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Figure S1: R1 does not affect contact inhibition during prostate cancer cell proliferation; Figure S2: R1 does not affect cell apoptosis in prostate cancer cells under both steady state and dynamic conditions; Figure S3: R1 promotes prostate cancer cell migration and invasion; Figure S4: R1/c-Myc physical interaction is not required for R1 regulation of c-Myc ubiquitination; Figure S5: R1 does not affect MIZ-1 and p53 protein expression in prostate cancer cells.

Androgen receptor (AR) is the primary oncogenic driver of prostate cancer, including aggressive castration-resistant prostate cancer (CRPC). The molecular mechanisms controlling AR activation in general and AR reactivation in CRPC remain elusive. Here we report that monoamine oxidase A (MAOA), a mitochondrial enzyme that degrades monoamine neurotransmitters and dietary amines, reciprocally interacts with AR in prostate cancer. MAOA was induced by androgens through direct AR binding to a novel intronic androgen response element of the MAOA gene, which in turn promoted AR transcriptional activity via upregulation of Shh/Gli-YAP1 signaling to enhance nuclear YAP1–AR interactions. Silencing MAOA suppressed AR-mediated prostate cancer development and growth, including CRPC, in mice. MAOA expression was elevated and positively associated with AR and YAP1 in human CRPC. Finally, genetic or pharmacologic targeting of MAOA enhanced the growth-inhibition efficacy of enzalutamide, darolutamide, and apalutamide in both androgen-dependent and CRPC cells. Collectively, these findings identify and characterize an MAOA–AR reciprocal regulatory circuit with coamplified effects in prostate cancer. Moreover, they suggest that cotargeting this complex may be a viable therapeutic strategy to treat prostate cancer and CRPC.Significance:MAOA and AR comprise a positive feedback loop in androgen-dependent and CRPC, providing a mechanistic rationale for combining MAOA inhibition with AR-targeted therapies for prostate cancer treatment.

This file include 11 Supplementary Figures, Supplementary Materials and Methods, and Supplementary References.

Emerging studies, including ours, have revealed the novel essential roles of monoamine oxidase A (MAO A) in mediating the growth and progression of several types of cancers. Recently, we presented the first evidence of MAO A’s ability to promote cancer cell perineural invasion, the neoplastic invasion of nerves widely recognized as a significant route for cancer metastasis. Here, we describe a perineural invasion in vitro assay using a 3D co-culture with a cancer cell line and an immortalized dorsal root ganglion neuronal cell line for rapid examination of MAO A’s roles in cancer-nerve cell crosstalk and evaluating the efficacy of MAO A inhibitors for disrupting perineural invasion. We also summarized the fundamental methods for determining MAO A’s effects on cancer cell proliferation in vitro and tumorigenesis in vivo.

Androgen receptor (AR) is the primary oncogenic driver of prostate cancer, including aggressive castration-resistant prostate cancer (CRPC). The molecular mechanisms controlling AR activation in general and AR reactivation in CRPC remain elusive. Here we report that monoamine oxidase A (MAOA), a mitochondrial enzyme that degrades monoamine neurotransmitters and dietary amines, reciprocally interacts with AR in prostate cancer. MAOA was induced by androgens through direct AR binding to a novel intronic androgen response element of the MAOA gene, which in turn promoted AR transcriptional activity via upregulation of Shh/Gli-YAP1 signaling to enhance nuclear YAP1–AR interactions. Silencing MAOA suppressed AR-mediated prostate cancer development and growth, including CRPC, in mice. MAOA expression was elevated and positively associated with AR and YAP1 in human CRPC. Finally, genetic or pharmacologic targeting of MAOA enhanced the growth-inhibition efficacy of enzalutamide, darolutamide, and apalutamide in both androgen-dependent and CRPC cells. Collectively, these findings identify and characterize an MAOA–AR reciprocal regulatory circuit with coamplified effects in prostate cancer. Moreover, they suggest that cotargeting this complex may be a viable therapeutic strategy to treat prostate cancer and CRPC. Significance: MAOA and AR comprise a positive feedback loop in androgen-dependent and CRPC, providing a mechanistic rationale for combining MAOA inhibition with AR-targeted therapies for prostate cancer treatment.

