCL8-dependent monocyte-enhanced PC3 invasion. Conclusions: Tumor associated macrophages (TAMs) are correlated with prostate cancer progression. Secretion of CXCL8 from PTEN-deficient prostate cancer cells activates macrophages, which then sustains cancer cell migration and invasion. Attenuating CXCL8 signaling within the TME and its downstream effects provides potential therapeutic targets to reduce metastasis in PTEN-deficient, CXCL8-high prostate cancer patients. Citation Format: Jessica L. Neisen, Nuala McCabe, Richard D. Kennedy, David JJ Waugh. Cabozantinib attenuates CXCL8-driven macrophage-dependent migration and invasion of PTEN-deficient prostate cancer. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A49. doi:10.1158/1538-7445.CHTME14-A49</jats:p

Ex Vivo Expansion of Human Outgrowth Endothelial Cells Leads to IL-8-Mediated Replicative Senescence and Impaired Vasoreparative Function

Harnessing outgrowth endothelial cells (OECs) for vasoreparative therapy and tissue engineering requires efficient ex vivo expansion. How such expansion impacts on OEC function is largely unknown. In this study, we show that OECs become permanently cell-cycle arrested after ex vivo expansion, which is associated with enlarged cell size, β-galactosidase activity, DNA damage, tumor suppressor pathway activation, and significant transcriptome changes. These senescence hallmarks were coupled with low telomerase activity and telomere shortening, indicating replicative senescence. OEC senescence limited their regenerative potential by impairing vasoreparative properties in vitro and in vivo. Integrated transcriptomeproteome analysis identified inflammatory signaling pathways as major mechanistic components of the OEC senescence program. In particular, IL8 was an important facilitator of this senescence; depletion of IL8 in OECs significantly extended ex vivo lifespan, delayed replicative senescence, and enhanced function. While the ability to expand OEC numbers prior to autologous or allogeneic therapy remains a useful property, their replicative senescence and associated impairment of vasorepair needs to be considered. This study also suggests that modulation of the senescence-associated secretory phenotype could be used to optimize OEC therapy.</p

Long-term safety of brazikumab in the open-label period of a randomized phase 2a study of patients with Crohn’s disease

Abstract Background Short-term efficacy and safety of brazikumab (MEDI2070), a human monoclonal antibody and anti-p19 subunit inhibitor of interleukin-23, was demonstrated in a phase 2a trial in patients with moderate-to-severe active Crohn’s disease (CD). We report brazikumab long-term safety and tolerability from the open-label period of this phase 2a study. Methods Patients who completed the 12-week, double-blind induction period were eligible for inclusion in an open-label period where all patients received subcutaneous brazikumab (210 mg) every 4 weeks for 100 weeks. Patients had moderate-to-severe active CD and had failed or were intolerant to ≥ 1 anti-tumour necrosis factor alpha (TNFα) agent. Safety assessments included treatment-emergent adverse events (TEAEs); further assessments were pharmacokinetics and immunogenicity. Results Of the 104 patients who entered the open-label period, 57 (54.8%) continued to the end of the open-label period and 47 (45.2%) discontinued brazikumab. The most common reasons for discontinuation were lack of response (14.4%), patient decision (12.5%), and TEAEs (11.5%). In total, 44 (84.6%) in the group switching from placebo to brazikumab (placebo/brazikumab) and 43 (82.7%) in the group continuing brazikumab (brazikumab/brazikumab) experienced 1 or more TEAEs. Most TEAEs were mild-to-moderate in severity. Common TEAEs included nasopharyngitis and headache. Numbers of treatment-emergent serious adverse events (TESAEs) were similar between groups. Infections occurred in 40.4% of patients in the placebo/brazikumab group and 50% in the brazikumab/brazikumab group. There were 5 TESAEs of infection, none of which were opportunistic. No major adverse cardiac events, malignancies, or deaths were reported. Conclusions Brazikumab was well tolerated with an acceptable safety profile over a 100-week period in patients with moderate-to-severe active CD who failed or were intolerant to 1 or more anti-TNFα agents. Trial registration NCT01714726; registered October 26, 2012

PTEN deficiency promotes macrophage infiltration and hypersensitivity of prostate cancer to IAP antagonist/radiation combination therapy

