Jason Boyang Wu

Regulation of monoamine oxidase A by the SRY gene on the Y chromosome

Monoamine oxidase A (MAO A), encoded by the X chromosome, catalyzes the oxidative deamination of monoamine neurotransmitters, such as serotonin, and plays a critically important role in brain development and functions. Abnormal MAO A activity has been implicated in several neuropsychiatric disorders, such as depression, autism, and attention deficit hyperactivity disorder, which show sexual dimorphism. However, the molecular basis for these disease processes is unclear. Recently, we found that MAO A was a putative target gene directly regulated by a transcription factor encoded by the sex‐determining region Y (SRY) gene located on the Y chromosome. We demonstrated that SRY activates both MAO A‐promoter and catalytic activities in a human male neuroblastoma BE(2)C cell line. A functional SRY‐binding site in the MAO A core promoter was identified and validated by electrophoretic mobility shift and chromatin immuno‐precipitation (ChIP) analyses. Coimmunoprecipitation and ChIP assays showed that SRY and Spl form a transcriptional complex and synergistically activate MAO A transcription. This is the first study demonstrating that the Y‐encoded transcription factor SRY is capable of regulating an X‐located gene, suggesting a novel molecular mechanism for sexual dimorphism in neural development, brain functions, and initiation/ progression of neural disorders associated with MAO A dysfunction.—Wu, J. B., Chen, K., Li, Y., Lau, Y.‐F. C., Shih, J. C. Regulation of monoamine oxidase A by the SRY gene on the Y chromosome. FASEB J. 23, 4029–4038 (2009). www.fasebj.org

Monoamine oxidase A mediates prostate tumorigenesis and cancer metastasis

Tumors from patients with high-grade aggressive prostate cancer (PCa) exhibit increased expression of monoamine oxidase A (MAOA), a mitochondrial enzyme that degrades monoamine neurotransmitters and dietary amines. Despite the association between MAOA and aggressive PCa, it is unclear how MAOA promotes PCa progression. Here, we found that MAOA functions to induce epithelial-to-mesenchymal transition (EMT) and stabilize the transcription factor HIF1α, which mediates hypoxia through an elevation of ROS, thus enhancing growth, invasiveness, and metastasis of PCa cells. Knockdown and overexpression of MAOA in human PCa cell lines indicated that MAOA induces EMT through activation of VEGF and its coreceptor neuropilin-1. MAOA-dependent activation of neuropilin-1 promoted AKT/FOXO1/TWIST1 signaling, allowing FOXO1 binding at the TWIST1 promoter. Importantly, the MAOA-dependent HIF1α/VEGF-A/FOXO1/TWIST1 pathway was activated in high-grade PCa specimens, and knockdown of MAOA reduced or even eliminated prostate tumor growth and metastasis in PCa xenograft mouse models. Pharmacological inhibition of MAOA activity also reduced PCa xenograft growth in mice. Moreover, high MAOA expression in PCa tissues correlated with worse clinical outcomes in PCa patients. These findings collectively characterize the contribution of MAOA in PCa pathogenesis and suggest that MAOA has potential as a therapeutic target in PCa.

Monoamine Oxidase A Inhibitor – Near-Infrared Dye Conjugate Reduces Prostate Tumor Growth

Development of anti-cancer agents with high tumor-targeting specificity and efficacy is critical for modern multidisciplinary cancer research. Monoamine oxidase A (MAOA), a mitochondria-bound enzyme, degrades monoamine neurotransmitters and dietary monoamines. Recent evidence suggests a correlation between increased MAOA expression and prostate cancer (PCa) progression with poor outcomes for patients. MAOA induces epithelial-mesenchymal transition (EMT) and augments hypoxic effects by producing excess reactive oxygen species. Thus, development of MAOA inhibitors which selectively target tumors becomes an important goal in cancer pharmacology. Here we describe the design, synthesis, and in vitro and in vivo evaluation of NMI, a conjugate that combines a near-infrared dye for tumor targeting with the moiety derived from the MAOA inhibitor clorgyline. NMI inhibits MAOA with low micromolar IC50, suppresses PCa cell proliferation and colony formation, and reduces migration and invasion. In mouse PCa xenografts, NMI targets tumors with no detectable accumulation in normal tissues, providing effective reduction of the tumor burden. Analysis of tumor specimens shows reduction in Ki-67(+) and CD31(+) cells, suggesting a decrease of cell proliferation and angiogenesis and an increase in M30(+) cells, indicating increased apoptosis. Gene expression profiles of tumors treated with NMI demonstrate reduced expression of oncogenes FOS, JUN, NFKB, and MYC and cell cycle regulators CCND1, CCNE1, and CDK4/6, along with increases in the levels of tumor suppressor gene TP53, cell cycle inhibitors CDKN1A and CDKN2A, and MAOA-downstream genes that promote EMT, tumor hypoxia, cancer cell migration, and invasion. These data suggest that NMI exerts its effect through tumor-targeted delivery of a MAOA-inactivating group, making NMI a valuable anti-tumor agent.

