Extracellular vesicles (EVs) provide a minimally invasive liquid biopsy source of tumor-specific markers for patients who have already undergone prostatectomies.Our laboratory has previously demonstrated enrichment of the cancer-type solute carrier organic anion transporter family 1B3 (ct-SLCO1B3) and the ATP Binding Cassette Subfamily Member C (ABCC3) in castration-resistant cell lines (CRPC).However, their expression in EVs has yet to be explored.Our study demonstrated that ct-SLCO1B3 and ABCC3 are highly detectable in CRPC cell line-derived EVs.We also showed that ct-SLCO1B3 and ABCC3 were detectable in a CRPC xenograft mouse model, both intratumorally and in plasma-derived EVs.Our results provide evidence for EV-contained ct-SLCO1B3 and ABCC3 as novel, EV-based tumor markers for prostate cancer progression.

Sustained androgen receptor (AR) signaling during relapse is a central driver of metastatic castration- resistant prostate cancer (mCRPC). Current AR antagonists, such as enzalutamide, fail to provide long- term benefit for the mCRPC patients who have dramatic increases in AR expression. Here, we report AR antagonists with efficacy in AR- overexpressing models. These molecules bind to the ligand- binding domain of the AR, promote AR localization to the nucleus, yet potently and selectively down- regulate AR- target genes. The molecules BG-15a and the pharmacokinetically optimized BG-15n elicit a decrease in cell and tumor growth in vitro and in vivo in models of mCRPC. BG-15a/n treatment causes the collapse of chromatin loops between enhancers and promoters at key genes in the AR- driven epigenome. AR binding in the promoter, as well as 3D chromatin clustering, is needed for genes to respond. BG-15a/n represent promising agents for treating patients with relapsed AR- driven mCRPC tumors.

S1. Expression of (A) doxycycline-inducible GFP-linked OATP1B3 was confirmed in the membrane and not cytosolic fractions of PC3 cells using a GFP-specific antibody Abcam goat antibody (#ab6673).

S3. t-SLCO1B3 expression was optimized using various concentrations of chetomin at different timepoints in (A) LNCaP and (B) 22Rv1 cell lines.

Supplemental Figure Legends 1-4

S4. Both (A) lt-SLCO1B3 and (B) ct-SLCO1B3 were plotted as a function of t-SLCO1B3 in 384 tissue and tumor samples.

S2. Ten hypoxia response elements were identified by sequence alignment of the RefSeq SLCO1B3 gene (NG_032071.1).

Background/Aim: Gonadotropin-releasing hormone 2 (GNRH2) is a poorly-studied peptide hormone that is widely distributed in the central nervous system and expressed in peripheral tissues of mammals. The non-synonymous rs6051545 variant in GNRH2 (A16V) has been linked to higher serum testosterone concentrations. This study investigated whether the A16V variant is associated with altered androgen-deprivation therapy (ADT) progression-free survival (PFS) and overall survival (OS). Patients and Methods: We examined the expression of GNRH2 in prostate tissue microarrays comprising normal tissue, prostatic hyperplasia, and prostate cancer using immunofluorescence. We also evaluated the GNRH2 genotype in 131 patients with prostate cancer who received ADT and compared PFS and OS between the variant and wild-type genotypes. Results: GNRH2 was detected in all prostate tissues, although expression did not vary with Gleason grade or disease stage (p=0.71). The GNRH2 A16V genotype was not associated with PFS or OS; however, univariate and multivariate analyses revealed Gleason score and definitive local therapy were each associated with PFS (p≤0.0074), whereas age and Gleason score were associated with OS (p≤0.0046). Conclusion: GNRH2 is expressed in normal, hyperplastic, and neoplastic prostate tissues; the A16V variant is not related to treatment outcome or survival.

