LSD1 promotes prostate cancer cell proliferation by upregulating PRAC1 expression

Abstract

PRAC1, which is specifically expressed in prostate, rectal, and distal colon tissues, plays a critical role in the maintenance and self-renewal of prostate epithelial stem cells. However, the role of PRAC1 in prostate cancer is unclear. In this study, we found that PRAC1 expression is upregulated in prostate cancer cells and that PRAC1 knockdown represses the proliferation of prostate cancer cells. Moreover, lysine-specific demethylase 1 (LSD1) promoted prostate cancer cell proliferation by upregulating PRAC1 expression. TAK-418, an LSD1 inhibitor, suppressed prostate cancer cell proliferation by downregulating PRAC1 expression. Our results highlight PRAC1 or LSD1 inhibition as promising avenues for prostate cancer treatment.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-42928-8.

Introduction

Prostate cancer is a leading malignancy in men worldwide that contributes substantially to global male mortality. Although its incidence rates vary considerably across regions, with a higher prevalence in developed countries, emerging evidence suggests rising trends in developing nations owing to aging populations and lifestyle changes^1,2. Prostate cancer poses substantial health risks, particularly for men aged > 50 years, those with a family history of the disease, and individuals of African descent, who face disproportionately higher morbidity and mortality^1,2. Although prostate cancer often progresses indolently, advanced stages can aggressively metastasize to bones and organs, causing debilitating complications^2,3. Advancing the understanding of prostate cancer pathogenesis and the identification of genes responsible for its malignant progression hold promise for the development of novel therapeutic targets.

PRAC1 encodes a 382 nucleotide RNA and a 6-kDa nuclear protein found in the prostate, rectum, and distal colon. The PRAC1 gene is located on chromosome 17 at position 17q21, approximately 4 kb downstream of the homeodomain of Hoxb-13⁴. Germline mutations in HOXB13 are associated with an increased risk of prostate cancer^5,6. PRAC1 mRNA is a specific marker distinguishing semen from other bodily fluids⁷. RNA in situ hybridization with a PRAC1 mRNA probe enables detection of exfoliated prostate cancer cells in urine^8,9. Research has shown that PRAC1 expression is lower in prostate cancer tissues than in benign prostatic hyperplasia tissues¹⁰ and that PRAC1 plays an essential role in the maintenance and self-renewal of prostate epithelial stem cells¹¹. However, the role of PRAC1 in prostate cancer remains unclear.

Therefore, we investigated the role of PRAC1 in prostate cancer in this study. We found that PRAC1 expression is upregulated in cancerous compared with non-cancerous prostate tissues, and that PRAC1 knockdown inhibits prostate cancer cell proliferation. Moreover, we found that lysine-specific demethylase 1 (LSD1) promotes PRAC1 expression in prostate cancer cells and that LSD1 inhibition represses prostate cancer cell proliferation by downregulating PRAC1 expression. Our findings highlight PRAC1 as a novel therapeutic target for prostate cancer therapy.

Results

PRAC1 knockdown inhibits proliferation, colony formation, migration, and invasion of prostate cancer cells

To investigate the role of PRAC1 in prostate cancer, we compared the expression levels of PRAC1 in prostate cancer and non-cancerous prostate tissues using The Cancer Genome Atlas and the Genotype-Tissue Expression Portal. PRAC1 expression was upregulated in cancerous compared with non-cancerous tissues (Fig. 1A). Next, to investigate the effect of PRAC1 on the proliferation, colony formation, migration, and invasion of prostate cancer cells, we knocked down PRAC1 in LNCaP and DU145 cells using two specific small interfering RNAs (siRNAs; Fig. 1B–C and Supplementary Fig. 1 A, B). The Cell Counting Kit-8 (CCK-8) assay and 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay revealed that PRAC1 knockdown reduced cell proliferation (Fig. 1D, E and Supplementary Fig. 1 C). Additionally, we found that PRAC1 knockdown repressed the colony formation, migration, and invasion of prostate cancer cells (Fig. 2A–D). To further examine the effect of PRAC1 on prostate tumor growth in vivo, we established LNCaP xenograft mouse models. Our results revealed that PRAC1 knockdown repressed xenograft tumor growth (Fig. 2E–G).

