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Supplementary Materials and Methods; Supplementary Tables (Table S1. Antibodies used in Western blotting, immunoprecipitation and IHC studies. Table S2. Sequences of primers used for real-time PCR analysis. Table S3. Combination index (CI) of bromocriptine and docetaxel treatment in C4-2 cells); Supplementary Figures ( Figure S1. Quantitative PCR analysis of mRNA expression of AR and survivin in C4-2 cells treated with varying concentrations of bromocriptine (24 h). Figure S2. Body weights of athymic nude mice bearing C4-2-Luc tumors and treated with the vehicle control, docetaxel, bromocriptine and the combination. *: p < 0.05. Figure S3. Western blot analysis of p-AR (Ser650) expression in C4-2 cells treated with bromocriptine for varying times)

Docetaxel resistance remains a major obstacle in the treatment of prostate cancer bone metastasis. In this study, we demonstrate that the dopamine D2 receptor (DRD2) agonist bromocriptine effectively enhances docetaxel efficacy and suppresses skeletal growth of prostate cancer in preclinical models. DRD2 is ubiquitously expressed in prostate cancer cell lines and significantly reduced in prostate cancer tissues with high Gleason score. Bromocriptine has weak to moderate cytotoxicity in prostate cancer cells, but effectively induces cell-cycle arrest. At the molecular level, bromocriptine inhibits the expression of c-Myc, E2F-1, and survivin and increases the expression of p53, p21, and p27. Intriguingly, bromocriptine markedly reduces androgen receptor levels, partially through Hsp90-mediated protein degradation. The combination of bromocriptine and docetaxel demonstrates enhanced in vitro cytotoxicity in prostate cancer cells and significantly retards the skeletal growth of C4-2-Luc tumors in mice. Collectively, these results provide the first experimental evidence for repurposing bromocriptine as an effective adjunct therapy to enhance docetaxel efficacy in prostate cancer. Mol Cancer Ther; 17(9); 1859–70. ©2018 AACR.

Supplementary Figure 2 from β2-Microglobulin Induces Epithelial to Mesenchymal Transition and Confers Cancer Lethality and Bone Metastasis in Human Cancer Cells

Supplementary Figure 3 from β2-Microglobulin Induces Epithelial to Mesenchymal Transition and Confers Cancer Lethality and Bone Metastasis in Human Cancer Cells

Supplementary Figure 1 from β2-Microglobulin Induces Epithelial to Mesenchymal Transition and Confers Cancer Lethality and Bone Metastasis in Human Cancer Cells

Supplementary Figure 4 from β2-Microglobulin Induces Epithelial to Mesenchymal Transition and Confers Cancer Lethality and Bone Metastasis in Human Cancer Cells

Bone metastasis is one of the predominant causes of cancer lethality. This study demonstrates for the first time how β2-microglobulin (β2-M) supports lethal metastasis in vivo in human prostate, breast, lung, and renal cancer cells. β2-M mediates this process by activating epithelial to mesenchymal transition (EMT) to promote lethal bone and soft tissue metastases in host mice. β2-M interacts with its receptor, hemochromatosis (HFE) protein, to modulate iron responsive pathways in cancer cells. Inhibition of either β2-M or HFE results in reversion of EMT. These results demonstrate the role of β2-M in cancer metastasis and lethality. Thus, β2-M and its downstream signaling pathways are promising prognostic markers of cancer metastases and novel therapeutic targets for cancer therapy. Cancer Res; 71(7); 2600–10. ©2011 AACR.

