Loss of MAOA in epithelia inhibits adenocarcinoma development, cell proliferation and cancer stem cells in prostate

Abstract

Monoamine oxidase A (MAOA) is a mitochondrial enzyme, which degrades monoamine neurotransmitters and dietary amines and produces H₂O₂. Recent studies have shown increased MAOA expression in prostate cancer (PCa), glioma, and classical Hodgkin lymphoma. However, the biological function of MAOA in cancer development remains unknown. In this study, we investigated the role of MAOA in the development of prostate adenocarcinoma by creating a prostate-specific Pten/MAOA knockout (KO) mouse model, in which MAOA-floxP mouse was crossed with the conditional Pten KO PCa mouse that develops invasive PCa. In contrast to Pten KO mice, age-matched Pten/MAOA KO mice exhibited a significant decrease in both prostate size and the incidence of invasive cancer. We observed a significant decline in AKT phosphorylation and Ki67 expression in Pten/MAOA KO mice, which reduced epithelial cell growth and proliferation. As cancer stem cells (CSCs) are required for tumor initiation and growth, we investigated expression of OCT4 and NANOG in the setting of decreased MAOA expression. We found that both OCT4 and NANOG were significantly attenuated in the prostate epithelia of Pten/MAOA KO mice compared to Pten KO mice, which was confirmed with targeted knockdown of MAOA with a short-hairpin(sh) vector targeting MAOA compared to cells transfected with a control vector. Expression of other markers associated with the a stem cell phenotype, including CD44, α2β1, and CD133 as well as HIF-1α⁺CD44⁺ stem cells were all decreased in shMAOA PCa cells compared with empty vector-transfected control cells. We also found spheroid formation ability in PCa cells was decreased when endogenous MAOA was suppressed by siRNA or MAOA inhibitor clorgyline in a colony formation assay. Using the TCGA database, elevated MAOA expression was associated with reduced Pten levels in high Gleason grade in patient samples. Further, we found that Pten-positive PCa cells were more resistant to clorgyline treatments than Pten-null cells in tumorigenicity and stemness. Taken together, these studies suggest that MAOA expression promotes PCa development by increasing cell proliferation and CSCs and highlights the potential use of MAOA inhibitors for the treatment of PCa.

Fig. 1

Generation of MAOA KO and Pten/MAOA KO mice. a Pten/MAOA KO mice were generated by crossing male Pten KO mice carrying Cre transgene and floxed Pten exon 5, with female MAOA KO mice carrying floxed-MAOA exon 12, which comprises the covalent FAD-binding site. b Schematic representation for ARR2PB-Cre and MAOA alleles. c Genotypes of mice were determined by PCR of genomic DNA isolated from both prostate and tail tissues. Upper: a 0.5-kb band represented the Cre gene in MAOA KO, Pten KO, and Pten/MAOA KO mice. Lower: the intact loxP-MAOA gene (1.5-kb band) was detected in the prostate and the tail of wild type and only in the tails of other three genotypes. The wild-type MAOA gene (1.2-kb band) was only detected in Pten KO. Finally, the MAOA recombinant (0.6-kb band) was detected in prostate in MAOA KO, Pten KO, and Pten/MAOA KO mice

Fig. 2

MAOA deletion attenuated the cancer morphology in Pten/MAOA KO prostates. Images of intact prostates dissected from all four genotypes at 4 and 6 months old were presented. a Ventral view. b Dorsal view. Prostate lobes were circled with dashed lines and marked. A anterior prostate, V ventral prostate, D dorsal–lateral prostate, B bladder, S seminal vesicle. Bar, 5 mm. Red arrow: blood vessels

Fig. 3

Histologic analysis of murine prostatic tissue sections. a H&E staining of paraffin-embedded DLP tissue sections dissected from all four genotypes at 4 and 6 months of age. Bar, 50 μm. Black arrow: invasive epithelial cells; red arrow: blood vessels. b, c The expression of PTEN (b) and MAOA (c) detected by IHC in 6-month-old DLP lobes of four genotypes (wild type, MAOA KO, Pten KO, and Pten/MAOA KO). Arrow: positive cells. Bar, 50 μm