PTEN loss is prognostic for patient relapse post-radiotherapy in prostate cancer (CaP). Infiltration of tumor-associated macrophages (TAMs) is associated with reduced disease-free survival following radical prostatectomy. However, the association between PTEN loss, TAM infiltration and radiotherapy response of CaP cells remains to be evaluated. Immunohistochemical and molecular analysis of surgically-resected Gleason 7 tumors confirmed that PTEN loss correlated with increased CXCL8 expression and macrophage infiltration. However PTEN status had no discernable correlation with expression of other inflammatory markers by CaP cells, including TNF-α. In vitro, exposure to conditioned media harvested from irradiated PTEN null CaP cells induced chemotaxis of macrophage-like THP-1 cells, a response partially attenuated by CXCL8 inhibition. Co-culture with THP-1 cells resulted in a modest reduction in the radio-sensitivity of DU145 cells. Cytokine profiling revealed constitutive secretion of TNF-α from CaP cells irrespective of PTEN status and IR-induced TNF-α secretion from THP-1 cells. THP-1-derived TNF-α increased NFκB pro-survival activity and elevated expression of anti-apoptotic proteins including cellular inhibitor of apoptosis protein-1 (cIAP-1) in CaP cells, which could be attenuated by pre-treatment with a TNF-α neutralizing antibody. Treatment with a novel IAP antagonist, AT-IAP, decreased basal and TNF-α-induced cIAP-1 expression in CaP cells, switched TNF-α signaling from pro-survival to pro-apoptotic and increased radiation sensitivity of CaP cells in co-culture with THP-1 cells. We conclude that targeting cIAP-1 can overcome apoptosis resistance of CaP cells and is an ideal approach to exploit high TNF-α signals within the TAM-rich microenvironment of PTEN-deficient CaP cells to enhance response to radiotherapy.</p

Potentiation of Inflammatory CXCL8 Signalling Sustains Cell Survival in PTEN-deficient Prostate Carcinoma

BACKGROUND: Inflammation and genetic instability are enabling characteristics of prostate carcinoma (PCa). Inactivation of the tumour suppressor gene phosphatase and tensin homolog (PTEN) is prevalent in early PCa. The relationship of PTEN deficiency to inflammatory signalling remains to be characterised. OBJECTIVE: To determine how loss of PTEN functionality modulates expression and efficacy of clinically relevant, proinflammatory chemokines in PCa. DESIGN, SETTING, AND PARTICIPANTS: Experiments were performed in established cell-based PCa models, supported by pathologic analysis of chemokine expression in prostate tissue harvested from PTEN heterozygous (Pten(+/−)) mice harbouring inactivation of one PTEN allele. INTERVENTIONS: Small interfering RNA (siRNA)–or small hairpin RNA (shRNA)–directed strategies were used to repress PTEN expression and resultant interleukin-8 (CXCL8) signalling, determined under normal and hypoxic culture conditions. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Changes in chemokine expression in PCa cells and tissue were analysed by real-time polymerase chain reaction (PCR), immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry; effects of chemokine signalling on cell function were assessed by cell cycle analysis, apoptosis, and survival assays. RESULTS AND LIMITATIONS: Transient (siRNA) or prolonged (shRNA) PTEN repression increased expression of CXCL8 and its receptors, chemokine (C-X-C motif) receptor (CXCR) 1 and CXCR2, in PCa cells. Hypoxia-induced increases in CXCL8, CXCR1, and CXCR2 expression were greater in magnitude and duration in PTEN-depleted cells. Autocrine CXCL8 signalling was more efficacious in PTEN-depleted cells, inducing hypoxia-inducible factor-1 (HIF-1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) transcription and regulating genes involved in survival and angiogenesis. Increased expression of the orthologous chemokine KC was observed in regions displaying atypical cytologic features in Pten(+/−) murine prostate tissue relative to normal epithelium in wild-type PTEN (Pten(WT)) glands. Attenuation of CXCL8 signalling decreased viability of PCa cells harbouring partial or complete PTEN loss through promotion of G1 cell cycle arrest and apoptosis. The current absence of clinical validation is a limitation of the study. CONCLUSIONS: PTEN loss induces a selective upregulation of CXCL8 signalling that sustains the growth and survival of PTEN-deficient prostate epithelium

CD44 enhances invasion of basal-like breast cancer cells by upregulating serine protease and collagen-degrading enzymatic expression and activity

INTRODUCTION: Basal-like breast cancers (BL-BCa) have the worst prognosis of all subgroups of this disease. Hyaluronan (HA) and the HA receptor CD44 have a long-standing association with cell invasion and metastasis of breast cancer. The purpose of this study was to establish the relation of CD44 to BL-BCa and to characterize how HA/CD44 signaling promotes a protease-dependent invasion of breast cancer (BrCa) cells. METHODS: CD44 expression was determined with immunohistochemistry (IHC) analysis of a breast cancer tissue microarray (TMA). In vitro experiments were performed on a panel of invasive BL-BCa cell lines, by using quantitative polymerase chain reaction (PCR), immunoblotting, protease activity assays, and invasion assays to characterize the basis of HA-induced, CD44-mediated invasion. RESULTS: Expression of the hyaluronan (HA) receptor CD44 associated with the basal-like subgroup in a cohort of 141 breast tumor specimens (P = 0.018). Highly invasive cells of the representative BL-BCa cell line, MDA-MB-231 (MDA-MB-231Hi) exhibited increased invasion through a basement membrane matrix (Matrigel) and collagen. In further experiments, HA-induced promotion of CD44 signaling potentiated expression of urokinase plasminogen activator (uPA) and its receptor uPAR, and underpinned an increased cell-associated activity of this serine protease in MDA-MB-231Hi and a further BL-BCa cell line, Hs578T cells. Knockdown of CD44 attenuated both basal and HA-stimulated uPA and uPAR gene expression and uPA activity. Inhibition of uPA activity by using (a) a gene-targeted RNAi or (b) a small-molecule inhibitor of uPA attenuated HA-induced invasion of MDA-MB-231Hi cells through Matrigel. HA/CD44 signaling also was shown to increase invasion of MDA-MB-231 cells through collagen and to potentiate the collagen-degrading activity of MDA-MB-231Hi cells. CD44 signaling was subsequently shown to upregulate expression of two potent collagen-degrading enzymes, the cysteine protease cathepsin K and the matrix metalloprotease MT1-MMP. RNAi- or shRNA-mediated depletion of CD44 in MDA-MB-231Hi cells decreased basal and HA-induced cathepsin K and MT1-MMP expression, reduced the collagen-degrading activity of the cell, and attenuated cell invasion through collagen. Pharmacologic inhibition of cathepsin K or RNAi-mediated depletion of MT1-MMP also attenuated MDA-MB-231Hi cell invasion through collagen. CONCLUSION: HA-induced CD44 signaling increases a diverse spectrum of protease activity to facilitate the invasion associated with BL-BCa cells, providing new insights into the molecular basis of CD44-promoted invasion