Fig. 1.

PRAC1 knockdown inhibits prostate cancer cell proliferation. (A) PRAC1 mRNA levels in cancerous and non-cancerous prostate tissues. Data were downloaded from The Cancer Genome Atlas. Red: prostate cancer tissue (n = 492); gray: non-cancerous prostate tissue (n = 52). (B–E) LNCaP cells were transfected with control or PRAC1 siRNA, and mRNA (B) and protein (C) levels of PRAC1 were determined using reverse transcription quantitative PCR and enzyme-linked immunosorbent assays, respectively, 48 h after transfection. (D, E) Cell proliferation was determined using the CCK8 (D) and EdU incorporation assays (E). Statistical analyses were performed using two-sided Student’s t-tests; *p < 0.05, ** p < 0.001.

Fig. 2.

PRAC1 knockdown inhibits colony formation, migration, and invasion of prostate cancer cells and represses xenograft tumor growth. (A-C) Colony formation (A), migration (B), and invasion (C) assays of control or PRAC1 knockdown cells. (D) Wound healing assay of control or PRAC1 knockdown cells. (E-G) Xenograft tumors of control or PRAC1 knockdown cells were photographed (E), the average volume (F) and weight (G) were determined. **P < 0.01 (two-sided Student’s t test).

LSD1 promotesPRAC1expression in prostate cancer cells

To investigate the mechanism underlying the upregulation of PRAC1 expression in prostate cancer cells, we searched GPSAdb, a database for exploring transcriptomic consequences of gene perturbations in human cell lines¹², to identify candidate genes that may regulate PRAC1 expression. As shown in Table 1, the knockdown of IQGAP3, HSF1, HSF2, PRRC2B, LSD1, POU5F1, ARPIN, RB1, ZZZ3, TP53, EWSR1, ALKBH5, BUB3, or RUVBL1 led to the downregulation of PRAC1 expression. Additionally, we found that the mRNA expression levels of PRAC1 were positively correlated with those of LSD1 in cancerous and non-cancerous prostate tissues (Fig. 3A; Table 1) and that the mRNA expression levels of LSD1 were upregulated in cancerous versus non-cancerous prostate tissues (Fig. 3B). These data suggest that LSD1 may promote PRAC1 expression in prostate cancer tissues.

Table 1.

Genes that can regulate PRAC1 expression.

pbgene	Method	Cell line	logFC	P value	*R value
IQGAP3	siRNA	NTERA-2 cl.D1	−8.184598395	9.16E-12	0.03
HSF1	siRNA	MCF 7.00	−5.067259342	3.41E-12	−0.2
HSF2	siRNA	MCF 7.00	−4.474306955	2.60E-05	−0.28
PRRC2B	shRNA	HEK293T	−3.979308572	3.44E-05	−0.15
LSD1	shRNA	A-673	−3.801029854	2.44E-121	0.27
POU5F1	shRNA	iPSCs	−3.787135872	1.15E-05	−0.3
ARPIN	shRNA	LoVo	−3.173206952	1.95E-08	0.089
RB1	shRNA	PC-3	−3.043327119	1.58E-06	−0.14
ZZZ3	siRNA	WA17	−2.609872037	2.89E-11	−0.04
TP53	shRNA	LNCaP clone FGC	−2.574998038	3.52E-142	0.083
RB1	shRNA	LNCaP C4-2	−2.39566212	1.92E-97	−0.14
EWSR1	siRNA	A-673	−2.138551717	4.62E-50	−0.24
ALKBH5	ko	hESCs	−2.126864494	5.03E-09	−0.16
BUB3	shRNA	LNCaP	−2.118112727	1.97E-30	0.06
EWSR1	siRNA	A-673	−2.082121605	6.99E-05	−0.24
RUVBL1	shRNA	BPH-1	−2.061111305	1.22E-19	0.089

* Correlation between the expression of PRAC1 and that of the indicated genes in cancerous and non-cancerous prostate tissues.