Figures S1, S2, S3, S4, and S5

Extracellular vesicles (EVs) provide a minimally invasive source of tumor-specific markers for liquid biopsy that can be utilized for cancer detection, diagnosis, or monitoring progression. Our laboratory has previously demonstrated the endogenous expression of all transporter systems in the common prostate cancer cell lines (LNCaP, 22Rv1, PC3, DU-145) using a medium throughput TaqMan-based qRT-PCR assay. We identified the two most differentially expressed transporter genes in castration-resistant prostate cancer cells (CRPC) as the cancer-type solute carrier organic anion transporter family 1B3 (ct-SLCO1B3) and the ATP Binding Cassette Subfamily Member C (ABBC3). Both transporters have been investigated as EV-derived tumor markers; however, their detection in prostate cancer-derived EVs remains to be determined. This study aims to investigate whether ct-SLCO1B31B3 and ABCC3 mRNA exists in prostate cancer-derived EVs and can be further detected using plasma specimens. We isolated EVs from prostate cancer (LNCaP, 22Rv1, PC3, DU-145) cell line supernatant and extracted exosomal RNA for qPCR analyses. Both ct-SLCO1B3 and ABBC3 transcripts were only detected in CRPC-derived EV cells vs androgen-responsive prostate cancer cells. To explore the possibility that both transcripts in CRPC-derived EVs can be detected in plasma, we isolated plasma EVs derived from human prostate cancer xenograft mice, and then performed digital droplet PCR analysis. We demonstrated that ct-SLCO1B3 and ABBC3 are detectable in the DU-145 xenograft mouse model, both in tissues and in plasma-derived EVs. Our results provide evidence that ct-SLCO1B3 and ABCC3 exist in EVs and can be detected (at least) in mouse plasma, potentially supporting their roles as novel, plasma-based biomarkers for monitoring prostate cancer progression. Citation Format: Erica L. Beatson, Kristi Y. Lee, Martina A. Knechel, Elijah R. Sommer, Roger Depaz, Emily N. Risdon, Andres F. Leon, Jonathan D. Strope, Douglas K. Price, Cindy H. Chau, William D. Figg. Exosomal cancer-type SLCO1B3 and ABCC3 as potential biomarkers of castration resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B044.

Modifications at the glycolate moiety of englerin A were made to explore variations at the most sensitive site on the molecule for activity in the NCI 60 screen, wherein englerin A is highly potent and selective for renal cancer cells. Replacement of the glycolate by other functionalities as well as esterification of the glycolate hydroxyl yielded compounds which displayed excellent selectivity and potency compared with the natural product. TRPC4/5 ion channel experiments with five compounds showed delayed or reduced agonism with TRPC5, at much higher concentrations than englerin A. With TRPC4, these compounds all had no effect at 10 μM. The same compounds were not detectable in mouse serum after a single oral dose of 12.5 mg/kg. At 100 mg/kg p.o., no toxicity was observed, and blood levels were barely detectable. Intravenous administration led to toxicity but at substantially lower doses than for englerin A.

Understanding mechanisms of resistance to abiraterone, one of the primary drugs approved for the treatment of castration resistant prostate cancer, remains a priority. The organic anion polypeptide 1B3 (OATP1B3, encoded by SLCO1B3 ) transporter has been shown to transport androgens into prostate cancer cells. In this study we observed and investigated the mechanism of induction of SLCO1B3 by abiraterone. Prostate cancer cells (22Rv1, LNCaP, and VCAP) were treated with anti-androgens and assessed for SLCO1B3 expression by qPCR analysis. Abiraterone treatment increased SLCO1B3 expression in 22Rv1 cells in vitro and in the 22Rv1 xenograft model in vivo. MicroRNA profiling of abiraterone-treated 22Rv1 cells was performed using a NanoString nCounter miRNA panel followed by miRNA target prediction. TargetScan and miRanda prediction tools identified hsa-miR-579-3p as binding to the 3′-untranslated region (3′UTR) of the SLCO1B3 . Using dual luciferase reporter assays, we verified that hsa-miR-579-3p indeed binds to the SLCO1B3 3′UTR and significantly inhibited SLCO1B3 reporter activity. Treatment with abiraterone significantly downregulated hsa-miR-579-3p, indicating its potential role in upregulating SLCO1B3 expression. In this study, we demonstrated a novel miRNA-mediated mechanism of abiraterone-induced SLCO1B3 expression, a transporter that is also responsible for driving androgen deprivation therapy resistance. Understanding mechanisms of abiraterone resistance mediated via differential miRNA expression will assist in the identification of potential miRNA biomarkers of treatment resistance and the development of future therapeutics.