Purpose: Despite numerous therapeutic modalities, including chemotherapy and next-generation androgen deprivation therapy, metastatic castration-resistant prostate cancer (mCRPC) remains a dreadful evolution and still bears a poor prognosis for most patients who develop metastases to bones and other organs, which have no cure. It is urgent to identify new molecular targets and develop novel therapeutic agents against lethal mCRPC. Methods: We developed a novel small-molecule anticancer compound named GH501 via a “molecular hybridization” approach. The in vitro cytotoxicity of GH501 was evaluated in the NCI-60 human cancer cell panel and human mCRPC cell lines. Molecular targets of GH501 were investigated using in silico docking, biolayer interferometry, cellular thermal shift assay, and other molecular and cellular approaches. The in vivo anticancer efficacy of GH501 against mCRPC was evaluated in clinically-relevant xenograft models. Results: GH501 effectively inhibited the in vitro proliferation of NCI-60 human cancer cell lines and established mCRPC cell lines, including African American prostate cancer (PCa) cells, at nanomolar potency by inducing cell cycle arrest and apoptosis. Mechanistically, GH501 might bind S-phase kinase-associated protein 1 (Skp1) and disrupt the physical interaction between Skp1 and S-phase kinase-associated protein 2 (Skp2), thereby affecting multiple oncogenic signals implicated in mCRPC progression. RNA-Seq analyses indicated that GH501 treatment in C4-2 cells significantly inhibited E2F-, G2/M checkpoint, c-Myc- and β-catenin-regulated genes, androgen receptor (AR) transcription, androgen-responsive genes, and enhancer of zeste homolog 2 (EZH2)-activated genes. Proteomic studies identified 109 proteins upregulated and 154 proteins downregulated significantly by GH501 in CRW22Rv1 cells. The top physiological processes and signaling pathways affected by GH501 included cell cycle, autophagy, steroid biosynthesis, DNA replication, p53, AMP-activated protein kinase (AMPK), and mitogen-activated protein kinase (MAPK). As a lead compound, GH501 had excellent preclinical safety. In animal studies, GH501 monotherapy effectively inhibited the skeletal and subcutaneous growth of four mCRPC xenografts with heterogeneous genetic backgrounds. Conclusion: These results support that pharmacological interruption of Skp1-Skp2 interaction is a promising therapeutic strategy for mCRPC. To our knowledge, GH501 is the first synthetic Skp1 inhibitor with a unique chemical structure that can serve as the basis for lead optimization and development. Citation Format: Xin Li, Kenza Mamouni, Rui Zhao, Yifei Wu, Zhong-Ru Xie, Giuseppe Sautto, Degang Liu, Nathan Bowen, Alira Danaher, Lajos Gera, Daqing Wu. A novel Skp1 inhibitor has potent preclinical efficacy against metastatic castration-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 519.

The aryl hydrocarbon receptor (AhR) is a nuclear transcription factor and xenobiotic sensor reported to mediate diet-induced obesity and is upregulated in high-grade prostate cancer (PCa). Previously published data from our lab shows AhR protein is overexpressed and constitutively active in castration-resistant PCa cells. AhR signaling is promoted by high fat diet-induced obesity, when circulating leptin levels are also high. Physiologically relevant leptin concentration is proportional to BMI and is secreted by adipose cells to inhibit hunger under normal circumstances. Our preliminary results suggest that both AhR and leptin independently desensitize PCa cells to clinically-relevant chemotherapeutics however, the underlying mechanisms must be elucidated and may require sustained AhR signaling. We also conducted an integrative bioinformatics analysis to explore the role of AHR and LEPR in PCa, which revealed AHR and LEPR mRNA expression positively correlates in prostate cancer (P Citation Format: Kofi K. Khamit-Kush, Joann B. Powell, Jeffrey A. Handy, Nathan J. Bowen, Valerie Odero-Marah, Daqing Wu. Aryl hydrocarbon receptor and leptin promote prostate cancer progression and may be associated with chemoresistance [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C023.

The aryl hydrocarbon receptor (AhR) is a nuclear transcription factor and xenobiotic sensor reported to mediate diet-induced obesity and is upregulated in high-grade prostate cancer (PCa). Previously published data from our lab shows that AhR protein is overexpressed and constitutively active in castration-resistant prostate cancer cells. Physiologically relevant leptin concentration is proportional to BMI and it is secreted by adipose cells to inhibit hunger under normal circumstances. Our preliminary results suggest that obesity-associated leptin concentrations desensitize PCa cells to cancer drugs however, the underlying mechanisms must be elucidated and may require sustained AhR signaling. In this study, we conducted an integrative bioinformatics analysis to explore the role of AHR and LEPR in PCa, which revealed AHR and LEPR mRNA expression positively correlates in prostate cancer (P Citation Format: Kofi Kyenku Khamit-Kush, Joann B. Powell, Jeffrey A. Handy, Nathan Bowen, Daqing Wu, Valerie Odero-Marah. Elevated aryl hydrocarbon receptor and leptin receptor expression correlates with prostate cancer progression and may be associated with obesity-associated leptin-mediated chemoresistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 394.

