Contribution of Macrophages and T Cells in Skeletal Metastasis

Bone is a common site for metastases with a local microenvironment that is highly conducive for tumor establishment and growth. The bone marrow is replete with myeloid and lymphoid linage cells that provide a fertile niche for metastatic cancer cells promoting their survival and growth. Here, we discuss the role of macrophages and T cells in pro- and anti-tumoral mechanisms, their interaction to support cancer cell growth, and their contribution to the development of skeletal metastases. Importantly, immunotherapeutic strategies targeting macrophages and T cells in cancer are also discussed in this review as they represent a great promise for patients suffering from incurable bone metastases

Unique Pro-Inflammatory Response of Macrophages during Apoptotic Cancer Cell Clearance

The clearance of apoptotic cells by macrophages (efferocytosis) is crucial to maintain normal tissue homeostasis; however, efferocytosis of cancer cells frequently results in inflammation and immunosuppression. Recently, we demonstrated that efferocytosis of apoptotic prostate cancer cells by bone marrow-derived macrophages induced a pro-inflammatory response that accelerated metastatic tumor growth in bone. To evaluate the microenvironmental impact of macrophages and their efferocytic function, we compared peritoneal macrophages (P-MΦ) versus bone marrow-derived macrophages (BM-MΦs) using an efferocytosis in vitro model. The capability to engulf apoptotic prostate cells was similar in BM-MΦs and P-MΦs. Ex vivo analysis of BM-MΦs showed an M2-like phenotype compared with a predominantly M1-like phenotype in P-MΦs. A distinct gene and protein expression profile of pro-inflammatory cytokines was found in BM-MΦs as compared with P-MΦs engulfing apoptotic prostate cancer cells. Importantly, the reprogramming of BM-MΦs toward an M1-like phenotype mitigated their inflammatory cytokine expression profile. In conclusion, BM-MΦs and P-MΦs are both capable of efferocytosing apoptotic prostate cancer cells; however, BM-MΦs exert increased inflammatory cytokine expression that is dependent upon the M2 polarization stage of macrophages. These findings suggest that bone marrow macrophage efferocytosis of apoptotic cancer cells maintains a unique pro-inflammatory microenvironment that may support a fertile niche for cancer growth. Finally, bone marrow macrophage reprogramming towards M1-type by interferon-γ (IFN-γ) induced a significant reduction in the efferocytosis-mediated pro-inflammatory signature

Epidermal YAP activity drives canonical WNT16/β-catenin signaling to promote keratinocyte proliferation in vitro and in the murine skin

The skin constantly self-renews throughout adult life. Wnt/β-catenin signaling plays a key role in promoting keratinocyte proliferation in the hair follicles and in the interfollicular epidermis. A recent report demonstrated that epidermal YAP activity drives β-catenin activation to promote keratinocyte proliferation in the murine skin. However, it remains unclear whether this is caused by paracrine activation of canonical Wnt signaling or through other YAP/β-catenin regulatory interactions. In the present study, we found that XAV939-inhibition of canonical WNT signaling in skin of YAP2-5SA-ΔC mice resulted in diminished β-catenin activation, reduced keratinocyte proliferation, and a mitigation of the hyperplastic abnormalities in the interfollicular epidermis, signifying a canonical WNT ligand-dependent mechanism. Our subsequent analyses determined that WNT16 is produced in response to YAP activity in keratinocytes both in vitro and in vivo, and that WNT16 drives HaCaT keratinocyte proliferation via canonical WNT16/β-catenin signaling. We conclude that under normal physiological conditions WNT16 is the paracrine WNT ligand secreted in response to epidermal YAP activity that promotes cell proliferation in the interfollicular epidermis. This study delineates a fundamental YAP-driven mechanism that controls normal skin regeneration, and that may be perturbed in human regenerative disease displaying increased YAP and WNT signaling activity

Transcriptome Analysis of Hyperplastic Skin of YAP2-5SA-?C Mice Identifies Molecular Mechanisms Associated with Skin Cancer and Atopic Dermatitis

Skin disease is the fourth leading cause of nonfatal disease burden worldwide. Eczema affects 20% of the world population, and 2 out of 3 Australians will develop skin cancer during their life time. These skin diseases arise due to perturbed skin homeostasis, but the exact mechanisms underlying their aetiology remain unknown. Oncoprotein Yes-associated protein (YAP) is a pivotal and highly conserved regulator of organ size. We generated a viable mouse model that overexpresses dominant active YAP2-5SA-?C in basal keratinocytes, resulting in ?-catenin activation and severe epidermal hyperplasia due to an expansion of the epidermal stem/progenitor cell population. In this project, I attempted to identify molecular pathways that are controlled by YAP in normal skin biology and in skin disease through YAP2-5SA-?C skin transcriptome analyses. The bioinformatics analyses, first of all, resulted in the identification of PLAU and TGFBR3 as potential downstream effectors of YAP activity in control of keratinocyte proliferation. These results suggest that pathways such as TGF-? signalling and PLAU/PLAUR signalling might be triggered by YAP activation to promote cell proliferation. I also identified WNT16 as a highly YAP-responsive WNT gene that activates canonical WNT/?-catenin signalling to drive keratinocyte proliferation both in vitro and in vivo, a pathway that may be perturbed in human skin carcinogenesis. Furthermore, I found a striking activation of the type 2 immune response in YAP2-5SA-?C skin resulting in atopic dermatitis development and the activation of the master cytokine IL-33 and of the itch mediator protein CTSS. In addition, I also found activation of YAP in skin of human eczema patients, where it co-localised with IL-33 and CTSS in diseased keratinocytes. These data suggest that YAP may drive IL-33 and CTSS production in atopic dermatitis development, possibly in response to mechanical stimuli and/or impaired barrier.This project reveals YAP as a putative therapeutic target to treat human skin cancer and atopic dermatitis, and other skin disease displaying activated YAP