Fig. 4

The distribution of three histological states, normal, PIN, and adenocarcinoma, in prostate lobes in each genotype at (a) 4 months and (b) 6 months of age. ▲: AP; ▽: VP; ■: DLP. n = 4 in each group, except in b Pten/MAOA KO, n = 5. Pten KO showed the presence of adenocarcinoma in all 4 mice in all prostate lobes (AP, VP, and DLP) at both 4 and 6 months of age. Pten/MAOA KO mice show PIN in all 4 mice in all prostate lobes at 4 months. Similar finding was observed in 6 months, except 1 mouse in VP showed adenocarcinoma out of 5 mice. c The incidence of adenocarcinoma in mouse from Pten KO and Pten/MAOA KO were compared. Data represent the mean ± SEM, *p < 0.01

Fig. 5

MAOA deletion in PCa suppressed AKT activity and cell proliferation but induced apoptosis in Pten/MAOA KO mice. IHC staining was performed on 6-month-old DLP sections collected from Pten KO and Pten/MAOA KO. a, c p-AKT-positive cells and proliferating prostate cells were identified by p-AKT (a) and Ki67 (c) antibodies, respectively. Black arrow: positive epithelial cell; red arrow: negative epithelial cell. Bar, 50 μm. e Detections of apoptotic cells by cleaved caspase-3 staining. Black arrow: cleaved caspase-3-positive epithelial cells. Bar, 50 μm. b, d, f Comparing the percentage of positive staining cells in epithelium between two genotypes. *p < 0.05. NS not significant

Fig. 6

MAOA deletion reduced cancer stem cell populations in epithelium in Pten/MAOA KO. a, c IHC images of (a) OCT4 and (c) NANOG staining on DLP, AP, and VP sections collected from 6-month-old Pten KO and DKO mice. Positive cells were detectable with nucleus staining. Black arrow: positive cells. Bar, 50 μm. b, d The quantitation of OCT4- and Nanog-positive stained epithelial cell in different prostate lobes in Pten/MAOA KO and Pten KO mice. Data represent the mean ± SD. *p < 0.05

Fig. 7

shMAOA PCa cell lines showed reduced stemness populations and cancer cell proliferation. a Reduced mRNA levels of Oct4 and Nanog in LNCaP-shMAOA compared to LNCaP-shControl. mRNA levels were determined by RT-PCR. b–d CD44 (b)-, α2β1 (c)-, and CD133 (d)-positive stem cell populations in LNCaP-shMAOA and -shControl cells determined by flow cytometry. Each panel includes both gating plot and quantification of positive cells in shMAOA and shControl cells. These experiments were performed at least three times. e, f Comparing proliferative stemness cell populations in LNCaP: Ki67⁺CD44⁺ cells (e) and Ki67⁺CD133⁺ cells (f). g, h Detection of HIF-1α⁺CD44⁺ stem cell populations in shMAOA as compared to shControl under hypoxia condition: LNCaP (g) and 22RV1 (h). Data represent the mean ± SD. *p < 0.05, **p < 0.01

Fig. 8

Loss of MAOA reduced the size of spheroid formed in 3D Matrigel cultures. a Representative bright-field images of spheroids formed from transformed LNCaP or 22RV1 (shControl or shMAOA) cells after 12 days of culture in 3D Matrigel. Bar, 100 μm. b, c Comparing the size (in diameter) (b) and number (c) of spheroid formed from two LNCaP cell lines. ***p < 0.001. d Representative images of spheroid forming of LNCaP cells with clorgyline treatments. Bar, 100 μm. e Comparing the number of LNCaP spheroid formed at different clorgyline concentrations. ***p < 0.001

Fig. 9

Patients with Gleason score 7 and above showed increase MAOA and decrease Pten expression. Two TCGA datasets with clinical annotation, including Gleason scores were used to interrogate the RNA expression of MAOA and Pten: a 236 patients from the dataset published in [50]; and b 491 patients from unpublished provisional PCa dataset [32, 33]. Data represent the mean ± SEM. *p < 0.05, **p < 0.01

Fig. 10

Clorgyline inhibitions in tumorigenesis and sternness were associated with Pten status in PCa cells. Comparing clorgyline treatments using PCa cells with Pten-null and -positive expressions. a 2D colony cultures of LNCaP (Pten-null) and 22RV1(Pten-positive) cells under treatments. Colonies were counted and compared (normalized by untreated controls). b Comparing cell viability of LNCaP and 22RV1 in MTT assay under treatments (normalized by untreated controls). Data represent the mean ± SD. *p < 0.05, **p < 0.01