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Breast cancer is the most common type of cancer in women worldwide, with the luminal subtype being the most widespread. Although characterized by better prognosis compared with other subtypes, luminal breast cancer is still considered a threatening disease due to therapy resistance, which occurs via both cell- and non–cell-autonomous mechanisms. Jumonji domain-containing 6, arginine demethylase and lysine hydroxylase (JMJD6) is endowed with a negative prognostic value in luminal breast cancer and, via its epigenetic activity, it is known to regulate many intrinsic cancer cell pathways. So far, the effect of JMJD6 in molding the surrounding microenvironment has not been explored. Here, we describe a novel function of JMJD6 showing that its genetic inhibition in breast cancer cells suppresses lipid droplet formation and ANXA1 expression, via estrogen receptor alpha and PPARα modulation. Reduction of intracellular ANXA1 results in decreased release in the tumor microenvironment (TME), ultimately preventing M2-type macrophage polarization and tumor aggressiveness. Implications: Our findings identify JMJD6 as a determinant of breast cancer aggressiveness and provide the rationale for the development of inhibitory molecules to reduce disease progression also through the remodeling of TME composition.

Breast cancer is the most common type of cancer in women worldwide, with the luminal subtype being the most widespread. Although characterized by better prognosis compared with other subtypes, luminal breast cancer is still considered a threatening disease due to therapy resistance, which occurs via both cell- and non–cell-autonomous mechanisms. Jumonji domain-containing 6, arginine demethylase and lysine hydroxylase (JMJD6) is endowed with a negative prognostic value in luminal breast cancer and, via its epigenetic activity, it is known to regulate many intrinsic cancer cell pathways. So far, the effect of JMJD6 in molding the surrounding microenvironment has not been explored.Here, we describe a novel function of JMJD6 showing that its genetic inhibition in breast cancer cells suppresses lipid droplet formation and ANXA1 expression, via estrogen receptor alpha and PPARα modulation. Reduction of intracellular ANXA1 results in decreased release in the tumor microenvironment (TME), ultimately preventing M2-type macrophage polarization and tumor aggressiveness.Implications:Our findings identify JMJD6 as a determinant of breast cancer aggressiveness and provide the rationale for the development of inhibitory molecules to reduce disease progression also through the remodeling of TME composition.

Significant enriched pathways in JMJD6-KO MCF7 and MDA-MB231 cells

JMJD6 regulates lipid metabolism in MCF7 cells. A) Immunohistochemistry staining for ANXA1 (brown) combined with Oil red-O staining (pink) for lipid droplets visualization in wild-type MCF7 cells B) Gene set enrichment plot for 'Hallmark of Adipogenesis' found to be significantly downregulated in JMJD6 KO MCF7 cells compared to scramble control. C) List of 'Reactome Pathways' found to be regulated by the genes included in the gene set enrichment pathway of 'Hallmark of Adipogenesis' and present in the DEGs list of JMJD6 KO versus scramble MCF7 cells.

Macrophages in the tumor microenvironment are causally linked with prostate cancer development and progression, yet little is known about their composition in neoplastic human tissue. By performing single cell transcriptomic analysis of human prostate cancer resident macrophages, three distinct populations were identified in the diseased prostate. Unexpectedly, no differences were observed between macrophages isolated from the tumorous and nontumorous portions of the prostatectomy specimens. Markers associated with canonical M1 and M2 macrophage phenotypes were identifiable, however these were not the main factors defining unique subtypes. The genes selectively associated with each macrophage cluster were used to develop a gene signature which was highly associated with both recurrence-free and metastasis-free survival. These results highlight the relevance of tissue-specific macrophage subtypes in the tumor microenvironment for prostate cancer progression and demonstrates the utility of profiling single-cell transcriptomics in human tumor samples as a strategy to design gene classifiers for patient prognostication. Implications: The specific macrophage subtypes present in a diseased human prostate have prognostic value, suggesting that the relative proportions of these populations are related to patient outcome. Understanding the relative contributions of these subtypes will not only inform patient prognostication, but will enable personalized immunotherapeutic strategies to increase beneficial populations or reduce detrimental populations.