In Sub-Saharan Africa, the cancer burden is predicted to increase by > 85% by 2030, the largest increase worldwide. This region has a large HIV-positive population. Drug-drug interactions (DDIs) from concomitant use of multiple drugs increase the risk of drug toxicities, sub-optimal therapy, and drug resistance. With the increase in polypharmacy, involving antiretroviral (ARV), and anticancer drugs, there is a greater need for an appreciation of clinically relevant DDIs. Anticancer and ARV drugs studied in this review were from The World Health Organization's Model List of Essential Medicines 2017. We reviewed; drug package inserts, www.drugbank.ca and www.UpToDate.com, to evaluate pharmacokinetic interactions with cytochrome P450 (CYP450) and ABCB1. The DDIs between drugs were assessed using the University Of Liverpool, UK HIV Drug Interactions Checker, and the LexiComp Drug Interaction tool of www.UpToDate.com. About 70% of ARVs studied interact with CYP450, all involve CYP3A4, and 55% interact with ABCB1. About 65% of anticancer drugs interact with CYP450, 44% of which do so through CYP3A4. About 75% of anticancer drugs interact with ARV drugs, with nine absolute contraindications to concomitant therapy. There exist a substantial number of DDIs between ARV and anticancer drugs, primarily mediated through CYP450 enzymes. Dolutegravir based regimens offer the safest DDI profile for concurrent use with anticancer drugs. However, there are substantial gaps in our knowledge, and this study serves to highlight the need for additional research to better define these interactions and their effect on drug exposure, as attention to these DDIs is a relatively simple intervention that could lead to optimizing disease treatment.

Abstract Effective treatments for patients with metastatic castration-resistant prostate cancer following disease progression on enzalutamide are currently an unmet clinical need. Simultaneous inhibition of the hypoxia-inducible factor (HIF)-1α and androgen receptor (AR) pathways has been previously shown to overcome enzalutamide resistance in vitro. Combination treatment with NLG207, a nanoparticle–drug conjugate of camptothecin and inhibitor of HIF-1α, and enzalutamide was evaluated in preclinical prostate cancer models of enzalutamide resistance. The effect of NLG207 and enzalutamide on average tumor volume and tumor re-growth after 3 weeks of treatment was evaluated in vivo using the subcutaneous 22Rv1 xenograft and castrated subcutaneous VCaP xenograft models. Correlative assessments of antitumor activity were evaluated in vitro using cell proliferation and qPCR assays. NLG207 8 mg/kg alone and in combination with enzalutamide reduced average tumor volume by 93% after 3 weeks of treatment (P < 0.05) in comparison with vehicle control in the subcutaneous 22Rv1 xenograft model. Notably, the addition of NLG207 also enhanced the efficacy of enzalutamide alone in the castrated subcutaneous VCaP xenograft model, decreasing the median rate of tumor growth by 51% (P = 0.0001) in comparison with enzalutamide alone. In vitro assessments of cell proliferation and gene expression further demonstrated antitumor activity via AR–HIF-1α crosstalk inhibition. Combination treatment with NLG207 and enzalutamide was shown to be effective in preclinical prostate cancer models of enzalutamide resistance. Clinical investigation of this treatment combination is ongoing (NCT03531827).

OATP1B3 is expressed de novo in primary prostate cancer tissue and to a greater degree in prostate cancer metastases. Gadoxetate disodium is a substrate of OATP1B3, and its uptake has been shown to correlate with OATP1B3 expression in other cancers. We aimed to evaluate use of gadoxetate disodium to image prostate cancer and to track its utility as a biomarker. A single center open-label non-randomized pilot study recruited men with (1) localized, and (2) metastatic castration resistant prostate cancer (mCRPC). Gadoxetate disodium-enhanced MRI was performed at four timepoints post-injection. The Wilcoxon signed rank test was used to compare MRI contrast enhancement ratio (CER) pre-injection and post-injection. OATP1B3 expression was evaluated via immunohistochemistry (IHC) and a pharmacogenomic analysis of OATP1B3, NCTP and OATP1B1 was conducted. The mCRPC subgroup ( n = 9) demonstrated significant enhancement compared to pre-contrast images at 20-, 40- and 60-min timepoints ( p < 0.0078). The localized cancer subgroup ( n = 11) demonstrated earlier enhancement compared to the mCRPC group, but no retention over time ( p > 0.05). OATP1B3 expression on IHC trended higher contrast enhancement between 20–40 min ( p ≤ 0.064) and was associated with contrast enhancement at 60 min ( p = 0.0422). OATP1B1 haplotype, with N130D and V174A substitutions, impacted enhancement at 40–60 min ( p ≤ 0.038). mCRPC lesions demonstrate enhancement after injection of gadoxetate disodium on MRI and retention over 60 min. As inter-individual variability in OATP1B3 expression and function has both predictive and prognostic significance, gadoxetate disodium has potential as a biomarker in prostate cancer.

