Monoamine oxidase isoenzymes: genes, functions and targets for behavior and cancer therapy

Abstract

Monoamine oxidase (MAO) catalyzes the oxidative deamination of monoamine neurotransmitters and dietary amines. Two pharmacological types with different substrate and inhibitor specificities were reported. Molecular cloning revealed that the two types of MAO were different genes expressed as different proteins with different functions. MAO A and B have identical intron-exon organization derived by duplication of a common ancestral gene thus they are termed isoenzymes. MAO A knockout mice exhibited aggression, the first clear evidence linking genes to behavior. MAO A KO mice exhibited autistic-like behaviors which could be prevented by reducing serotonin levels at an early developmental age (P1-P7) providing potential therapy. MAO B KO mice were non-aggressive and resistant to Parkinsongenic neurotoxin. More recently it was found that MAO A is overexpressed in prostate cancer and correlates with degree of malignancy. The oncogenic mechanism involves a ROS-activated AKT/FOXO1/TWIST1 signaling pathway. Deletion of MAO A reduced prostate cancer stem cells and suppressed invasive adenocarcinoma. MAO A was also overexpressed in classical Hodgkin lymphoma and glioma brain tumors. MAO B was overexpressed in glioma and non-small cell lung cancer. MAO A inhibitors reduce the growth of prostate cancer, drug sensitive and resistant gliomas and classical Hodgkin lymphoma, and enhance standard chemotherapy. Currently, we are developing NIR dye-conjugated clorgyline (MAO A inhibitor) as a novel dual therapeutic/diagnostic agent for cancer. A phase II clinical trial of MAO inhibitor for biochemical recurrent prostate cancer is ongoing. The role of MAO A and B in several cancer types opens new avenues for cancer therapies.

Keywords: Autism; Behavior; Cancer; Gene; Isoenzyme; Metastasis; Monoamine oxidase; Survival; Therapy.

Fig. 1. The alignment of deduced polypeptide sequences shows that MAO A and B are encoded by different genes with 70% amino acid identity with identical intron-exon organizations and thus termed isoenzymes.

Amino acids are numbered on the left side. Identical amino acids are indicated by asterisks. Dashes mark spaces introduced for maximum alignment of identical residues. Pink or blue shade denotes amino acids in each exon. Identical or divergent amino acids at the 5’ or 3’ boundary of the introns are indicated by bold asterisks or filled circles, respectively. Triangles denote potential glycosylation sites. (Adapted from Bach et al., 1988; Grimsby et al., 1991)

Fig. 2. Substrate and inhibitor profiles of human MAO A and B expressed from cDNAs.

(A) MAO activity in transfected COS cells. The human MAO A and B cDNAs containing the full coding sequences of MAO A and B were cloned into pECE vector in either the sense or antisense orientation and expressed under the control of SV40 early promoter. The resultant plasmid was transiently transfected into COS cells and the MAO A and B activities in the lysates were determined using [¹⁴C]serotonin or [¹⁴C]phenylethylamine as the substrate for MAO A or B, respectively. (B) Inhibition of transiently expressed MAO A and B by clorgyline (○) or deprenyl (●). The lysate of COS cells transfected with either human MAO A or B cDNA were assayed in the presence of various concentrations of inhibitors. The inhibition of the expressed MAO A and B by clorgyline and deprenyl was determined by preincubating the inhibitors with the whole-cell homogenates at 37°C for 1 h before addition of the substrates. (Adapted from Lan et al., 1989a)

Fig. 3. Partial structural map of the MAO A and MAO B genes showing the location of exons and sequencing strategy.

Filled bars represent coding regions, and unfilled bars represent untranslated regions of the exons. Exon numbers are presented below the bars. The horizontal arrows represent the regions sequenced. The prefix “A” denotes λ bacteriophage clones, and the prefix “p” denotes pUC19 subclones of λ bacteriophage DNA. E and H, EcoRI and HindIII restriction sites, respectively; “⇊”, equivocal assignment of an exon to either restriction fragment; “//”, intron gap. (Adapted from Grimsby et al, 1991)

Fig. 4. pCPA treatment during P1 to P7 rescued the autistic-like behaviors in adult MAO A KO mice.

Effects of pCPA treatment from postnatal day 1 through 7 on (A) marble-burying behavior and (B) spontaneous alternations in a T-maze of MAO A KO mice. pCPA, an inhibitor of trypophan hydroxylase that reduces 5-HT level, was injected (30 mg/kg/day, IP) daily from P1 to P7. Values represent mean ±SEM; *P<0.05, ***P<0.001 compared to untreated wild-type mice; ^#P<0.05, ^###P<0.001 compared to untreated MAO A deficient mice; ^†P<0.05, ^††P<0.01 compared to untreated saline-treated MAO A KO mice; ^δδP<0.01, ^δP<0.05 compared to saline treated wild-type mice. (Adapted from Bortolato et al., 2013b)

Fig. 5. Elevated expression of MAO A in various human cancers.

