Prostate Cancer Peripheral Blood NK Cells Show Enhanced CD9, CD49a, CXCR4, CXCL8, MMP-9 Production and Secrete Monocyte-Recruiting and Polarizing Factors

Natural killer (NK) cells, effector lymphocytes of the innate immunity, have been shown to be altered in several cancers, both at tissue and peripheral levels. We have shown that in Non-Small Cell Lung Cancer (NSCLC) and colon cancer, tumour associated circulating NK (TA-NK) and tumour infiltrating NK (TI-NK) exhibit pro-angiogenic phenotype/functions. However, there is still a lack of knowledge concerning the phenotype of peripheral blood (PB) NK (pNK) cells in prostate cancer (PCa). Here, we phenotypically and functionally characterized pNK from PCa patients (PCa TA-NKs) and investigated their interactions with endothelial cells and monocytes/macrophages. NK cell subset distribution in PB of PCa patients was investigated, by multicolor flow cytometry, for surface antigens expression. Protein arrays were performed to characterize the secretome on FACS-sorted pNK cells. Conditioned media (CM) from FACS-sorted PCa pTA-NKs were used to determine their ability to induce pro-inflammatory/pro-angiogenic phenotype/functions in endothelial cells, monocytes, and macrophages. CM from three different PCa (PC-3, DU-145, LNCaP) cell lines, were used to assess their effects on human NK cell polarization in vitro, by multicolor flow cytometry. We found that PCa pTA-NKs acquire the CD56brightCD9+CD49a+CXCR4+ phenotype, increased the expression of markers of exhaustion (PD-1, TIM-3) and are impaired in their degranulation capabilities. Similar effects were observed on healthy donor-derived pNK cells, exposed to conditioned media of three different PCa cell lines, together with increased production of pro-inflammatory chemokines/chemokine receptors CXCR4, CXCL8, CXCL12, reduced production of TNFα, IFNγ and Granzyme-B. PCa TA-NKs released factors able to support inflammatory angiogenesis in an in vitro model and increased the expression of CXCL8, ICAM-1, and VCAM-1 mRNA in endothelial cells. Secretome analysis revealed the ability of PCa TA-NKs to release pro-inflammatory cytokines/chemokines involved in monocyte recruitment and M2-like polarization. Finally, CMs from PCa pTA-NKs recruit THP-1 and peripheral blood CD14+ monocyte and polarize THP-1 and peripheral blood CD14+ monocyte-derived macrophage towards M2-like/TAM macrophages. Our results show that PCa pTA-NKs acquire properties related to the pro-inflammatory angiogenesis in endothelial cells, recruit monocytes and polarize macrophage to an M2-like type phenotype. Our data provides a rationale for a potential use of pNK profiling in PCa patients.

Abstract 14078: Ros-dependent Sumoylation of Cu Chaperone Atox1 Drives Its Nuclear Translocation to Promote Inflammatory Angiogenesis Induced by Ischemic Injury

Introduction: Neovascularization in response to ischemia depends on inflammation, angiogenesis and reactive oxygen species (ROS). Copper (Cu) is implicated in inflammation and angiogenesis. We reported that cytosolic Cu chaperone Atox1 activates secretory Cu enzymes lysyl oxidase (LOX), while nuclear Atox1 functions as a Cu-dependent transcription factor to promote ROS/NFkB-dependent inflammation in endothelial cells (ECs). However, mechanism of Atox1 nuclear translocation as well as role of endothelial Atox1 in inflammatory angiogenesis in vivo remain unknown. SUMOylation and its deSUMOylation by SENPs regulates transcription factor function. Silica analysis identified a conserved putative SUMOylation motif at Lys(K3) of Atox1. Results: Atox1 expression was dramatically increased in angiogenic ECs in mice hindlimb ischemia model. EC-specific Atox1-deficient mice significantly reduced angiogenesis (CD31+, 67%) and Mac+ inflammatory cells in ischemic tissues. In cultured ECs, inflammatory cytokine TNFα or hypoxia promoted Atox1 nuclear translocation and Atox1 SUMOylation (3.6-fold), which were inhibited by antioxidant NAC or overexpression of “SUMO-dead” Atox1K3R. Mechanistically, TNFα induced Cys603 oxidation/inactivation of SENP1 in cytosol, which in turn increased Atox1 SUMOylation and nuclear translocation. Functionally, siAtox1or Atox1K3R inhibited TNFα-induced inflammatory/angiogenic genes VCAM/ICAM, IL-15 and RANTES. In nucleus with reduced state, ChIP assay using SUMO-Atox1 revealed that Atox1 deSUMOylation by nuclear SENP1 increases Atox1 transcriptional activity for inflammatory genes. In parallel, Atox1K3R which maintains Cu chaperone function inhibited TNFα-induced EC permeability by activating LOX. In vivo, Atox1 SUMOylation was increased after hindlimb ischemia while CRISPR/Cas9-generated SUMO-dead Atox1K3R knock-in mice showed impaired angiogenesis in hindlimb ischemia model. Conclusion: Atox1 SUMOylation via oxidative/inactivation of SENP1 in cytosol promotes: 1) its translocation to nucleus where deSUMOylated Atox1 can function as Cu-dependent transcription factor to drive inflammatory angiogenesis and 2) EC barrier dysfunction in inflamed/hypoxic ECs after ischemic injury.