Neuroendocrine prostate cancer (NEPC), a lethal subset of prostate cancer, is characterized by loss of AR signaling and resulting resistance to AR-targeted therapy during neuroendocrine transdifferentiation, for which the molecular mechanisms remain unclear. Here, we report that neuropilin 2 (NRP2) is upregulated in both de novo and therapy-induced NEPC, which induces neuroendocrine markers, neuroendocrine cell morphology, and NEPC cell aggressive behavior. NRP2 silencing restricted NEPC tumor xenograft growth. Mechanistically, NRP2 engages in reciprocal crosstalk with AR, where NRP2 is transcriptionally inhibited by AR, and in turn suppresses AR signaling by downregulating the AR transcriptional program and confers resistance to enzalutamide. Moreover, NRP2 physically interacts with VEGFR2 through the intracellular SEA domain to activate STAT3 phosphorylation and subsequently SOX2, thus driving NEPC differentiation and growth. Collectively, these results characterize NRP2 as a driver of NEPC and suggest NRP2 as a potential therapeutic target in NEPC.

Prostate cancer is a prevalent public health problem, especially because noncutaneous advanced malignant forms significantly affect the lifespan and quality of life of men worldwide. New therapeutic targets and approaches are urgently needed. The current study reports elevated expression of R1 (CDCA7L/RAM2/JPO2), a c-Myc–interacting protein and transcription factor, in human prostate cancer tissue specimens. In a clinical cohort, high R1 expression is associated with disease recurrence and decreased patient survival. Overexpression and knockdown of R1 in human prostate cancer cells indicate that R1 induces cell proliferation and colony formation. Moreover, silencing R1 dramatically reduces the growth of prostate tumor xenografts in mice. Mechanistically, R1 increases c-Myc protein stability by inhibiting ubiquitination and proteolysis through transcriptional suppression of HUWE1, a c-Myc–targeting E3 ligase, via direct interaction with a binding element in the promoter. Moreover, transcriptional repression is supported by a negative coexpression correlation between R1 and HUWE1 in a prostate cancer clinical dataset. Collectively, these findings, for the first time, characterize the contribution of R1 to prostate cancer pathogenesis. Implications: These findings provide evidence that R1 is a novel regulator of prostate tumor growth by stabilizing c-Myc protein, meriting further investigation of its therapeutic and prognostic potential.

G-protein-coupled receptors (GPCRs), especially chemokine receptors, are ideal targets for monoclonal antibody drugs. Considering the special multi-pass transmembrane structure of GPCR, it is often a laborious job to obtain antibody information about off-targets and epitopes on antigens. To accelerate the process, a rapid and simple method needs to be developed. The split-ubiquitin-based yeast two hybrid system (YTH) was used as a blue script for a new method. By fusing with transmembrane peptides, scFv antibodies were designed to be anchored on the cytomembrane, where the GPCR was co-displayed as well. The coupled split-ubiquitin system transformed the scFv-GPCR interaction signal into the expression of reporter genes. By optimizing the topological structure of scFv fusion protein and key elements, including signal peptides, transmembrane peptides, and flexible linkers, a system named Antigen-Antibody Co-Display (AACD) was established, which rapidly detected the interactions between antibodies and their target GPCRs, CXCR4 and CXCR5, while also determining the off-target antibodies and antibody-associated epitopes. The AACD system can rapidly determine the association between GPCRs and their candidate antibodies and shorten the research period for off-target detection and epitope identification. This system should improve the process of GPCR antibody development and provide a new strategy for GPCRs antibody screening.

Perineural invasion (PNI), a pathologic feature defined as cancer cell invasion in, around, and through nerves, is an indicator of poor prognosis and survival in prostate cancer (PC). Despite widespread recognition of the clinical significance of PNI, the molecular mechanisms are largely unknown. Here, we report that monoamine oxidase A (MAOA) is a clinically and functionally important mediator of PNI in PC. MAOA promotes PNI of PC cells in vitro and tumor innervation in an orthotopic xenograft model. Mechanistically, MAOA activates SEMA3C in a Twist1-dependent transcriptional manner, which in turn stimulates cMET to facilitate PNI via autocrine or paracrine interaction with coactivated PlexinA2 and NRP1. Furthermore, MAOA inhibitor treatment effectively reduces PNI of PC cells in vitro and tumor-infiltrating nerve fiber density along with suppressed xenograft tumor growth and progression in mice. Collectively, these findings characterize the contribution of MAOA to the pathogenesis of PNI and provide a rationale for using MAOA inhibitors as a targeted treatment for PNI in PC.