(A) High Gleason grade prostate cancers associated with increased MAO A expression. Normal prostatic epithelium and Gleason grade 3 and 5 prostate cancer specimens from a tissue microarray were stained for MAO A. Magnification, 400x; bars, 20 μm. (Adapted from Wu et al., 2014) (B) MAO A expression is increased in human glioma. Non-malignant brain and glioma tissue specimens. Scale bars represent 100 μm (Adapted from Kushal et al., 2016) (C) MAO A is highly expressed by Hodgkin/Reed–Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL). MAO A was detected by immunohistochemistry on formalin-fixed, paraffin-embedded (FFPE) patient material. Double staining for CD30 (red) and MAO A (brown) confirms the localization of MAO A in HRS cells (original magnification 1000×) (Adapted from Li et al., 2017)

Fig. 6. A proposed mechanism that MAO A regulates prostate tumorigenesis, progression, and metastasis.

MAO A generates ROS that inhibits PHD activity and stabilizes HIF1α to induce epithelial to mesenchymal transition. Generation of ROS by MAO A is elevated by hypoxic conditions that are characteristic of the tumor microenvironment. MAO A’s activation of VEGF-A/NRP1 signaling upregulates the AKT/FOXO1 pathway, resulting in the nuclear export of FOXO1 transcription repressor to increase the expression of nuclear TWIST1. Overall, increasing the MAO A expression promotes epithelial to mesenchymal transition, hypoxia and ROS production to drive prostate tumorigenesis, progression and metastasis. (Adapted from Wu et al., 2014)

Fig. 7. Deletion of MAO A gene suppresses the initiation of prostate cancer.

Immunohistochemistry stain for the cell proliferation marker Ki67 detected high percentage of proliferative cells in Pten KO mouse prostate tissue whereas significantly less cells express Ki67 in Pten/MAO A KO mice. (Arrows point to Ki67⁺ cells; Bar: 100 μm) (Adapted from Liao et al., 2018)

Fig. 8. MAO A gene knockdown in human prostate cancer cells (LNCaP) significantly reduced cultured stem cell spheroid size and number.

Light microscope view of spheroid culture with prostate cancer cells expressing a scrambled shRNA (shControl, left side panel) versus a MAO A-targeting shRNA (shMAO A, right side panel). Loss of MAO A gene function reduced the size of spheroids formed after 12 days of culture in 3D Matrigel. Bar, 100 μm. (Adapted from Liao et al., 2018)

Fig. 9. Clorgyline, NMI or the combination with TMZ increases the survival of TMZ-resistant tumors.

Athymic/nude mice were implanted intracranially with TMZ-resistant human glioma cells (U251R). Animals were then treated daily with: vehicle, TMZ (1 mg/kg), clorgyline (10 mg/kg), TMZ (1 mg/kg) + clorgyline (10 mg/kg), NMI (5 mg/kg), or TMZ (1 mg/kg) + NMI (5 mg/kg). MAO A inhibitors were administered subcutaneously daily for 21 days; TMZ treatment was given for the first 10 days only. (A) The Kaplan-Meier survival curve shows that clorgyline significantly prolonged survival. (B) The KaplanMeier survival curve for NMI and NMI+TMZ shows that NMI was effective alone as compared to the vehicle, and the combination of NMI and TMZ was significantly more effective (*p < 0.001). (Adapted from Kushal et al., 2016)

Fig. 10. MAO A inhibitor-near infrared dye conjugate inhibits the growth of hu5man prostate tumor xenograft in mice.

The structure of NIR dye−MAO A inhibitor conjugate (NMI) comprised of clorgyline and a near-infrared (NIR) dye is shown. (upper right panel) In vivo NIR imaging of the whole body clearly shows NMI localization within the tumor. Mice bearing C4–2B tumor xenografts were intraperitoneally injected with NMI (50 nmol/mouse) and subjected to NIR imaging 24 h later. (lower right panel) NMI inhibits the growth of C4–2B tumor xenografts in mice. C4–2B cells were subcutaneously injected into male nude mice (n = 5 for each group) to establish tumor xenografts. After tumor size reached 200 mm³, mice were given intratumoral treatments (saline and NMI; 750 nmol/mouse) every other day for a 21-day period. (Adapted from Wu et al., 2015)