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The next-generation antiandrogen drugs such as enzalutamide and abiraterone extend survival times and improve quality of life in patients with advanced prostate cancer. However, resistance to both drugs occurs frequently through mechanisms that are incompletely understood. Wnt signaling, particularly through Wnt5a, plays vital roles in promoting prostate cancer progression and induction of resistance to enzalutamide and abiraterone. Development of novel strategies targeting Wnt5a to overcome resistance is an urgent need. In this study, we demonstrated that Wnt5a/FZD2-mediated noncanonical Wnt pathway is overexpressed in enzalutamide-resistant prostate cancer. In patient databases, both the levels of Wnt5a and FZD2 expression are upregulated upon the development of enzalutamide resistance and correlate with higher Gleason score, biochemical recurrence, and metastatic status, and with shortened disease-free survival duration. Blocking Wnt5a/FZD2 signal transduction not only diminished the activation of noncanonical Wnt signaling pathway, but also suppressed the constitutively activated androgen receptor (AR) and AR variants. Furthermore, we developed a novel bioengineered BERA-Wnt5a siRNA construct and demonstrated that inhibition of Wnt5a expression by the BERA-Wnt5a siRNA significantly suppressed tumor growth and enhanced enzalutamide treatment in vivo. These results indicate that Wnt5a/FZD2 signal pathway plays a critical role in promoting enzalutamide resistance, and targeting this pathway by BERA-Wnt5a siRNA can be developed as a potential therapy to treat advanced prostate cancer.

Supplementary Data from Bioengineered BERA-Wnt5a siRNA Targeting Wnt5a/FZD2 Signaling Suppresses Advanced Prostate Cancer Tumor Growth and Enhances Enzalutamide Treatment

Supplemental Tables 1, 2, 3 and 4, Supplemental Figures 1 through 7 and Supplemental Figure legends

miR-127 is downregulated in breast cancer, where it has been shown to suppress the proliferation, migration, and invasion of breast cancer cells. In triple-negative breast cancer (TNBC), miR-127 downregulation correlates with decreased disease-free and overall patient survival. Tumor suppressor miRNAs may hold therapeutic promise but progress has been limited by several factors, including the lability and high cost of miRNA mimics. Here, we take a novel approach to produce a miR-127 prodrug (miR-127PD), which we demonstrate is processed to mature, functional miR-127-3p in TNBC tumor cells. miR-127PD decreased the viability and motility of TNBC cells, sensitized TNBC cells to chemotherapy, and restricted the TNBC stem cell population. Furthermore, systemic delivery of miR-127PD suppressed tumor growth of MDA-MB-231 and MDA-MB-468 TNBC cells and spontaneous metastasis of MDA-MB-231 cells. In addition, CERK, NANOS1, FOXO6, SOX11, SOX12, FASN, and SUSD2 were identified as novel, functionally important targets of miR-127. In conclusion, our study demonstrates that miR-127 functions as a tumor and metastasis suppressor in TNBC and that delivery of miR-127 may hold promise as a novel therapy.Significance:Exogenous administration of miR-127, which is functionally activated in target cells, inhibits growth and spontaneous metastasis of triple-negative breast cancer.