Positive regulatory interactions between YAP and Hedgehog signalling in skin homeostasis and BCC development in mouse skin in vivo

Skin is a highly plastic tissue that undergoes tissue turnover throughout life, but also in response to injury. YAP and Hedgehog signalling play a central role in the control of epidermal stem/progenitor cells in the skin during embryonic development, in postnatal tissue homeostasis and in skin carcinogenesis. However, the genetic contexts in which they act to control tissue homeostasis remain mostly unresolved. We provide compelling evidence that epidermal YAP and Hedgehog/GLI2 signalling undergo positive regulatory interactions in the control of normal epidermal homeostasis and in basal cell carcinoma (BCC) development, which in the large majority of cases is caused by aberrant Hedgehog signalling activity. We report increased nuclear YAP and GLI2 activity in the epidermis and BCCs of K14-CreER/Rosa-SmoM2 transgenic mouse skin, accompanied with increased ROCK signalling and ECM remodelling. Furthermore, we found that epidermal YAP activity drives GLI2 nuclear accumulation in the skin of YAP2-5SA-ΔC mice, which depends on epidermal β-catenin activation. Lastly, we found prominent nuclear activity of GLI2, YAP and β-catenin, concomitant with increased ROCK signalling and stromal fibrosis in human BCC. Our work provides novel insights into the molecular mechanisms underlying the interplay between cell signalling events and mechanical force in normal tissue homeostasis in vivo, that could potentially be perturbed in BCC development

Plau and Tgfbr3 are YAP-regulated genes that promote keratinocyte proliferation

Yes-associated protein (YAP) is a mechanosensor protein and a downstream effector of the Hippo kinase pathway, which controls organ growth, cell proliferation, survival, maintenance and regeneration. Unphosphorylated YAP translocates to the nucleus where it acts as a cofactor of primarily the TEAD transcription factors to activate target gene transcription and cell proliferation. Perturbed YAP activation results in tumorigenesis. The pathways downstream of activated YAP that drive cell proliferation remain relatively unexplored. In this study, we employed YAP2-5SA-∆C transgenic mice, which overexpress a mildly activated YAP mutant protein in basal keratinocytes leading to increased proliferation of the epidermal stem/progenitor cell populations. We performed massively-parallel sequencing of skin biopsy mRNA (RNA-Seq) and found dysregulation of 1491 genes in YAP2-5SA-∆C skin, including many with roles in cell activation and proliferation. Furthermore, we found that 150 of these dysregulated genes harbored YAP/TEAD binding motifs in the 3' UTR, suggesting that these may be direct YAP/TEAD target genes in the control of epidermal regeneration. Further validation and functional characterization assays identified Plau and Tgfbr3 as prime candidate genes that may be activated by epidermal YAP activity in the mouse skin in vivo to promote keratinocyte proliferation. This study provides novel insights into the mechanisms regulated by YAP that control tissue homeostasis, and in particular in conditions where YAP is aberrantly activated such as in neoplastic and regenerative skin disease

Novel and known MYOC exon 3 mutations in an admixed Peruvian primary open-angle glaucoma population

Purpose: The aim of this study was to characterize a representative sample of the Peruvian population suffering openangle glaucoma (OAG) with respect to the myocilin gene (MYOC) mutations, glaucoma phenotype, and ancestry for future glaucoma risk assessment. Methods: DNA samples from 414 unrelated Peruvian subjects, including 205 open-angle glaucoma cases (10 juvenile glaucoma [JOAG], 19 normal-tension glaucoma [NTG], and 176 POAG) and 209 randomly sampled controls, were screened for nucleotide changes in MYOC exon 3 by conformational sensitive gel electrophoresis (CSGE) and mutation screening. Results: We identified a probable causative novel MYOC missense mutation, Gly326Ser, in one POAG case and found a consistent genotype-phenotype correlation in eight of his relatives. We also found the known causative MYOC mutation Trp286Arg in one JOAG case and one POAG case. A known causative single base MYOC deletion, T1357, was found in one POAG case. Two previously reported silent polymorphisms, Thr325Thr and Tyr347Tyr, were found in both the case and the control populations. A novel missense variant, Met476Arg, was identified in two unrelated controls. Conclusions: The screening of exon 3 of MYOC in a representative sample of 205 independent POAG patients from Peru and 209 matched controls identified novel and previously reported mutations (both pathogenic and nonpathogenic) from other global regions. These results reflect the complex admixture of Amerindian and Old World ancestry in urban populations of Latin America, in general, and in Peru, in particular. It will be important to gather information about the ancestral origin of MYOC and other POAG gene mutations to develop screening panels and risk assessment for POAG in Peru.National Eye Institute, National Institutes of Health, Bethesda, MD (J.E.R.), Research to Prevent Blindness (EY011671- J.E.R.), Universidad de San Martín de Porres Funds E10012009016, E10012009011, E10012009027, E10012012011, Consejo Nacional de Ciencia y Tecnología (Concytec) proyecto OAJ-2003

