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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.

Primary prostate cancer lesions are clonally heterogeneous and often arise independently. In contrast, metastases were reported to share a monoclonal background. Because prostate cancer mortality is the consequence of distant metastases, prevention of metastatic outgrowth by primary tumor ablation is the main focus of treatment for localized disease. Focal therapy is targeted ablation of the primary index lesion, but it is unclear whether remaining primary lesions metastasize at a later stage. In this study, we compared copy number aberration profiles of primary prostate cancer lesions with matching pelvic lymph node metastases of 30 patients to establish clonality between a lymph node metastasis and multiple primary lesions within the same patient. Interestingly, in 23.3% of the cases, the regional metastasis was not clonally linked to the index primary lesion. These findings suggest that focal ablation of only the index lesion is potentially an undertreatment of a significant proportion of prostate cancer patients.

// 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

International audience; MUC2, the major mucin in the intestine, is expressed early during development and shows an altered expression pattern in intestinal bowel diseases. However, the mechanisms responsible for MUC2 expression in the intestine during these events are largely unknown. Having found putative GATA binding sites in the murine Muc2 promoter and that GATA-4 is expressed in Muc2-expressing goblet cells of the mouse small intestine, we undertook to study its regulation by this transcription factor. A panel of deletion mutants made in pGL3 vector and covering 2.2 kb of the promoter were used to transfect the murine CMT-93 colorectal cancer cell line. The role of GATA-4 on Muc2 gene regulation was investigated by RT-PCR and co-transfections in the presence of expression vectors encoding either wild-type or mutated GATA-4 or by mutating the GATA-4 site identified within Muc2 promoter. Four GATA-4 cis-elements were identified in the promoter by EMSA and Muc2 promoter was efficiently activated when GATA-4 was overexpressed in the cells with a loss of transactivation when those sites were either mutated or a mutated form of GATA-4 was used. Altogether, these results identify Muc2, a goblet cell marker, as a new target gene of GATA-4 and point out an important role for this factor in Muc2 expression in the intestine. The mucus layer in the lumen of the gut forms a physical barrier against microorganisms and insoluble material [1]. Goblet cells are a hallmark of the intestinal epithelium, and are known to secrete mucins that participate in mucus formation to protect the underlying mucosa. The secretory mucin Muc2, a very large O-gly-coprotein, is particularly relevant for the study of goblet cell biology as it is exclusively and abundantly expressed by intestinal goblet cells [2,3]. It is produced throughout the gastro-intestinal tract increasing from small intestine to colon. Muc2 is expressed early during embryonic development of the intestine [4], which suggests that Muc2 transcription is under the influence of transcription factors responsible for intestinal development and cell differentiation. The early expression of mucin before mucus cell differentiation or during the process of differentiation indicates that they may be the targets of transcription factors responsible for those programs [5]. This hypothesis has been recently confirmed in a previous study

