br Tumor associated inflammation is involved in aggressivene
Tumor-associated inflammation is involved in aggressiveness and metastatic progression in several types of cancer , including pros-tate cancer [21,22]. It has been suggested that inducing the expressions of inflammatory response genes may contribute to the development of prostate cancer. Prostaglandin endoperoxide synthase 1 (PTGS1), also known as cyclooxygenase 1 (COX1), was shown to regulate angiogen-esis in endothelial cells . Activation of PTGS1 is involved in the inflammatory response, cell proliferation, and fatty Liproxstatin-1 metabolism during tumor progression . However, the association of abundant PTGS1 with prostate cancer progression and the possible involvement of PTGS1 in AR signaling regulation remain unclear. There is a growing body of evidence linking the inflammatory response to ADT resistance in advanced prostate cancer [25,26]. We hypothesized that the abun-dance of PTGS1 may be involved in NEPC diﬀerentiation following ADT. Therefore, elucidating the regulatory pathway of PTGS1 might lead to a better understanding of the therapeutic targets for NEPC.
We previously identified that a novel prostatic tumor promoter, zinc finger and BTB domain containing 46 (ZBTB46), is negatively regulated by the AR functions via microRNA-1-mediated downregulation . ZBTB46 is a lineage-specific transcription factor that is crucial for dif-ferent stages of dendritic cell (DC) development . Downregulation of ZBTB46 aﬀects macrophages and DCs in the microenvironment, leading to a decrease in tumor burden . In prostate cancer, ZBTB46 is associated with epithelial-to-mesenchymal transition (EMT) and prostate cancer metastasis by transcriptionally regulating SNAI1 . ZBTB46 overexpression promotes androgen- and AR-independent pro-liferation , which provides insights into the oncogenic role of ZBTB46 to ADT resistance. Although several types of immune responses are induced in prostate cancer following ADT [25,30,31], we hy-pothesized that the abundance of ZBTB46 is connected to the induction of inflammatory response genes. We investigated the role of ZBTB46 in promoting NEPC progression and its association with PTGS1 expression in ADT-treated prostate cancer.
Our results demonstrated that ADT induces ZBTB46 expression through the downregulation of the androgen-responsive SAM pointed domain containing ETS transcription factor (SPDEF), leading to in-creased expression of PTGS1 and contributing to NE diﬀerentiation of prostate cancer cells. The addition of PTGS1 inhibitor treatment can restore enzalutamide (MDV3100) sensitivity and reduce tumor growth, whereas overexpression of ZBTB46 disrupts the tumor-suppressive ef-fect of this combination treatment and induces PTGS1- and NEPC-as-sociated genes. Our findings provide a novel link between NE diﬀer-entiation and the expressions of inflammatory response genes as well as new biomarkers and therapeutic targets for NEPC.
2. Materials and methods
2.1. Cell lines and cell culture
The AR-positive C4-2B and LNCaP-AR (parental LNCaP over-expressing wild-type AR ) and AR-negative RasB1 (an aggressive cell line expressing a constitutively active Ras in the DU145 cells and isolated from a bone metastasis [27,33–40]) cell lines were obtained from Dr. Kathleen Kelly (NCI/NIH, MD, USA) and maintained as pre-viously described. The PC3, LNCaP, C4-2, and 22Rv1 cell lines were from ATCC (VA, USA), and they were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). The NE-like NE-1-8 (a subclone cell from the LNCaP cell line that is treated with long-term ADT ) and TRAMP-C1 (a more NE mouse prostate cancer model [42–46]) cell lines were purchased from ATCC and separately cultured in RPMI 1640 medium supplemented with 5% charcoal-stripped serum (CSS) and DMEM medium supplemented with 10% FBS, 0.005 mg/ml
bovine insulin (Sigma-Aldrich), and 10 nM dehydroisoandrosterone (Sigma-Aldrich). The AR antagonist, enzalutamide (MDV3100) (Selleck), and the PTGS inhibitor, NS-398 (Sigma-Aldrich), were used separately to treat the cells at 10 μM and 1.77 or 75 μM for 24 h in 10% FBS-containing medium. Dihydrotestosterone (DHT) (Sigma-Aldrich) was used to treat cells at 10 nM for 24 h in 10% CSS-containing medium.
2.2. Chromatin immunoprecipitation (ChIP) assay
ChIP assays were performed using an EZ magna ChIP A kit (Millipore) with a modified protocol. C4-2B and LNCaP-AR cells were treated with DHT (Sigma-Aldrich) at 10 nM for 4 h in 10% CSS-con-taining medium or MDV3100 (Selleck) at 10 μM for 4 h in 10% FBS-containing medium, as described in Supplementary Methods.
2.3. Promoter reporter assay
For the promoter reporter assays, LNCaP, RasB1, or C4-2B cells in 12-well plates (5 × 104 cells/well) were transiently transfected with 1 μg of the PTGS1- or ZBTB46-green fluorescent protein (GFP) reporter containing ZBTB46- or SPDEF-binding sites. The cells were treated with DHT (Sigma-Aldrich) and MDV3100 (Selleck) or small interfering (si) RNA and DNA (empty vector (EV), ZBTB46- or SPDEF-expressing vector) by transfection, as described in Supplementary Methods.