Bone Marrow Macrophages Induce Inflammation by Efferocytosis of Apoptotic Prostate Cancer Cells via HIF-1α Stabilization

The clearance of apoptotic cancer cells by macrophages, known as efferocytosis, fuels the bone-metastatic growth of prostate cancer cells via pro-inflammatory and immunosuppressive processes. However, the exact molecular mechanisms remain unclear. In this study, single-cell transcriptomics of bone marrow (BM) macrophages undergoing efferocytosis of apoptotic prostate cancer cells revealed a significant enrichment in their cellular response to hypoxia. Here, we show that BM macrophage efferocytosis increased hypoxia inducible factor-1alpha (HIF-1α) and STAT3 phosphorylation (p-STAT3 at Tyr705) under normoxic conditions, while inhibitors of p-STAT3 reduced HIF-1α. Efferocytosis promoted HIF-1α stabilization, reduced its ubiquitination, and induced HIF-1α and p-STAT3 nuclear translocation. HIF-1α stabilization in efferocytic BM macrophages resulted in enhanced expression of pro-inflammatory cytokine MIF, whereas BM macrophages with inactive HIF-1α reduced MIF expression upon efferocytosis. Stabilization of HIF-1α using the HIF-prolyl-hydroxylase inhibitor, Roxadustat, enhanced MIF expression in BM macrophages. Furthermore, BM macrophages treated with recombinant MIF protein activated NF-κB (p65) signaling and increased the expression of pro-inflammatory cytokines. Altogether, these findings suggest that the clearance of apoptotic cancer cells by BM macrophages triggers p-STAT3/HIF-1α/MIF signaling to promote further inflammation in the bone tumor microenvironment where a significant number of apoptotic cancer cells are present

Activated ROCK-signalling, increased dermal fibroblast numbers, and dermal fibrosis in the skin of K14-CreER/Rosa-SmoM2 transgenic mice.

<p>(A-G) Immunofluorescence staining and area coverage analysis of dorsal skin tamoxifen- and vehicle-treated K14-CreER/Rosa-SmoM2 transgenic littermate mice detecting Fsp1 (A & B) and Vimentin (C), Phalloidin (G), DIAPH3 (H), Thr696-phosphorylated MYPT1 (I & J), Thr18/Ser19-phosphorylated MLC2 (K & L). (D) Masson’s trichrome histological staining of sections through the dorsal neck skin of tamoxifen- and vehicle-treated K14-CreER/Rosa-SmoM2 mice. (E & J) Dual two-photon SHG and monochromatic transmission (Trans; grayscale in merge) images showing collagen (white in single channel, magenta in merged) in tamoxifen- and vehicle-treated K14-CreER/Rosa-SmoM2 skin sections. Area coverage analysis (5 fields/sample from three mice per genotype) of SHG is quantified. Basement membranes and hair follicles are demarcated with dashed lines. DAPI, 4, 6-diamidino-2-phenylindole. Scale bars = 20 μm.</p

A model outlining the cross-regulatory interactions between epidermal YAP, ROCK, β-catenin and Hh signalling.

<p>(A) Epidermal SmoM2 activates YAP, ROCK signalling and dermal fibroblasts in the dorsal skin of K14-CreER/Rosa-SmoM2 transgenic mice (based on Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.g001" target="_blank">1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.g002" target="_blank">2</a>). (B) Epidermal YAP activates GLI2 mediated by β-catenin activation in the dorsal skin of YAP2-5SA-ΔC transgenic mice (based on Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.g004" target="_blank">4</a>). (C) A model outlining the proposed regulatory interactions between epidermal YAP, Hedgehog and ROCK-dependent mechanosignalling to balance skin regeneration based on our findings (red arrows) and on cited studies [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref025" target="_blank">25</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref027" target="_blank">27</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref029" target="_blank">29</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref034" target="_blank">34</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref039" target="_blank">39</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref040" target="_blank">40</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref045" target="_blank">45</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref047" target="_blank">47</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref048" target="_blank">48</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref063" target="_blank">63</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref064" target="_blank">64</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref065" target="_blank">65</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref066" target="_blank">66</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0183178#pone.0183178.ref069" target="_blank">69</a>].</p