Background: It is imperative to develop novel therapeutics to overcome chemoresistance, a major obstacle in the clinical management of prostate cancer (PCa) and other cancers. Drug repositioning is being pursued as an attractive approach due to its potentially lower overall development costs and shorter timelines. The purpose of this study is to investigate the preclinical efficacy and mechanism of action of nicardipine, an FDA-approved hypertension drug, as a new treatment against chemoresistant prostate cancer. Methods: A panel of FDA-approved non-oncology drugs was screened for their selectivity and potency to inhibit chemoresistant PCa cells using a two-tier phenotypic screening platform (Theranostics, 2021, 11(14): 6873-6890). The mechanism of action of potential candidate(s) was evaluated using in silico docking, cellular and molecular assays in ARCaPE-shEPLIN and C4-2B-TaxR, two independent chemoresistant PCa cell lines. The in vivo efficacy was evaluated in clinically-relevant xenograft models of PCa. Results: Nicardipine exhibited high selectivity and potency against chemoresistant PCa cells via effectively inducing apoptosis and cell cycle arrest. The IC50 of nicardipine was determined as 0.5 μM and 2.1 μM in ARCaPE-shEPLIN and C4-2B-TaxR cells, respectively. Mechanically, nicardipine targeted a novel non-canonical enhancer of zeste homolog 2 (EZH2) signal pathway in chemoresistant PCa cells. Specifically, nicardipine might bind embryonic ectoderm development (EED), interrupt the integrity and stability of the polycomb repressive complex 2 (PRC2), reduce the phosphorylation and expression of EZH2, and subsequently inhibit downstream effectors including signal transducer and activator of transcription 3 (Stat3), S-phase kinase-associated protein 2 (SKP2), ATP binding cassette B1 (ABCB1) and survivin. In animal models, as a monotherapy, nicardipine significantly suppressed the intratibial growth of chemoresistant C4-2B-TaxR xenografts, with an average PSA level of 43.47 ng/ml, 36.63 ng/ml, and 28.78 ng/ml for the vehicle control, docetaxel, and nicardipine groups at the endpoint, respectively (p < 0.05 between the nicardipine group and the vehicle control or docetaxel groups). As a combination regime, nicardipine synergistically enhanced the efficacy of docetaxel against the intratibial growth of C4-2 xenografts, with an average PSA level of 79.17 ng/ml, 49.04 ng/ml, 42.80 ng/ml, and 23.23 ng/ml for the vehicle control, docetaxel, nicardipine, and combination groups at the endpoint, respectively (p < 0.0001 or 0.01 between the combination group and the vehicle control or docetaxel groups, respectively). Conclusion: These results demonstrated that, for the first time, nicardipine could be a novel EED inhibitor that has the potential to be promptly tested in PCa patients to overcome chemoresistance and improve clinical outcomes. Citation Format: Xin Li, Alira Danaher, Jason M. Wu, Yifei Wu, Zhong-Ru Xie, Nicholas Cook, Nathan Bowen, Daqing Wu. Repositioning nicardipine as a novel targeted therapy for chemoresistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5365.

Docetaxel resistance remains a major obstacle in the treatment of prostate cancer bone metastasis. In this study, we demonstrate that the dopamine D2 receptor (DRD2) agonist bromocriptine effectively enhances docetaxel efficacy and suppresses skeletal growth of prostate cancer in preclinical models. DRD2 is ubiquitously expressed in prostate cancer cell lines and significantly reduced in prostate cancer tissues with high Gleason score. Bromocriptine has weak to moderate cytotoxicity in prostate cancer cells, but effectively induces cell-cycle arrest. At the molecular level, bromocriptine inhibits the expression of c-Myc, E2F-1, and survivin and increases the expression of p53, p21, and p27. Intriguingly, bromocriptine markedly reduces androgen receptor levels, partially through Hsp90-mediated protein degradation. The combination of bromocriptine and docetaxel demonstrates enhanced in vitro cytotoxicity in prostate cancer cells and significantly retards the skeletal growth of C4-2-Luc tumors in mice. Collectively, these results provide the first experimental evidence for repurposing bromocriptine as an effective adjunct therapy to enhance docetaxel efficacy in prostate cancer. Mol Cancer Ther; 17(9); 1859–70. ©2018 AACR.