The androgen receptor (AR) is the master regulator of prostate cancer (PCa) development, and inhibition of AR signalling is the most effective PCa treatment. AR is expressed in PCa cells and also in the PCa-associated stroma, including infiltrating macrophages. Macrophages have a decisive function in PCa initiation and progression, but the role of AR in macrophages remains largely unexplored. Here, we show that AR signalling in the macrophage-like THP-1 cell line supports PCa cell line migration and invasion in culture via increased Triggering Receptor Expressed on Myeloid cells-1 (TREM-1) signalling and expression of its downstream cytokines. Moreover, AR signalling in THP-1 and monocyte-derived macrophages upregulates IL-10 and markers of tissue residency. In conclusion, our data suggest that AR signalling in macrophages may support PCa invasiveness, and blocking this process may constitute one mechanism of anti-androgen therapy., Anti-androgen therapy inhibits prostate cancer (PC) progression, and is thought to act directly on cancer cells. Here the authors show that androgen receptor is expressed on normal and PC-associated macrophages, and its stimulation alters macrophage secretome to promote migration of cultured PC cell lines.

Fibroblasts are abundantly present in the prostate tumor microenvironment (TME), including cancer-associated fibroblasts (CAFs) which play a key role in cancer development. Androgen receptor (AR) signaling is the main driver of prostate cancer (PCa) progression, and stromal cells in the TME also express AR. High-grade tumor and poor clinical outcome are associated with low AR expression in the TME, which suggests a protective role of AR signaling in the stroma against PCa development. However, the mechanism of this relation is not clear. In this study, we isolated AR-expressing CAF-like cells. Testosterone (R1881) exposure did not affect CAF-like cell morphology, proliferation, or motility. PCa cell growth was not affected by culturing in medium from R1881-exposed CAF-like cells; however, migration of PCa cells was inhibited. AR chromatin immune precipitation sequencing (ChIP-seq) was performed and motif search suggested that AR in CAF-like cells bound the chromatin through AP-1-elements upon R1881 exposure, inducing enhancer-mediated AR chromatin interactions. The vast majority of chromatin binding sites in CAF-like cells were unique and not shared with AR sites observed in PCa cell lines or tumors. AR signaling in CAF-like cells decreased expression of multiple cytokines; most notably CCL2 and CXCL8 and both cytokines increased migration of PCa cells. These results suggest direct paracrine regulation of PCa cell migration by CAFs through AR signaling.

Background Human papillomavirus (HPV)‐positive oropharyngeal squamous cell carcinoma (OPSCC) is a highly immunogenic tumor and differences in tumor microenvironment might contribute to the improved survival of HPV‐positive OPSCC patient. Methods A comprehensive multivariate analysis with clinical and immune variables (human leukocyte antigen [HLA] I/II, programmed death ligand 1 (PD‐L1), programmed death receptor 1 (PD1), T cells, and macrophages) was performed in 142 OPSCC patients. Results We found an inverse correlation between the expression of HLA class II molecules on tumor cells and CD68+ CD163+ tumor‐associated macrophages (TAMs). High HLA‐DP/DQ/DR expression and low number of TAMs were associated with longer disease‐specific survival and disease‐free survival (DFS). Furthermore, a new population of CD8+ FoxP3+ T cells was correlated with shorter DFS in multivariate analysis. Conclusions \We identified new prognostic markers for patients with oropharyngeal cancer, which can be used for selecting patients that can benefit from immunotherapy.