A randomised trial of anti-GM-CSF otilimab in severe COVID-19 pneumonia (OSCAR).

BACKGROUND: Granulocyte-macrophage colony-stimulating factor (GM-CSF) and dysregulated myeloid cell responses are implicated in the pathophysiology and severity of COVID-19. METHODS: In this randomised, sequential, multicentre, placebo-controlled, double-blind study, adults aged 18-79 years (Part 1) or ≥70 years (Part 2) with severe COVID-19, respiratory failure and systemic inflammation (elevated C-reactive protein/ferritin) received a single intravenous infusion of otilimab 90 mg (human anti-GM-CSF monoclonal antibody) plus standard care (NCT04376684). The primary outcome was the proportion of patients alive and free of respiratory failure at Day 28. RESULTS: In Part 1 (n=806 randomised 1:1 otilimab:placebo), 71% of otilimab-treated patients were alive and free of respiratory failure at Day 28 versus 67% who received placebo; the model-adjusted difference of 5.3% was not statistically significant (95% CI -0.8-11.4%, p=0.09). A nominally significant model-adjusted difference of 19.1% (95% CI 5.2-33.1%, p=0.009) was observed in the predefined 70-79 years subgroup, but this was not confirmed in Part 2 (n=350 randomised) where the model-adjusted difference was 0.9% (95% CI -9.3-11.2%, p=0.86). Compared with placebo, otilimab resulted in lower serum concentrations of key inflammatory markers, including the putative pharmacodynamic biomarker CC chemokine ligand 17, indicative of GM-CSF pathway blockade. Adverse events were comparable between groups and consistent with severe COVID-19. CONCLUSIONS: There was no significant difference in the proportion of patients alive and free of respiratory failure at Day 28. However, despite the lack of clinical benefit, a reduction in inflammatory markers was observed with otilimab, in addition to an acceptable safety profile

A randomised trial of anti-GM-CSF otilimab in severe COVID-19 pneumonia (OSCAR).

BACKGROUND: Granulocyte-macrophage colony-stimulating factor (GM-CSF) and dysregulated myeloid cell responses are implicated in the pathophysiology and severity of COVID-19. METHODS: In this randomised, sequential, multicentre, placebo-controlled, double-blind study, adults aged 18-79 years (Part 1) or ≥70 years (Part 2) with severe COVID-19, respiratory failure and systemic inflammation (elevated C-reactive protein/ferritin) received a single intravenous infusion of otilimab 90 mg (human anti-GM-CSF monoclonal antibody) plus standard care (NCT04376684). The primary outcome was the proportion of patients alive and free of respiratory failure at Day 28. RESULTS: In Part 1 (n=806 randomised 1:1 otilimab:placebo), 71% of otilimab-treated patients were alive and free of respiratory failure at Day 28 versus 67% who received placebo; the model-adjusted difference of 5.3% was not statistically significant (95% CI -0.8-11.4%, p=0.09). A nominally significant model-adjusted difference of 19.1% (95% CI 5.2-33.1%, p=0.009) was observed in the predefined 70-79 years subgroup, but this was not confirmed in Part 2 (n=350 randomised) where the model-adjusted difference was 0.9% (95% CI -9.3-11.2%, p=0.86). Compared with placebo, otilimab resulted in lower serum concentrations of key inflammatory markers, including the putative pharmacodynamic biomarker CC chemokine ligand 17, indicative of GM-CSF pathway blockade. Adverse events were comparable between groups and consistent with severe COVID-19. CONCLUSIONS: There was no significant difference in the proportion of patients alive and free of respiratory failure at Day 28. However, despite the lack of clinical benefit, a reduction in inflammatory markers was observed with otilimab, in addition to an acceptable safety profile