Bone metastasis is one of the predominant causes of cancer lethality. This study demonstrates for the first time how β2-microglobulin (β2-M) supports lethal metastasis in vivo in human prostate, breast, lung, and renal cancer cells. β2-M mediates this process by activating epithelial to mesenchymal transition (EMT) to promote lethal bone and soft tissue metastases in host mice. β2-M interacts with its receptor, hemochromatosis (HFE) protein, to modulate iron responsive pathways in cancer cells. Inhibition of either β2-M or HFE results in reversion of EMT. These results demonstrate the role of β2-M in cancer metastasis and lethality. Thus, β2-M and its downstream signaling pathways are promising prognostic markers of cancer metastases and novel therapeutic targets for cancer therapy. Cancer Res; 71(7); 2600–10. ©2011 AACR.

Purpose: β2-Microglobulin (β2M), a soluble protein secreted by cancer and host inflammatory cells, has various biological functions, including antigen presentation. Because aberrant expression of β2M has been reported in human renal cell carcinoma, we investigated the effects of β2M overexpression on cancer cell growth and analyzed its molecular signaling pathway. Experimental Design: We established clonal cell lines that overexpressed β2M in human renal cell carcinoma (SN12C) cells and then examined cell growth in vitro and in vivo and studied the β2M-mediated downstream cell signaling pathway. Results: Our results showed that β2M expression positively correlates with (a) in vitro growth on plastic dishes and as Matrigel colonies, (b) cell invasion and migration in Boyden chambers, and (c) vascular endothelial growth factor (VEGF) expression and secretion by cells. We found, in addition, that β2M mediates its action through increased phosphorylation of cyclic AMP–responsive element-binding protein (CREB) via the protein kinase A-CREB axis, resulting in increased VEGF expression and secretion. In convergence with this signal axis, β2M overexpression also activated both phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways. β2M overexpression induced accelerated growth of SN12C in mouse subcutis and bone. Interrupting the β2M signaling pathway using small interfering RNA led to apoptosis with increased activation of caspase-3 and caspase-9 and cleaved poly(ADP-ribose) polymerase. Conclusions: Our results showed for the first time that the β2M-protein kinase A-CREB-VEGF signaling axis plays a crucial role in support of renal cell carcinoma growth and progression and reveals a novel therapeutic target.

A standard treatment for prostate cancer (PCa) is androgen deprivation therapy (ADT) that suppresses androgen receptor (AR) signaling axis. Although initially responsive, most patients receiving ADT eventually develop metastatic castration-resistant prostate cancer (CRPC), with more than 90% of them exhibiting bone metastases. A mechanism by which CRPC cells evade ADT is the expression of constitutively active AR variants (AR-Vs), such as the well-characterized AR-V7. Recently we developed GH501, a flurbiprofen-modified small-molecule compound, and investigated its anti-cancer activity and mechanism of action in pre-clinical models of CRPC. At nanomolar range, GH501 effectively induces cell cycle arrest and apoptosis in CRPC cells regardless of their resistance status to enzalutamide treatment. RNA-seq analysis of GH501 combined with Western blotting analysis identified key targets implicated in CRPC progression, including the full-length AR, AR-V7 variant, and other important genes implicated in CRPC progression. Importantly, low doses of GH501 effectively inhibit the skeletal growth of CRPC in a xenograft model without obvious in vivo toxicities. These preclinical results indicate that GH501 is a promising small-molecule compound that can be further developed for the treatment of lethal prostate cancer. Citation Format: Kenza Mamouni, Lajos Gera, Xin Li, Daqing Wu. A nanomolar potency small-molecule compound against castration-resistant and bone metastatic prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3205. doi:10.1158/1538-7445.AM2017-3205