Monoamine oxidases (MAOs) degrade a number of biogenic and dietary amines, including monoamine neurotransmitters, and play an essential role in many biological processes. Neurotransmitters and related neural events have been shown to participate in the development, differentiation, and maintenance of diverse tissues and organs by regulating the specialized cellular function and morphological structures of innervated organs such as the prostate. Here we show that mice lacking both MAO isoforms, MAOA and MAOB, exhibit smaller prostate mass and develop epithelial atrophy in the ventral and dorsolateral prostates. The cellular composition of prostate epithelium showed reduced CK5+ or p63+ basal cells, accompanied by lower Sca-1 expression in p63+ basal cells, but intact differentiated CK8+ luminal cells in MAOA/B-deficient mouse prostates. MAOA/B ablation also decreased epithelial cell proliferation without affecting cell apoptosis in mouse prostates. Using a human prostate epithelial cell line, we found that stable knockdown of MAOA and MAOB impaired the capacity of prostate stem cells to form spheres, coinciding with a reduced CD133+/CD44+/CD24− stem cell population and less expression of CK5 and select stem cell markers, including ALDH1A1, TROP2, and CD166. Alternative pharmacological inhibition of MAOs also repressed prostate cell stemness. In addition, we found elevated expression of MAOA and MAOB in epithelial and/or stromal components of human prostate hyperplasia samples compared with normal prostate tissues. Taken together, our findings reveal critical roles for MAOs in the regulation of prostate basal progenitor cells and prostate maintenance.

// Jiaze An 1, 2, * , Ningning Zhao 1, * , Caiqin Zhang 1 , Yong Zhao 1 , Dengxu Tan 1 , Ya Zhao 1 , Bing Bai 1 , Hai Zhang 1 , Boyang Jason Wu 3, ** and Changhong Shi 1, ** 1 Laboratory Animal Center, The Fourth Military Medical University, Xi’an, Shaanxi 710032, China 2 Department of Hepatobiliary and Pancreaticosplenic Surgery, Xijing Hospital, The Fourth Military Medical University, Xi’an, Shaanxi 710032, China 3 Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Spokane, WA 99210, USA * These authors contributed equally to this work ** Joint corresponding authors Correspondence to: Changhong Shi, email: changhong@fmmu.edu.cn Boyang Jason Wu, email: boyang.wu@wsu.edu Keywords: heptamethine carbocyanine, hepatocellular carcinoma, HIF1α, near-infrared fluorescence, organic anion-transporting polypeptide Received: February 28, 2017 Accepted: April 17, 2017 Published: May 24, 2017 ABSTRACT Near-infrared fluorescence (NIRF) dyes have recently emerged as promising tools for non-invasive imaging of different types of cancers. Here, we explored the potential utility of a NIRF DZ-1 dye, with dual imaging and tumour targeting functions, in hepatocellular carcinoma (HCC). We showed the preferential uptake of DZ-1 by HCC cells in vitro and in derived subcutaneous/orthotopic tumour xenografts, accompanied by a minimal effect on normal cells. DZ-1 simplified tumour growth profiling as well, since we were able to correlate NIRF signals with tumour volume and/or tumour-emitting luminescence in mice. Using both orthotopic tumour transplantation and cirrhosis models in parallel, we demonstrated the ability of DZ-1 to differentiate liver tumour from cirrhosis. DZ-1 showed superiority in HCC imaging over indocyanine green by demonstrating significantly enhanced tumour-targeting specificity. At the cellular level, DZ-1 was mainly retained in mitochondria and lysosomes. Additionally, DZ-1 fluorescence spectroscopy has been used for the intraoperative navigation of rabbit liver cancer, to determine surgical margins. We showed that tumor hypoxia and select organic anion-transporting polypeptide genes mediate NIRF dye uptake in HCC, which was supported by clinical evidence. All these findings represent the first evidence that DZ-1 is an effective molecular probe for tumour-specific imaging in HCC, and provide insights into the development of a new generation of imaging agents for intraoperative guidance of cancer surgery.

The tumor microenvironment plays a critical role in prostate cancer (PC) development and progression. Inappropriate activation of the stroma potentiates the growth and transformation of epithelial tumor cells. Here, we show that upregulation of monoamine oxidase A (MAOA), a mitochondrial enzyme that degrades monoamine neurotransmitters and dietary amines, in stromal cells elevates production of reactive oxygen species, triggers an inflammatory response including activation of IL-6, and promotes tumorigenesis in vitro and in vivo. Mechanistically, MAOA enhances IL-6 transcription through direct Twist1 binding to a conserved E-box element at the IL-6 promoter. MAOA in stromal fibroblasts provides tumor cell growth advantages through paracrine IL-6/STAT3 signaling. Tissue microarray analysis revealed co-expression correlations between individual pairs of proteins of the stromal MAOA-induced Twist1/IL-6/STAT3 pathway in clinical specimens. Downstream of stromal MAOA, STAT3 also promotes cell stemness and transcriptionally activates expression of cancer stem cell marker CD44 in PC cells. MAOA inhibitor treatment effectively suppressed prostate tumor growth in mice in a stroma-specific targeted manner. Collectively, these findings characterize the contribution of MAOA to stromal activation in PC pathogenesis and provide a rationale for targeting MAOA in stromal cells to treat PC.