mRNA Sequencing Data Matrix Table

sRNA Sequencing Data Matrix Table

Introduction and Objectives: Androgen receptor (AR) blockade using antiandrogens is a mainstay for the treatment of castration-resistant prostate cancer (CRPC). However, resistance to antiandrogens occurs unavoidably due to the escaping mechanisms of tumor cells that are not completely understood. Upregulation of non-canonical Wnt ligand Wnt5a is identified from circulating tumor cells from CRPC patients after being treated with enzalutamide. FZD2, the cognate frizzled receptor for Wnt5a, is the most commonly co-upregulated non-canonical Wnt signaling molecule in prostate cancer. In this study, we determined the role of non-canonical Wnt5a/FZD2 to enzalutamide resistance and neural lineage plasticity, and explore the potential of targeting Wnt5a/FZD2 to overcome resistance in advanced prostate cancer. Methods: Wnt5a and FZD2 expression was determined in enzalutamide-resistant C4-2B MDVR cells. Wnt5a and FZD2 expressions were modulated using specific siRNAs. Cell growth, colony formation, and migration were studied in vitro. Transcriptomic analysis was performed on C4-2B MDVR cells treated with FZD2 knocked down; gene program of non-canonical Wnt signaling, hallmark androgen response and AR-V7 associated genes, and neural lineage pathways were analyzed. A novel tRNA bioengineered Wnt5a siRNA was developed to target Wnt5a/FZD2 signaling. The effect of tRNA-siWnt5a on tumor growth, sensitivity to enzalutamide treatment, and neural lineage plasticity was evaluated in vitro and in vivo. Results: Wnt5a and FZD2 are highly upregulated in castration-resistant prostate cancer patients, which is verified overexpression in enzalutamide-resistant C4-2B MDVR cells. Knocking down Wnt5a and FZD2 diminishes the enrichment of the non-canonical Wnt signaling pathway. Downregulating Wnt5a and FZD2 expression by specific siRNAs suppresses prostate cancer cell proliferation and resensitized C4-2B MDVR cells to enzalutamide treatment. Suppressing Wnt5a and FZD2 abrogated the enrichment of gene programs regulating cancer cell survival/proliferation, and neural lineage pathways including neuron differentiation and embryonic stem cell markers. Using the bioengineered tRNA-Wnt5a siRNA effectively inhibited the growth of enzalutamide-resistant prostate cancer cells and resensitized tumor cells to enzalutamide treatment in vitro, and resistant CRPC PDX LuCaP35CR tumor growth in vivo. Conclusions: Our results suggest that activation of noncanonical Wnt5a/FZD2 signaling confers to enzalutamide resistance and neural lineage plasticity. Targeting the Wnt5a signal could provide benefits for CRPC patients with tumors expressing a high level of Wnt5a, FZD2, and lineage markers, not only enhancing the anti-tumor effects of enzalutamide but suppressing the potential of neuroendocrine differentiation in advanced prostate cancer patients. Citation Format: Shu Ning, Wei Lou, Chengfei Liu, Joy C. Yang, Alan P. Lombard, Leandro S. Abronzo, Neelu Batra, Aiming Yu, Christopher P. Evans, Allen C. Gao. Targeting noncanonical Wnt5a signaling suppresses the neural lineage network and overcomes enzalutamide resistance in advanced 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 3069.

Background: We previously showed that Nrdp1, an E3 ubiquitin ligase, is transcriptionally regulated by the androgen receptor (AR) in prostate cancer (CaP) cells and that Nrdp1 can post-translationally regulate ErbB3 (an EGFR family member) levels via ubiquitination and subsequent proteasomal degradation. Increased levels of ErbB3 are associated with worse CaP patient outcome. The goal of the current study was to determine whether dysregulation of the AR-Nrdp1-ErbB3 axis contributes to prostate cancer health disparities for African American (AA) men, and to identify the underlying mechanisms involved. Methods: Expression levels and localization of AR, Nrdp1, and ErbB3 were assessed in a total of 208 CaP patient samples (50 African American (AA) and 158 Caucasian (CA) CaP patients) and two cell lines (LNCaP (CA) and MDAPCa2b (AA)). Expression levels and localization of these molecules were determined by immunohistochemistry (IHC) and subcellular fractionation and western blot. A combination of knockdown (siRNA), forced overexpression (Nrdp1-FLAG construct), and treatment with enzalutamide or synthetic androgen (R1881) experiments were employed to investigate the relationship between AR, Nrdp1, and ErbB3 expression levels and localization and to identify differences in AA and CA cell lines. Immunoprecipitation allowed for investigation of the mechanisms which facilitate nuclear transport of Nrdp1. Results: A statistically significant negative association between cytoplasmic levels of AR (AR C) and nuclear Nrdp1 (Nrdp1 N) exists in both Caucasian (CA) and African American (AA) prostate cancer (CaP) patients, but this association was stronger in AA patients compared to CA patients (Spearman correlation coefficient of -0.62 for AA patients, and -0.36 for CA patients). Increased nuclear Nrdp1 expression levels were associated with increased 5-year survival rates, and reduced nuclear Nrdp1 expression levels predicted biochemical recurrence (AUC 0.63). Dysregulation of the AR-Nrdp1-ErbB3 axis in AA CaP cells was also observed in cell line studies; AA CaP cells expressed significantly lower levels of both total and nuclear Nrdp1 compared to their CA CaP counterparts. Manipulation of AR expression levels and localization via knockdown and inhibition with enzalutamide had a lesser impact on Nrdp1 expression levels and localization in AA versus CA CaP cells. Immunoprecipitation studies demonstrated that Nrdp1 can bind to AR in CA CaP cells but not AA CaP cells. Conclusions: Our combined data indicate that dysregulation of the AR-Nrdp1-ErbB3 axis occurs to a larger extent in AA than CA CaP cells and that this can contribute to worse patient outcomes. Our data also indicate that lack of binding between AR and Nrdp1 in AA CaP cells may account for the lower levels of nuclear Nrdp1 observed in AA CaP cells. Further understanding of the mechanisms which regulate Nrdp1 levels and localization may allow for the development of treatments which help abrogate cancer health disparities. Citation Format: Anhao Sam, Shawna Evans, Elizabeth Browning, Sheryl Krig, Neelu Batra, Thomas Steele, Salma Siddqui, Paramita Ghosh, Ruth Vinall. Dysregulation of the AR-Nrdp1-ErbB3 axis occurs in African American prostate cancer patients and is associated with worse outcomes [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B083.