Increasing evidence from epidemiological and pathological studies indicates a role of the immune system in initiation and progression of multiple cancers, including prostate cancer. Throughout carcinogenesis of the prostate, immune cells are found in the microenvironment. Overexpression of MYC or loss of PTEN are frequent genetic lesions in human prostate cancer. It has been shown that these genetic lesions induce chemokine expression and attraction of immune cells. In previous studies the adaptive system has shown to play a pivotal role in disease progression, however the role of the adaptive immune system in prostate carcinogenesis has been inadequately studied. In this study we address the role of the adaptive immune system in the development of prostate cancer in two independent spontaneous prostate cancer mouse models; the FVB/HiMYC mouse model in which prostate cancer formation is driven by transgenic expression of human MYC under control of the ARR2/probasin promoter, and the PB-Cre;PTENF/F mouse model in which prostate cancer formation is initiated by loss of PTEN expression. Both mouse models develop Prostatic Intraepithelial Neoplasia (PIN) from the age of 4 weeks on. FVB/HiMYC mice develop adenocarcinoma of the prostate from the age of 24 weeks and PB-Cre;PTENF/F mice from the age of 12 weeks on. To address the functional significance of lymphocytes in prostate cancer development, FVB/HiMYC and PB-Cre;PTENF/F were crossed to lymphocyte deficient RAG-1-/- mice. Analysis of the PTEN model is ongoing, however in the HiMYC model a small but significant delay (p Marked inflammation and increased nuclear BrdU incorporation was found in concert with prostate cancer development. Flow cytometry confirmed an increase in CD45 positive cells in the prostate already in pre-cancerous lesions, which was not observed in the peripheral blood. A significant increase in CD45+ cells in HiMYCRAG-1+/- was found compared to HiMYCRAG-1-/- at 24 weeks, with the majority being CD11b positive cells. This finding was strengthen by increased expression of cytokines involved in the chemo-attraction of macrophages and neutrophils, including CXCL1, CXCL2, CXCL5 and CCL5 which was observed in HiMYCRAG-1+/- mice compared to HiMYCRAG-1-/- In conclusion: Our study provides insight into the role of the adaptive immune system in prostate cancer development. In the absence of T and B cells, the composition of infiltrating immune cells and cytokine expression in the microenvironment is altered, which might contribute to the delay of prostate cancer development. This research was funded by a grant from the Dutch Cancer Society (KWF grant 2009-4356) Citation Format: Monique H.M Melis, J. van Burgsteden, H J.T van Zeeburg, J Zevenhoven, J-Y Song, K E. de Visser, A M. Bergman. The adaptive immune system promotes spontaneous prostate carcinogenesis in a transgenic mouse model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1080. doi:10.1158/1538-7445.AM2014-1080

Increasing evidence from epidemiological and pathology studies indicates a role of the immune system in initiation and progression of multiple cancers, including prostate cancer. However, reports on the specific role of the immune system are contradictive since both suppression and acceleration of disease progression have been described. Overexpression of MYC and loss of PTEN are frequent genetic lesions in prostate cancer. It has been shown that these lesions induce chemokine expression, attraction of immune cells and subsequent angiogenesis and disease progression in cancer models. In this study we address the role of the adaptive immune system in prostate cancer progression in two independent spontaneous prostate cancer mouse models; the FVB/HiMYC mouse model in which prostate cancer formation is driven by transgenic expression of human MYC under control of the ARR2/probasin promoter, and the PB-Cre;PTENF/F mouse model in which prostate cancer formation is initiated by loss of PTEN expression. Both mouse models develop Prostatic Intraepithelial Neoplasia (PIN) from the age of 4 weeks on. FVB/HiMYC mice develop invasive carcinoma from the age of 24 weeks and PB-Cre;PTENF/F mice from the age of 12 weeks on. To address the functional significance of lymphocytes in prostate cancer development, FVB/HiMYC and PB-Cre;PTENF/F were crossed to lymphocyte deficient RAG-1−/− mice. Preliminary data suggest that both mouse models crossed to RAG-1−/− have a longer latency of invasive carcinoma development and less extensive lesions than RAG-1+/− mice, suggesting an important role of the adaptive immune system in prostate cancer initiation and/or progression. Marked inflammation and increased nuclear BrdU incorporation was found in concert with prostate cancer development using immunohistochemistry. Flow cytometry confirmed an increase in CD45 positive cells in the prostate, which was not observed in blood. Studies to identify the lymphocyte population with a key role in prostate cancer development and to dissect the exact mechanism of growth promotion are currently ongoing. In addition, we will verify whether the identified immune cells and soluble mediators in the in vivo models are also found in human prostate cancer samples. In conclusion: Our study provides insight into the role of the adaptive immune system in prostate cancer development. These data might serve as a basis to develop intervention strategies that intersect with the supportive role of the immune system in human prostate carcinogenesis. Citation Format: Monique H.M Melis, Johan van Burgsteden, Hester J.T. van Zeeburg, John Zevenhoven, Ji-Ying Song, Karin E. de Visser, Andre M. Bergman. The role of the adaptive immune system in initiation and progression in two independent spontaneous mouse models for prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1268. doi:10.1158/1538-7445.AM2013-1268