SOX2 control activation of dormant prostate cancer cells in bone metastases by promoting CCNE2 gene expression.

Am J Clin Exp Urol 2024;12(6):375-388 www.ajceu.us /ISSN:2330-1910/AJCEU0160381 Original Article SOX2 control activation of dormant prostate cancer cells in bone metastases by promoting CCNE2 gene expression Min Deng1*, Pei-Zheng Huang1,2*, Ze-Yu Huang1*, Ting-Ting Chen1, Xing Luo1, Chao-Yu Liao1, Wen-Hao Xu1, Jiang Zhao1, Qing-Jian Wu1#, Ji Zheng1,3# Department of Urology, The Second Affiliated Hospital, Army Military Medical University, Chongqing, China; School of Medicine, Chongqing University, Chongqing, China; 3State Key Laboratory of Trauma and Chemical Poisoning, Daping Hospital, Third Military Medical University, Chongqing, China. *Equal contributors. #Equal contributors. 1 2 Received September 6, 2024; Accepted December 12, 2024; Epub December 15, 2024; Published December 30, 2024 Abstract: Background: Cancer stem cells (CSCs) have a powerful tumor initiation ability, which can promote the early dissemination of single disseminated tumor cells (DTCs), leading to tumor progression. SOX2, a pluripotent inducible transcription factor, is key to maintaining self-renewal and pluripotency of prostate cancer stem cells. However, there is a lack of comprehensive understanding of how SOX2 regulates DTCs dormancy and proliferation in the bone marrow microenvironment. Methods and Results: By constructing a mouse bone metastasis model to simulate the progression of prostate cancer with bone metastasis, the bone tissue immunofluorescence showed that SOX2 expression increased with the progression of prostate cancer in the bone marrow microenvironment. We validated this phenomenon with publicly available single-cell and transcriptome datasets and found that SOX2 is involved in multiple phenotypes associated with prostate cancer dormancy, proliferation, and invasion. Further, CCNE2, a potential target downstream of SOX2, was identified through multiple transcription factor databases and protein interaction networks. Conclusion: The expression of SOX2 affects multiple phenotypes related to dormancy, proliferation and invasion of prostate cancer, and may indirectly activate the dormant prostate cancer cells through the downstream target gene CCNE2, thus affecting the progression and bone metastasis of prostate cancer. Keywords: Prostate cancer, SOX2, CCNE2, dormancy, bone metastasis Introduction Prostate cancer is the second leading cause of cancer-related death in men [1]. At present, androgen deprivation therapy (ADT) is the main treatment for prostate cancer. Although the tumor was well controlled in the early stage, castration resistance still appeared in some patients in the late stage, eventually leading to bone metastasis and recurrence [2]. A large number of studies have shown that most tumor cells that metastasise from the primary site to distant organs through blood are cleared by the immune system or undergo apoptosis, but a small number of residual DTCs colonize in the body and remain in a dormant state. These DTCs that survive immune system attack and drug killing form cancer metastases. These dormant DTCs are generally CSCs with stem cell activity, which are generally considered to be the root cause of resistance to conventional chemoradiotherapy and cause tumor recurrence and metastasis [3, 4]. After CSCs adapt to the microenvironment or receive some kind of stimulation, they will reactivate, divide and proliferate, and eventually become clinical metastatic lesions. However, the interaction between tumor cells and the bone marrow microenvironment is still unclear. Therefore, further understanding of the molecular mechanism of DTCs dormancy in tumor microenvironment will lay a theoretical foundation for elucidating the molecular mechanism of prostate cancer metastasis, which has important scientific significance and clinical guiding value. https://doi.org/10.62347/ASCY2532 Control activation of dormant prostate cancer SOX2 is an important pluripotent inducible transcription factor that plays a key role in maintaining self-renewal and pluripotency of embryonic stem cells [5]. Moreover, it is expressed in both benign and malignant prostate tissues, and is also associated with various tumor functions in non-stem cell prostate cancer cells [5-9]. These findings imply a potential link between SOX2 and prostate cancer progression, although whether it is involved in regulating and activating dormant tumor cells has not been clearly defined. fare and Ethics Committee of the Army Medical University. Intracardiac injection (I.C.) was used to construct a bone metastasis model. 100 μL cells were injected into the left ventricle of mice using a 26G needle [10]. Bioluminescence signals were evenly distributed throughout the body through BLI imaging within 24 hours, confirming the successful injection. Bone metastasis load was monitored weekly with BLI imaging. The mice were euthanized, and their hindlimbs were collected within 24 hours, at week 1, and at week 5. Here, we simulated the process of prostate cancer bone metastasis by constructing an intracardial bone metastasis model in mice, verified the close correlation between SOX2 and prostate cancer progression through multiple transcriptome data and transcription factor databases, and speculated that SOX2 may regulate the cell cycle of prostate cancer cells through the downstream target gene CCNE2, thereby regulating dormant cancer cell activation. Histological analysis and immunofluorescence (IF) staining Materials and methods Cell culture PC3 (RRID: CVCL_0035) cells were purchased from Prenoxel. The cells were tested for mycoplasma before treatment. As previously mentioned, the cells were cultured in DMEM (Gibco) supplemented with 10% heat-inactivated FBS (Hyclone) and 1% penicillin/streptomycin (Corning). To generate stable transfection cell lines, a lentiviral vector based on GFPLuciferase-Puromycin (Genechem, GV633) was used, and the lentivirus was packaged into PC3 cells. The GFP expression efficiency was initially measured by fluorescence microscopy 3 days after transfection, and the recipient cell lines were exposed to complete medium supplemented with 10 μg/mL purinomycin (Beyotime) to produce stable cell lines. Animal studies Male BALB/c nude mice aged 4 to 6 weeks were purchased from Vitonlihua and raised in SPF animal centres. All mice were exposed to 12 hours of light and 12 hours of darkness, eating and drinking freely. All procedures and experimental protocols involving mice have been approved by the Laboratory Animal Wel- 376 After skin and muscle removal, the tibia of mice were fixed with 4% paraformaldehyde, decalcified with EDTA for 4 weeks, and then paraffin embedding was performed to make continuous coronal sections (5 mm thick) of the entire tibia. Permeate with 0.2% Triton X-100 for 5 minutes, block with 5% BSA in PBS for 1 hour, incubate with appropriate primary antibody at 4°C overnight, then incubate with secondary antibody at 4°C in the dark for 1 hour. The antibodies used for IF stain (...truncated)