Androgen receptor (AR) signaling is vital for the normal development of the prostate and is critically involved in prostate cancer (PCa). AR is not only found in epithelial prostate cells but is also expressed in various cells in the PCa-associated stroma, which constitute the tumor microenvironment (TME). In the TME, AR is expressed in fibroblasts, macrophages, lymphocytes and neutrophils. AR expression in the TME was shown to be decreased in higher-grade and metastatic PCa, suggesting that stromal AR plays a protective role against PCa progression. With that, the functionality of AR in stromal cells appears to deviate from the receptor’s classical function as described in PCa cells. However, the biological action of AR in these cells and its effect on cancer progression remains to be fully understood. Here, we systematically review the pathological, genomic and biological literature on AR actions in various subsets of prostate stromal cells and aim to better understand the consequences of AR signaling in the TME in relation to PCa development and progression.

// Monique H.M. Melis 1 , Ekaterina Nevedomskaya 1 , Johan van Burgsteden 1 , Bianca Cioni 1 , Hester J.T. van Zeeburg 1 , Ji-Ying Song 2 , John Zevenhoven 1 , Lukas J.A.C Hawinkels 3 , Karin E. de Visser 4 and Andries M. Bergman 1, 5 1 Division of Molecular Genetics, Netherlands Cancer Institute, The Netherlands 2 Division of Experimental Animal Pathology, Netherlands Cancer Institute, The Netherlands 3 Division of Gastroenterology-Hepatology and Molecular Cell biology, Leiden university medical center, (LUMC), Netherlands 4 Division of Immunology, Netherlands Cancer Institute, The Netherlands 5 Division of Medical Oncology, Netherlands Cancer Institute, The Netherlands Correspondence to: Andries M. Bergman, email: a.bergman@nki.nl Keywords: prostate cancer, GEMM, adaptive immune system Received: April 07, 2017 Accepted: August 26, 2017 Published: September 28, 2017 ABSTRACT Increasing evidence from epidemiological and pathological studies suggests a role of the immune system in the initiation and progression of multiple cancers, including prostate cancer. Reports on the contribution of the adaptive immune system are contradictive, since both suppression and acceleration of disease development have been reported. This study addresses the functional role of lymphocytes in prostate cancer development using a genetically engineered mouse model (GEMM) of human c-Myc driven prostate cancer (Hi-Myc mice) combined with B and T cell deficiency (RAG1 -/- mice). From a pre-cancerous stage on, Hi-Myc mice showed higher accumulation of immune cells in their prostates then wild-type mice, of which macrophages were the most abundant. The onset of invasive adenocarcinoma was delayed in Hi-MycRAG1 -/- compared to Hi-Myc mice and associated with decreased infiltration of leukocytes into the prostate. In addition, lower levels of the cytokines CXCL2, CCL5 and TGF-β1 were detected in Hi-MycRAG1 -/- compared to Hi-Myc mouse prostates. These results from a GEMM of prostate cancer provide new insights into the promoting role of the adaptive immune system in prostate cancer development. Our findings indicate that the endogenous adaptive immune system does not protect against de novo prostate carcinogenesis in Hi-Myc transgenic mice, but rather accelerates the formation of invasive adenocarcinomas. This may have implications for the development of novel treatment strategies.