Fig. 3.

The expression of LSD1 is correlated with that of PRAC1 in prostate cancer tissues. (A) Pearson correlation analysis of PRAC1 and LSD1 mRNA expression levels in 492 cancerous and 52 non-cancerous prostate tissues. Data were downloaded from The Cancer Genome Atlas. (B) LSD1 mRNA levels in prostate cancer tissues and non-cancerous prostate tissues. Data were downloaded from The Cancer Genome Atlas. Red: prostate cancer tissue (n = 492); gray: non-cancerous prostate tissue (n = 52).

To investigate the regulatory role of LSD1 in PRAC1 expression, we knocked down LSD1 in prostate cancer cells, which inhibited PRAC1 expression (Fig. 4A–C and Supplementary Fig. 2A–C). Additionally, we found that LSD1 overexpression enhanced the promoter activity of PRAC1 and promoted PRAC1 expression (Fig. 4D). Furthermore, treatment with TAK-418, a specific LSD1 inhibitor, suppressed PRAC1 expression in a dose-dependent manner (Fig. 4E, F and Supplementary Fig. 2D, E). Taken together, these data indicate that LSD1 promotes PRAC1 expression in prostate cancer cells.

Fig. 4.

LSD1 promotes PRAC1 expression in prostate cancer cells. (A–C) LNCaP cells were transfected with control or LSD1 siRNAs. (A) LSD1 and PRAC1 mRNA levels were determined using reverse transcription quantitative PCR 48 h after transfection, (B) LSD1 protein levels were determined using western blotting 48 h after transfection, and (C) PRAC1 protein levels were determined using an enzyme-linked immunosorbent assay 48 h after transfection. (D) Luciferase activities of the PRAC1 promoter reporter in LNCaP cells transfected with a LSD1 expression plasmid or control plasmid. (E, F) PRAC1 mRNA (E) and protein (F) levels in LNCaP cells were determined using reverse transcription quantitative PCR and an enzyme-linked immunosorbent assay, respectively, 72 h after treatment with different doses of TAK-418. **P < 0.01 (two-sided Student’s t test).

TAK-418 represses prostate cancer cell proliferation by inhibitingPRAC1expression

Given that LSD1 promotes the expression of PRAC1 and that inhibiting PRAC1 expression suppresses the proliferation of prostate cancer cells, we investigated whether LSD1 inhibition can repress prostate cancer cell proliferation by suppressing PRAC1 expression. The specific knockdown of LSD1 in prostate cancer cells significantly inhibited cellular proliferation (Fig. 5A–D), whereas PRAC1 overexpression partially rescued this effect (Fig. 5E, F). Moreover, treatment with TAK-418 repressed prostate cancer cell proliferation (Fig. 6A, B); PRAC1 overexpression partially rescued this effect (Fig. 6C, D).

Fig. 5.

LSD1 knockdown represses prostate cancer cell proliferation by inhibiting PRAC1 expression. LNCaP (A) and DU145 (B) cells were transfected with control or LSD1 siRNAs, and cell proliferation assays were performed at different time points after transfection. (c–f) LNCaP cells were transfected with LSD1 siRNAs or co-transfected with LSD1 siRNAs and PRAC1-expressing plasmid; LSD1 protein (C) and mRNA (D) levels were determined using reverse transcription quantitative PCR and western blotting, respectively, at 48 h after transfection. (E) Protein levels of PRAC1 were determined using an enzyme-linked immunosorbent assay 48 h after transfection, and (F) cell proliferation assays were performed at different time points after transfection. Statistical analysis was performed using two-sided Student’s t-tests; *p < 0.05, ** p < 0.001.

Fig. 6.