Review on near-infrared heptamethine cyanine dyes as theranostic agents for tumor imaging, targeting, and photodynamic therapy

Abstract. A class of near-infrared fluorescence (NIRF) heptamethine cyanine dyes that are taken up and accumulated specifically in cancer cells without chemical conjugation have recently emerged as promising tools for tumor imaging and targeting. In addition to their fluorescence and nuclear imaging-based tumor-imaging properties, these dyes can be developed as drug carriers to safely deliver chemotherapy drugs to tumors. They can also be used as effective agents for photodynamic therapy with remarkable tumoricidal activity via photodependent cytotoxic activity. The preferential uptake of dyes into cancer but not normal cells is co-operatively mediated by the prevailing activation of a group of organic anion-transporting polypeptides on cancer cell membranes, as well as tumor hypoxia and increased mitochondrial membrane potential in cancer cells. Such mechanistic explorations have greatly advanced the current application and future development of NIRF dyes and their derivatives as anticancer theranostic agents. This review summarizes current knowledge and emerging advances in NIRF dyes, including molecular characterization, photophysical properties, multimodal development and uptake mechanisms, and their growing potential for preclinical and clinical use.

Near-infrared fluorescence imaging of cancer mediated by tumor hypoxia and HIF1α/OATPs signaling axis.

Near-infrared fluorescence (NIRF) imaging agents are promising tools for noninvasive cancer imaging. Here, we explored the mechanistic properties of a specific group of NIR heptamethine carbocyanines including MHI-148 dye we identified and synthesized, and demonstrated these dyes to achieve cancer-specific imaging and targeting via a hypoxia-mediated mechanism. We found that cancer cells and tumor xenografts exhibited hypoxia-dependent MHI-148 dye uptake inxa0vitro and inxa0vivo, which was directly mediated by hypoxia-inducible factor 1α (HIF1α). Microarray analysis and dye uptake assay further revealed a group of hypoxia-inducible organic anion-transporting polypeptides (OATPs) responsible for dye uptake, and the correlation between OATPs and HIF1α was manifested in progressive clinical cancer specimens. Finally, we demonstrated increased uptake of MHI-148 dye in situ in perfused clinical tumor samples with activated HIF1α/OATPs signaling. Our results establish these NIRF dyes as potential tumor hypoxia-dependent cancer-targeting agents and provide a mechanistic rationale for continued development of NIRF imaging agents for improved cancer detection, prognosis and therapy.

Retinoic Acid Activates Monoamine Oxidase B Promoter in Human Neuronal Cells

Monoamine oxidase (MAO) B deaminates a number of biogenic and dietary amines and plays an important role in many biological processes. Among hormonal regulations of MAO B, we have recently found that retinoic acid (RA) significantly activates both MAO B promoter activity and mRNA expression in a human neuroblastoma BE(2)C cell line. RA activates MAO B promoter in both concentration- and time-dependent manners, which is mediated through retinoic acid receptor α (RARα) and retinoid X receptor α (RXRα). There are four retinoic acid response elements (RAREs) as identified in the MAO B 2-kb promoter, and mutation of the third RARE reduced RA-induced MAO B promoter activation by 50%, suggesting this element is important. Electrophoretic mobility shift analysis and chromatin immunoprecipitation assay demonstrated that RARα specifically binds to the third RARE both in vitro and in vivo. Moreover, transient transfection and luciferase assays revealed that Sp1 enhances but not essentially required for the RA activation of MAO B through two clusters of Sp1-binding sites in the MAO B promoter. RARα physically interacts with Sp1 via zinc finger domains in Sp1 as determined by co-immunoprecipitation assay. Further, RARα was shown to be recruited by Sp1 and to form a transcriptional regulation complex with Sp1 in the Sp1-binding sites of natural MAO B promoter. Taken together, this study provides evidence for the first time showing the stimulating effect of RA on MAO B and new insight into the molecular mechanisms of MAO B regulation by hormones.

Transcription Factor E2F-Associated Phosphoprotein (EAPP), RAM2/CDCA7L/JPO2 (R1), and Simian Virus 40 Promoter Factor 1 (Sp1) Cooperatively Regulate Glucocorticoid Activation of Monoamine Oxidase B