Increasing evidence from epidemiologic and pathologic studies suggests a role of macrophages in the initiation and progression of prostate cancer (PCa). During PCa development, macrophages (CD68+) are recruited into the human prostate cancer microenvironment, including inflammation-associated M1 and cancer-promoting M2 macrophages. Like other prostate cancer-associated stromal cells, macrophages express the androgen receptor (AR); however, its functionality is not known. CD68+ cells generated in vitro from peripheral white blood cells and PMA activated and IFN and LPS polarized human acute monocytic leukemia THP-1 cells also expressed AR. In these cells AR is an 80KD splice variant, lacking parts of exon 1 and exon 2. AR translocated to the nucleus upon testosterone stimulation, which was counteracted by the AR signaling inhibitor RD162. In vitro generated M1 macrophages expressed the M2 markers CD163 and CD206 upon testosterone stimulation, which was reversed by RD162. AR-ChIP sequencing suggested AR regulated Macrophage Triggering Receptor 1 (TREM1) signaling. Expression of key cytokines (e.g., CCL2, CXCL8, CCL7, IL10, IL1B) involved in TREM1 signaling was upregulated upon testosterone stimulation. Actions of these cytokines is generally associated with a protumor phenotype of macrophages. Conditioned medium from macrophages stimulated with testosterone for 8 hrs resulted in enhanced proliferation and migration of human prostate LNCap and CWRR1 cells. In conclusion, AR signaling might affect differentiation of prostate cancer-associated macrophages into cancer-promoting M2, which affects prostate cancer cell proliferation and migration. Maintaining macrophages in M1 might be a novel mechanism of action of androgen receptor inhibitors. Citation Format: Bianca Cioni, Ekatarina Nevedomskaya, Suzan Stelloo, Monique Melis, Johan van Burgsteden, Jeroen de Jong, Henk van der Poel, Jan Paul de Boer, Wilbert Zwart, Andries M. Bergman. Androgen receptor signaling affects macrophage differentiation in the human prostate cancer microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5198.

Background: Prostate cancer is a heterogeneous disease both genetically and in its clinical behavior. It is widely accepted that a tumor does not only consist of cancer cells but also contains many other non-malignant cells, including immune cells and cancer associated fibroblasts. These non-malignant cells create the tumor microenvironment. It is previously suggested that the tumor microenvironment modulates the behavior of various cancers, including prostate cancer. Little is known about the exact interactions between prostate cancer cells and its micro environment. In this study we aim to gain insight into the composition of the microenvironment and the interaction between malignant and non-malignant cells. Moreover, these interactions might hold a predictive signature for disease progression and metastatic disease. Methods: Biopsies were obtained from Formalin Fixed Paraffin Embedded (FFPE) human prostate cancers from 10 patients with pelvic lymph node metastases and 10 gleason score (7-8-9) matched patients with non-metastatic disease. RNA was isolated from the FFPE samples and sequenced. A tissue micro array was constructed from the same prostate cancers. Fresh prostate cancer biopsies were taken right after radical prostatectomy, and fibroblast cells were isolated for short term culture. Results: The RNA sequencing of FFPE prostate biopsies revealed an increased expression of cancer related stroma genes in tumor tissue compared to non-tumorous tissue. Presence of various cell types was confirmed by immunohistochemistry (IHC). The subtypes of various immune cell populations could be identified, including regulatory T cells and pro-tumorous M2-like macrophages. The percentage of M2-like macrophages identified by the CD163 marker showed a significant increase in tumor tissue compared to non-tumorous tissue, which was unique for patients with metastatic disease. Furthermore we could confirm that decreased stromal Androgen Receptor (AR) expression correlated with disease progression and also correlated with metastatic disease. To investigate which stromal cells were AR positive we performed double stainings of PDGFR beta and AR. Costaining of both markers confirmed AR expression in fibroblasts. The stromal cells cultured from the fresh prostate cancer biopsies were morphologically fibroblasts and expressed AR, Smooth Muscle Actin and PDGFR beta, suggesting a cancer associated phenotype. Testosterone stimulation of these fibroblasts showed increased chromatin binding of the AR. This suggests that the AR is functional in these cancer associated fibroblasts. Conclusions: This study gained insight into the composition of the prostate cancer microenvironment. The increased expression of CD163 and the decreased expression of stromal AR was related to pelvic lymph node metastases. Citation Format: Monique Melis, Bianca Cioni, Ekaterina Nevedomskaya, Johan van Burgsteden, Emma Hodel, Annegien Broeks, Henk van der Poel, Jeroen de Jong, Andre Bergman. The composition and interactions in the microenvironment of human prostate cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5075. doi:10.1158/1538-7445.AM2015-5075