TAK-418 represses prostate cancer cell proliferation by inhibiting PRAC1 expression. LNCaP (A) and DU145 (B) cells were treated with different concentrations of TAK-418, and cell proliferation assays were performed at different time points. (C, D) LNCaP cells were transfected with PRAC1-expressing plasmid or control plasmid and treated with or without 100 µM TAK-418. (C) Protein levels of PRAC1 were determined using enzyme-linked immunosorbent assay. (D) Cell proliferation assays were performed at different time points after transfection. Statistical analysis was performed using two-sided Student’s t-tests; *p < 0.05, ** p < 0.001.

Discussion

PRAC1 is specifically expressed in prostate, rectal, and distal colon tissues and plays a critical role in the maintenance and self-renewal of prostate epithelial stem cells. Notably, PRAC1 mRNA serves as a semen-specific biomarker for forensic identification, enabling the non-invasive detection of exfoliated prostate cancer cells in urine^4,7–9. In this study, we found that PRAC1 expression is upregulated in prostate cancer cells, and that PRAC1 knockdown represses prostate cancer cell proliferation. These findings identify PRAC1 as a potential therapeutic target in prostate cancer.

LSD1 is an evolutionarily conserved histone demethylase that dynamically regulates the chromatin structure and gene transcription by removing methyl groups from histone H3 lysine residues at positions 4 (H3K4) and 9 (H3K9) via oxidative reactions. As a core component of multiprotein complexes (e.g., CoREST/histone deacetylase complexes), LSD1 mediates transcriptional repression via H3K4 demethylation and participates in stem cell differentiation, embryonic development, and hematopoietic cell maturation^10,13–16. In nuclear receptor signaling pathways such as androgen receptor signaling, LSD1 removes repressive H3K9 methylation marks to activate target gene transcription and promote prostate cancer cell proliferation^17,18. LSD1 also exhibits non-histone substrate specificity, regulating the methylation status of proteins such as p53 and DNMT1 to influence DNA damage response and methylation homeostasis^17,19,20. The activity of LSD1 is dynamically regulated via post-translational modifications and combinatorial associations with corepressors, such as ZNF198 or REST^21,22. By integrating epigenetic modifications with signaling networks, LSD1 serves as a key spatiotemporal regulator of gene expression. Aberrant LSD1 activity contributes to tumorigenesis, viral latency, and other pathological processes^11,20,23. LSD1 also promotes cancer progression via multiple epigenetic and non-epigenetic mechanisms²⁴. In this study, we demonstrated that LSD1 promotes prostate cancer cell proliferation by upregulating PRAC1 expression.

TAK-418 is a reversible brain-penetrating inhibitor that selectively targets LSD1 to block histone H3K4me1/me2 demethylation. Structurally optimized to minimize the off-target effects on monoamine oxidases, TAK-418 exhibits superior safety to earlier LSD1 inhibitors, avoiding hematological toxicity^25–28. Although primarily investigated in neurodevelopmental disorders^{26,27,29–31}, the ability of TAK-418 to modulate cancer-related epigenetic plasticity makes it a promising candidate for precision oncology, particularly in tumors with LSD1 amplification or aberrant chromatin remodeling. In solid tumors, TAK-418 synergizes with DNA-demethylating agents or histone deacetylase inhibitors to enhance epigenetic reprogramming and overcome resistance to therapy^32,33. Early-phase trials explored the use of TAK-418 in combination with immune checkpoint inhibitors, leveraging LSD1 inhibition to enhance tumor immunogenicity by upregulating endogenous retroviral elements and antigen presentation³². We found that TAK-418 inhibits prostate cancer cell proliferation by repressing PRAC1 expression.

In conclusion, we found that LSD1 promotes prostate cancer cell proliferation by upregulating PRAC1 expression. Therefore, we propose PRAC1 or LSD1 inhibition as a promising therapeutic avenue for prostate cancer treatment.