Glucocorticoid steroid hormones play important roles in many neurophysiological processes such as responses to stress, behavioral adaption, and mood. One mechanism by which glucocorticoids exert functions in the brain is via the modulation of neurotransmission systems. Glucocorticoids are capable of inducing the activities of monoamine oxidases (MAOs), which degrade monoamine neurotransmitters including serotonin, norepinephrine, phenylethylamine, and dopamine. However, the molecular mechanisms for such induction are not yet fully understood. Here, we report that dexamethasone, a synthetic glucocorticoid hormone, stimulates MAO B (an isoform of MAOs) promoter and catalytic activities via both the fourth glucocorticoid response element (GRE) and simian virus 40 promoter factor 1 (Sp1) binding sites in MAO B promoter. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation analysis demonstrated that glucocorticoid receptor binds to the fourth GRE in vitro and in vivo. Using Sp1-binding motifs as bait in a yeast one-hybrid system, we identified two novel transcriptional repressors of MAO B, E2F-associated phosphoprotein (EAPP) and R1 (RAM2/CDCA7L/JPO2), that down-regulate MAO B via MAO B core promoter, which contains Sp1 sites. EMSA suggested that EAPP and R1 competed with Sp1 for binding to the Sp1 site in vitro. Moreover, EAPP and R1 reduced Sp1-activated glucocorticoid activation of MAO B promoter. In response to dexamethasone, lower occupancy by EAPP and R1 and higher occupancy by Sp1 were shown at the natural MAO B core promoter. Together, this study uncovers for the first time the molecular mechanisms for glucocorticoid activation of MAO B gene and provides new insights into the hormonal regulation of MAO.

Near-infrared fluorescence heptamethine carbocyanine dyes mediate imaging and targeted drug delivery for human brain tumor

Brain tumors and brain metastases are among the deadliest malignancies of all human cancers, largely due to the cellular blood-brain and blood-tumor barriers that limit the delivery of imaging and therapeutic agents from the systemic circulation to tumors. Thus, improved strategies for brain tumor visualization and targeted treatment are critically needed. Here we identified and synthesized a group of near-infrared fluorescence (NIRF) heptamethine carbocyanine dyes and derivative NIRF dye-drug conjugates for effective imaging and therapeutic targeting of brain tumors of either primary or metastatic origin in mice, which is mechanistically mediated by tumor hypoxia and organic anion-transporting polypeptide genes. We also demonstrate that these dyes, when conjugated to chemotherapeutic agents such as gemcitabine, significantly restricted the growth of both intracranial glioma xenografts and prostate tumor brain metastases and prolonged survival in mice. These results show promise in the application of NIRF dyes as novel theranostic agents for the detection and treatment of brain tumors.

Combined cell surface carbonic anhydrase 9 and CD147 antigens enable high-efficiency capture of circulating tumor cells in clear cell renal cell carcinoma patients

Circulating tumor cells (CTCs) have emerged as promising tools for noninvasive cancer detection and prognosis. Most conventional approaches for capturing CTCs use an EpCAM-based enrichment strategy, which does not work well in cancers that show low or no expression of EpCAM, such as renal cell carcinoma (RCC). In this study, we developed a new set of cell surface markers including CA9 and CD147 as alternative CTC-capture antigens specifically designed for RCC patients. We showed that the expression of both CA9 and CD147 was prevalent in a RCC patient cohort (n=70) by immunohistochemical analysis, with both molecules in combination covering 97.1% of cases. The NanoVelcro platform combined with CA9-/CD147-capture antibodies demonstrated significantly higher efficiency for capturing both CTC-mimicking renal cancer cells and RCC CTCs in peripheral blood, compared to the conventional EpCAM-based method. Using immunofluorescence cytological validation at the single-cell level, we were able to identify bona fide CTCs in RCC patient blood following the well-accepted criteria in our CTC-capture system. We further demonstrated a significant association of CTC numbers as well as the CTC expression status of Vimentin, a mesenchymal marker, with disease progression, including pathologic features and clinical staging. These results provide new insights into developing novel, effective targets/approaches for capturing CTCs, making CTCs a valuable tool for improved cancer detection, prognosis and treatment in RCC.

SREBP-2 promotes stem cell-like properties and metastasis by transcriptional activation of c-Myc in prostate cancer.

Sterol regulatory element-binding protein-2 (SREBP-2) transcription factor mainly controls cholesterol biosynthesis and homeostasis in normal cells. The role of SREBP-2 in lethal prostate cancer (PCa) progression remains to be elucidated. Here, we showed that expression of SREBP-2 was elevated in advanced pathologic grade and metastatic PCa and significantly associated with poor clinical outcomes. Biofunctional analyses demonstrated that SREBP-2 induced PCa cell proliferation, invasion and migration. Furthermore, overexpression of SREBP-2 increased the PCa stem cell population, prostasphere-forming ability and tumor-initiating capability, whereas genetic silencing of SREBP-2 inhibited PCa cell growth, stemness, and xenograft tumor growth and metastasis. Clinical and mechanistic data showed that SREBP-2 was positively correlated with c-Myc and induced c-Myc activation by directly interacting with an SREBP-2-binding element in the 5′-flanking c-Myc promoter region to drive stemness and metastasis. Collectively, these clinical and experimental results reveal a novel role of SREBP-2 in the induction of a stem cell-like phenotype and PCa metastasis, which sheds light on translational potential by targeting SREBP-2 as a promising therapeutic approach in PCa.