Despite a recent decrease in overall incidence of prostate cancer (CaP), CaP incidence continues to be much higher among men of African origin living in the United States (223.9 men per 100,000) compared to those of European origin (139.9 men per 100,000). Also, the CaP age-specific mortality rates are 2.4 times greater among African-American (AA) compared with European American (EA) men. The causes for these differences are multifactorial, but include genetic effects that contribute to CaP-associated health disparity. We previously showed that the androgen receptor (AR) can suppress levels of the receptor tyrosine kinase ErbB3, a molecule that is known to drive CaP progression, by stimulating the E3 ubiquitin ligase Nrdp1 (also called RNF41 or FLRF). The goals of the current study were to determine whether differential expression and/or localization of Nrdp1 contribute to CaP-associated health disparities, and to further elucidate the mechanisms by which regulation of Nrdp1 levels and localization occurs. Immunohistochemistry (IHC) in prostate tissue determined that nuclear Nrdp1 levels are significantly lower in AA CaP patients (n=19) versus CA CaP patients (n=121) with localized disease (p=0.008). A similar association was observed in CaP cell lines; immunofluorescence (IF) analyses demonstrated MDAPCa2b and E006 (cell lines derived from AA CaP patients) express significantly lower levels of nuclear Nrdp1 compared to LNCaP, CWR22Rv1, C4-2, and C4-2B (cell lines derived from CA CaP patients). Western blot and qPCR determined that Nrdp1 levels are lower in AA versus CA CaP cells (MDAPCa2b express ~2-fold less Nrdp1 protein and mRNA levels compared to LNCaP), and that androgen withdrawal has a bigger impact on Nrdp1 expression levels in AA CaP cells (2-fold decrease in MDAPCa2b compared to ~1.2-fold decrease in LNCaP). Proteasomal regulation of Nrdp1 also occurs; treatment with MG132 resulted in accumulation of Nrdp1 in both AA and CA CaP cell lines. Interestingly, altering androgen and/or AR levels also affected Nrdp1 localization (IF analyses) as well as the ability of Nrdp1 to mediate CaP cell apoptosis (flow cytometry). In summary, we demonstrate that Nrdp1 is differentially expressed in AA versus CA CaP patients as well as in AA versus CA CaP-patient derived cell lines, and that androgen withdrawal has a bigger impact on Nrdp1 expression in AA CaP cell lines. We also demonstrate that androgen/AR is involved in determining Nrdp1 localization, and that Nrdp1 can mediate CaP cell apoptosis in the presence of AR. The combined data, and the knowledge that Nrdp1 regulates ErbB3 levels, suggest that differential expression of Nrdp1 in AA versus CA CaP may contribute to CaP-associated health disparities and may occur as a result of dysregulation of the regulation of Nrdp1 by androgen/AR. Citation Format: Neelu Batra, Sheryl Krig, Anhao Sam, Katelyn Macias, Rosalinda Savoy, Salma Siddiqui, Frank Melgoza, Leandro D'Abronzo, Sidhartha Hazari, Blythe Durbin-Johnson, Paramita Ghosh, Ruth L. Vinall. Differential expression and localization of the E3 ubiquitin ligase Nrdp1 in African American versus Caucasian American prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3535.