Materials and methods

Cell culture and treatment

LNCaP and DU145 cells were obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China) and cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 1% antibiotics (100 U/mL penicillin and 100 µg/mL streptomycin sulfate; Sigma-Aldrich, Burlington, MA, USA) and 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA). The cells were maintained at 37 °C in a humidified incubator with 5% CO2. To inhibit LSD1 activity, the cells were exposed to different concentrations of TAK-418 (MedChemExpress, Monmouth Junction, NJ, USA). To induce apoptosis, the cells were treated with 0.5 µM Docetaxel (MedChemExpress).

Plasmids and siRNA transfection

The siRNAs were purchased from General Biol (Anhui, China). Cells were transfected with siRNAs using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. The siRNA sequences were as follows: si-PRAC1-1: 5’-CATCTTACTACCTCCAAGAGT-3’; si-PRAC1-2:5’-GCTCAGCCTGTAATTCTGGAA-3’; siLSD1-1:5’-TGAATTAGCTGAAACACAATT-3’; siLSD1-2:5’-GCCTAGACATTAAACTGAATA-3’.

Plasmid transfection was performed using Lipofectamine 8000 (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. The PRAC1 or LSD1 expression plasmid was engineered by cloning the full-length open reading frame of PRAC1 or LSD1 into the pCDH-CMV-MCS-EF1-Puro lentiviral vector. The shPRAC1-1 and shPRAC1-2 plasmids were generated by cloning hairpin oligonucleotides into the pLVX-shRNA2-puro vector, with targeting sequences identical to those of siPRAC1-1 and siPRAC1-2, respectively.

Colony formation

LNCaP-shNC, LNCaP-shPRAC1-1, and LNCaP-shPRAC1-2 cells were seeded in 6-well plates and cultured for 12 days. Wells were rinsed three times with PBS, and cell colonies were stained with a 0.5% crystal violet solution for 10 min. After air drying at room temperature, the plates were photographed.

Mouse xenograft tumor model

Female BALB/c athymic nude mice (6–8 weeks old; Vital River Experimental Animal Center, Beijing, China) were subcutaneously injected with 5 × 10⁶ cells to generate tumor xenografts. All animal procedures were approved by the Animal Care Committee of Chongqing Medical University and were performed in accordance with all relevant guidelines and regulations.

Reverse transcription and quantitative polymerase chain reaction

Total RNA was isolated using TRIzol reagent (Life Technologies), incubated with RNase-free DNase I (Promega, Madison, WI, USA) for 30 min, and reverse-transcribed using M-MLV Reverse Transcriptase (Promega). SYBR Green real-time polymerase chain reaction (PCR) was performed using the ChamQ Universal SYBR^® qPCR Master Mix (Vazyme Biotech Co., Ltd.) and ABI 7500 FAST sequence detection system (Life Technologies). The expression levels of all samples were normalized to the signal generated by glyceraldehyde-3-phosphate dehydrogenase. The primer sequences used were as follows: GAPDH: 5’-AACGGGAAGCTTGTCATCAA-3’ and 5’-TGGACTCCACGACGTACTCA-3’; LSD1: 5’-GTGGACGAGTTGCCACATTTC-3’ and 5’-TGACCACAGCCATAGGATTCC-3’; PRAC1: 5’-GCCCATTTCTCAGATCAAGG-3’ and 5’-GGTCTCGCCCAGTAGATGTT-3’.

Western blotting analysis

Cells were lysed using radioimmunoprecipitation assay buffer supplemented with protease inhibitor cocktail tablets (Roche, Basel, Switzerland), and the total protein content was measured using a BCA kit (Beyotime Biotechnology, Shanghai, China). Thirty to fifty micrograms of total protein were resolved using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a PVDF membrane (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was sequentially incubated with primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibody, followed by incubation with Clarity Western enhanced chemiluminescence substrate (Bio-Rad Laboratories). Protein signals were then visualized by exposing the membrane to X-ray films (FujiFilm, Tokyo, Japan) in a dark room. The primary antibodies used were anti-GAPDH (Abcam, Waltham, MA, USA) and anti-LSD1 (Abcam).