Evidence for a non-stochastic two-field hypothesis for persistent skin cancer risk

With recurring carcinogen exposures, individual tumors develop in a field of genetic mutations through a stepwise process of clonal expansion and evolution. Once established, this “cancer field” persists in the absence of continued carcinogen exposures, resulting in a sustained risk for cancer development. Using a bioimaging approach, we previously demonstrated that a dermal premalignant field characterized by inflammatory angiogenesis persists following the cessation of ultraviolet light exposures and accurately predicts future overlying epidermal tumor formation. Following ultraviolet light treatments, others have observed that patches of p53 immunopositive cells persist stochastically throughout the epidermal stem cell population. However, these studies were done by random biopsies, introducing sampling bias. We now show that, rather than being randomly distributed, p53+ epidermal cells are enriched only in areas overlying this multi-focal dermal field. Moreover, we also show that the dermal field is characterized by a senescent phenotype. We propose that persistence of the overlying epithelial cancerization field in the absence of exogenous carcinogens or promoters requires a two-field composite consisting of a dermal senescent field driving the persistence of the overlying epidermal cancer field. These observations challenge current models that suggest that persistence of cancer risk in the absence of continued carcinogen exposures is simply a function of stochastically arranged, long-lived but dormant epithelial clonal stem cells mutants. The model proposed here could provide new insights into how cancer risk persists following cessation of carcinogenic exposures.

Macrophage 11β-HSD-1 deficiency promotes inflammatory angiogenesis

11β-Hydroxysteroid dehydrogenase-1 (11β-HSD1) predominantly converts inert glucocorticoids into active forms, thereby contributing to intracellular glucocorticoid levels. 11β-HSD1 is dynamically regulated during inflammation, including in macrophages where it regulates phagocytic capacity. The resolution of inflammation in some disease models including inflammatory arthritis is impaired by 11β-HSD1 deficiency or inhibition. However, 11β-HSD1 deficiency/inhibition also promotes angiogenesis, which is beneficial in mouse models of surgical wound healing, myocardial infarction or obesity. The cell types responsible for the anti-inflammatory and anti-angiogenic roles of 11β-HSD1 have not been characterised. Here, we generated Hsd11b1MKO mice with LysM-Cre mediated deletion of Hsd11b1 to investigate whether 11β-HSD1 deficiency in myeloid phagocytes is pro-angiogenic and/or affects the resolution of inflammation. Resolution of inflammatory K/BxN-induced arthritis was impaired in Hsd11b1MKO mice to a similar extent as in mice globally deficient in 11β-HSD1. This was associated with >2-fold elevation in levels of the endothelial marker Cdh5 mRNA, suggesting increased angiogenesis in joints of Hsd11b1MKO mice following arthritis. A pro-angiogenic phenotype was confirmed by measuring angiogenesis in subcutaneously implanted polyurethane sponges, in which Hsd11b1MKO mice showed 20% greater vessel density than their littermate controls, associated with higher expression of Cdh5. Thus, 11β-HSD1 deficiency in myeloid phagocytes promotes angiogenesis. Targeting 11β-HSD1 in macrophages may be beneficial in tissue repair.