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Noncoding RNAs (ncRNAs), including microRNAs (miRNAs), small interfering RNAs (siRNAs), and long noncoding RNAs (lncRNAs), regulate target gene expression and can be used as tools for understanding biological processes and identifying new therapeutic targets. Currently, ncRNA molecules for research and therapeutic use are limited to ncRNA mimics made by chemical synthesis. We have recently established a high-yield and cost-effective method of producing bioengineered or biologic ncRNA agents (BERAs) through bacterial fermentation, which is based on a stable tRNA/pre-miR-34a carrier (~ 180 nt) that accommodates target small RNAs. Nevertheless, it remains a challenge to heterogeneously express longer ncRNAs (e.g., > 260 nt), and it is unknown if single BERA may carry multiple small RNAs. To address this issue, we hypothesized that an additional human pre-miR-34a could be attached to the tRNA/pre-miR-34a scaffold to offer a new tRNA/pre-miR-34a/pre-miR-34a carrier (~ 296 nt) for the accommodation of multiple small RNAs. We thus designed ten different combinatorial BERAs (CO-BERAs) that include different combinations of miRNAs, siRNAs, and antagomirs. Our data showed that all target CO-BERAs were successfully expressed in Escherichia coli at high levels, greater than 40% in total bacterial RNAs. Furthermore, recombinant CO-BERAs were purified to a high degree of homogeneity by fast protein liquid chromatography methods. In addition, CO-BERAs exhibited strong anti-proliferative activities against a variety of human non-small cell lung cancer cell lines. These results support the production of long ncRNA molecules carrying different warhead small RNAs for multi-targeting which may open avenues for developing new biologic RNAs as experimental, diagnostic, and therapeutic tools.

Lung cancer remains the leading cause of cancer deaths worldwide and accounts for more than 22% of all cancer-related deaths in the US. Developing new therapies is essential to combat against deadly lung cancer, especially the most common type, non-small cell lung cancer (NSCLC). With the discovery of genome-derived functional small noncoding RNA (ncRNA), namely microRNAs (miRNA or miR), restoration of oncolytic miRNAs lost or downregulated in NSCLC cells represents a new therapeutic strategy. Very recently, we have developed a novel technology that achieves in vivo fermentation production of bioengineered miRNA agents (BERA) for research and development. In this study, we aimed at simultaneously introducing two miRNAs into NSCLC cells by using single recombinant "combinatorial BERA" (CO-BERA) molecule. Our studies show that single CO-BERA molecule (e.g., let-7c/miR-124) was successfully processed to two miRNAs (e.g., let-7c-5p and miR-124-3p) to combinatorially regulate the expression of multiple targets (e.g., RAS, VAMP3 and CDK6) in human NSCLC cells, exhibiting greater efficacy than respective BERA miRNAs in the inhibition of cell viability and colony formation. Furthermore, we demonstrate that CO-BERA let-7c/miR-124-loaded lipopolyplex nanomedicine was the most effective among tested RNAs in the control of tumor growth in NSCLC patient-derived xenograft mouse models. The anti-tumor activity of CO-BERA let-7c/miR-124 was associated with the suppression of RAS and CDK6 expression, and enhancement of apoptosis. These results support the concept to use single ncRNA agent for dual-targeting and offer insight into developing new RNA therapeutics for the treatment of lethal NSCLC.

Our group and others have previously shown that genistein combined polysaccharide (GCP), an aglycone isoflavone-rich extract with high bioavailability and low toxicity, can inhibit prostate cancer (CaP) cell growth and survival as well as androgen receptor (AR) activity. We now elucidate the mechanism by which this may occur using LNCaP and PC-346C CaP cell lines, GCP can inhibit intracrine androgen synthesis in CaP cells. UPLC-MS/MS and qPCR analyses demonstrated that GCP can mediate a ~3-fold decrease in testosterone levels (p &lt, 0.001) and cause decreased expression of intracrine androgen synthesis pathway enzymes (~2.5-fold decrease of 3&beta, HSD (p &lt, 0.001), 17&beta, 0.001), CYP17A (p &lt, 0.01), SRB1 (p &lt, 0.0001), and StAR (p &lt, 0.01)), respectively. Reverse-phase HPLC fractionation and bioassay identified three active GCP fractions. Subsequent NMR and LC-MS analysis of the fraction with the highest level of activity, fraction 40, identified genistein as the primary active component of GCP responsible for its anti-proliferative, pro-apoptotic, and anti-AR activity. GCP, fraction 40, and genistein all mediated at least a ~2-fold change in these biological activities relative to vehicle control (p &lt, 0.001). Genistein caused similar decreases in the expression of 17&beta, HSD and CYP17A (2.5-fold (p &lt, 0.001) and 1.5-fold decrease (p &lt, 0.01), respectively) compared to GCP, however it did not cause altered expression of the other intracrine androgen synthesis pathway enzymes, 3&beta, HSD, SRB1, and StAR. Our combined data indicate that GCP and/or genistein may have clinical utility and that further pre-clinical studies are warranted.

The coronavirus disease of 2019 (COVID-19) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is a global pandemic with increasing incidence and mortality rates. Recent evidence based on the cytokine profiles of severe COVID-19 cases suggests an overstimulation of macrophages and monocytes associated with reduced T-cell abundance (lymphopenia) in patients infected with SARS-CoV-2. The SARS-CoV-2 open reading frame 3 a (ORF3a) protein was found to bind to the human HMOX1 protein at a high confidence through high-throughput screening experiments. The HMOX1 pathway can inhibit platelet aggregation, and can have anti-thrombotic and anti-inflammatory properties, amongst others, all of which are critical medical conditions observed in COVID-19 patients. Here, we review the potential of modulating the HMOX1-ORF3a nexus to regulate the innate immune response for therapeutic benefits in COVID-19 patients. We also review other potential treatment strategies and suggest novel synthetic and natural compounds that may have the potential for future development in clinic.

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.

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.

Lung cancer is the second most common cancer among both men and women in the United States. The majority of lung cancer cases are classified as non-small cell lung cancer (NSCLC). Current NSCLC tre...

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.

Gallic acid (GA), a key intermediate in the synthesis of plant hydrolysable tannins, is also a primary anti-inflammatory, cardio-protective agent found in wine, tea, and cocoa. In this publication, we reveal the identity of a gene and encoded protein essential for GA synthesis. Although it has long been recognized that plants, bacteria, and fungi synthesize and accumulate GA, the pathway leading to its synthesis was largely unknown. Here we provide evidence that shikimate dehydrogenase (SDH), a shikimate pathway enzyme essential for aromatic amino acid synthesis, is also required for GA production. Escherichia coli (E. coli) aroE mutants lacking a functional SDH can be complemented with the plant enzyme such that they grew on media lacking aromatic amino acids and produced GA in vitro. Transgenic Nicotianatabacum lines expressing a Juglans regia SDH exhibited a 500% increase in GA accumulation. The J. regia and E. coli SDH was purified via overexpression in E. coli and used to measure substrate and cofactor kinetics, following reduction of NADP+ to NADPH. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (RP-LC/ESI–MS) was used to quantify and validate GA production through dehydrogenation of 3-dehydroshikimate (3-DHS) by purified E. coli and J. regia SDH when shikimic acid (SA) or 3-DHS were used as substrates and NADP+ as cofactor. Finally, we show that purified E. coli and J. regia SDH produced GA in vitro. Electronic supplementary material The online version of this article (doi:10.1007/s11103-011-9739-3) contains supplementary material, which is available to authorized users.