Cell proliferation assay

Cells were seeded into 96-well plates (2 × 103 cells/well), and cell proliferation was assessed using CCK-8 (MedChemExpress) and the BeyoClick™ EdU Cell Proliferation Kit (Beyotime Biotechnology) according to the manufacturers’ instruction.

Luciferase assays

Cells were co-transfected with 0.5 µg of firefly luciferase reporter vector, 0.05 µg of Renilla luciferase control vector (pRL-CMV), and 0.5 µg of LSD1 expression plasmid or empty vector using Lipofectamine 8000 in a 24-well plate. Luciferase assays were performed 48 h after transfection using the dual-luciferase reporter assay system (Promega). Firefly luciferase activity was normalized to the Renilla luciferase activity.

Cell migration and invasion assays

Cell migration and invasion were assessed using Transwell assays. Inserts with 8-µm pores, either uncoated (for migration) or coated with Matrigel (for invasion; Costar, High Wycombe, UK), were placed into wells of a 24-well plate. The lower chamber was filled with 600 µL of RPMI 1640 medium supplemented with 10% fetal bovine serum. LNCaP cells were washed once with Hank’s Balanced Salt Solution and resuspended in 100 µL of serum-free RPMI 1640 medium at a density of 1 × 10⁵ cells, which were then seeded into the upper chamber. Following an 18-h incubation at 37 °C at 5% CO₂, non-migrated cells on the upper surface of the membrane were carefully removed using a cotton swab. Cells that had migrated to the lower side of the membrane were fixed with cold methanol for 10 min and subsequently stained with 0.01% crystal violet in 20% ethanol.

Scratch wound healing assay

LNCaP cells were seeded in a culture plate and allowed to form a confluent monolayer. A straight scratch was then carefully created in the cell layer using a sterile pipette tip. The detached cells were washed away with phosphate-buffered saline (PBS), and fresh medium was added. The plate was placed in an incubator, and images of the scratch were captured at 0 and 24 h using a microscope.

Enzyme-Linked Immunosorbent Assays

Given its size of 6 kDa, the PRAC1 protein is difficult to detect using western blotting. Therefore, we performed enzyme-linked immunosorbent assays (ELISA) to measure PRAC1 protein expression. To this end, we coated the wells of a microtiter plate with anti-PRAC1 antibody (abx310485, Abbexa Ltd, Cambridge, UK) at a concentration of 10 µg/ml. After blocking, we added 100 µL of total cell lysate (1 µg/µL) to each well and incubated the plate at room temperature for 2 h. The plate was washed three times with PBS, 100 µL of HRP-conjugated anti-PRAC1 antibody was added (abx310486, Abbexa Ltd), and the plate was incubated at room temperature for 2 h. The plate was washed three times with PBS, and substrate working solution was prepared using the SignalUp™ Super Sensitive ELISA Assay Kit with Fluorescent HRP Substrate according to the manufacturer’s instructions (Beyotime Biotechnology). One hundred microliters of this substrate working solution was then added to each well, and the fluorescence intensity was measured using a microplate reader.

Gene expression analysis

The expression levels of PRAC1 and LSD1 and their correlation in cancerous and non-cancerous prostate tissues were retrieved from the Gene Expression Profiling Interactive Analysis (http://gepia.cancer-pku.cn/index.html) database³⁴. Genes potentially regulating PRAC1 expression were identified by searching the GPSAdb database (https://www.gpsadb.com/).

Statistical analysis

Data are presented as the mean ± SD of values from at least three independent experiments. Statistical analyses were performed using Student’s t-test. A p-value < 0.05 was considered to indicate statistically significant intergroup differences. Statistical analyses were conducted using GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA; RRID: SCR_002798).

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

YL performed the experiments, conducted data analysis, and drafted the manuscript. CL designed and supervised the study. All authors reviewed and approved the final manuscript.

Funding

No funding was received for this research.

Data availability

The datasets generated during and/or analyzed in the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.

Ethics declarations

This study did not involve human or animal subjects, thus ethical approval was not required.