Cereblon (CRBN), the substrate receptor of an E3 ubiquitin ligase complex, is a target of thalidomide and thalidomide-derived immunomodulatory drugs (IMiDs). The binding of these IMiDs to CRBN alters the substrate specificity of the ligase, thereby mediating multiple effects that are exploited in cancer therapy. However, to date, it is not clear which other possible targets might be involved in the efficacy of IMiDs. One especially prominent effect of a number of thalidomide analogs is their ability to inhibit angiogenesis, which is typically enhanced in fluorinated analogs. So far, the involvement of CRBN in antiangiogenic effects is under debate. Here, starting from a systematic set of thalidomide analogs and employing a quantitative in vitro CRBN-binding assay, we study the correlation of fluorination, CRBN binding and antiangiogenic effects. We clearly identify fluorination to correlate both with CRBN binding affinity and with antiangiogenic effects, but do not find a correlation between the latter two phenomena, indicating that the main target for the antiangiogenic effects of thalidomide analogs still remains to be identified.

Introduction: Transporters comprising the blood-brain barrier complicate delivery of many therapeutics to the central nervous system. The present study ascertained whether the natural product botryllamide G is viable for in vivo inhibition of ABCG2 using lapatinib as a probe for ABCB1 and ABCG2-mediated efflux from the brain. Methods: Wild-type and Mdr1a/Mdr1b (-/-) mice were treated with botryllamide G and lapatinib (“doublet therapy”), and while a separate cohort of wild-type mice was treated with botryllamide, tariquidar and lapatinib (“triplet therapy”). Results: Botryllamide G demonstrates biphasic elimination with a rapid distribution, decreasing below the in vitro IC50 of 6.9 µM within minutes, yet with a relatively slower terminal half-life (4.6 h). In Mdr1a/Mdr1b (-/-) mice, doublet therapy resulted in a significant increase in brain lapatinib AUC at 8 h (2058 h*ng/mL vs 4007 h*ng/mL; P = .031), but not plasma exposure (P = .15). No significant differences were observed after 24 h. Lapatinib brain exposure was greater through 1 h when wild-type mice were administered triplet therapy (298 h*pg/mg vs 120 h*pg/mg; P < .001), but the triplet decreased brain AUC through 24 h vs. mice administered lapatinib alone (2878 h*pg/mg vs 4461hr*ng/mL; P < .001) and did not alter the brain:plasma ratio. Conclusions: In summary, the ABCG2 inhibitor, botryllamide G, increases brain exposure to lapatinib in mice lacking Abcb1, although the combination of botryllamide G and tariquidar increases brain exposure in wild-type mice only briefly (1 h). Additional research is needed to find analogs of this compound that have better pharmacokinetics and pharmacodynamic effects on ABCG2 inhibition.

Background Immunosuppressive cytokines have the potential to promote prostate cancer progression. Assessing their longitudinal changes may implicate mechanisms of progression, treatment resistance, and suggest new therapeutic targets. Methods Thirty-seven men with biochemically recurrent (BCR) prostate cancer who received 6 months of androgen deprivation therapy (ADT) and were monitored until the time to prostate-specific antigen progression (TTPP) were identified from a completed phase III trial (NCT00020085). Serum samples were archived at baseline, 3 months after ADT, and at TTPP. Cytokine concentrations were quantified using a 36-parameter electrochemiluminescence assay. The Wilcoxon signed-rank sum test was used to compare observations between time points. Kaplan-Meier analysis was used to calculate TTPP dichotomized by cytokine values above or below the median. Pearson's rank correlation coefficient was used to compare continuous variables. Results Median TTPP was 399 days (range, 114-1641). Median prostate-specific antigen (PSA) at baseline and progression were 8.5 and 5.3 ng/mL, respectively. Twenty-three patients (62%) achieved undetectable PSA with ADT. Castrate levels of testosterone (<50 ng/dL) after 3 months of ADT occurred in 35 patients (95%). TNF-alpha (P = .002), IL-23 (P = .002), and CXCL10 (P = .001) significantly increased from baseline to post ADT. Certain cytokines correlated longitudinally: TNF-alpha correlated with IL-23 (r = .72; P < .001) and IL-8 (r = .59; P < .001) from baseline to post ADT and to PSA progression. Neutrophil-to-lymphocyte ratio correlated with IL-27 (r = .57; P < .001) and MIP-3 alpha (r = .56; P < .001). Patients with a detectable PSA after ADT had elevated levels of IL-6 (P = .049) and IL-8 (P = .013) at PSA progression as compared with those with an undetectable PSA. There was a trend toward shorter TTPP in patients with TNF-alpha levels above the median (P = .042). Conclusions Several innate cytokines were associated with biochemically recurrent prostate cancer.

COVID-19 has a clear sex disparity in clinical outcome. Globally, infection rates between men and women are similar; however, men are more likely to have more severe disease and are more likely to die. The causes for this disparity are currently under investigation and are most likely multifactorial. Sex hormones play an important role in the immune response with estrogen seen as immune boosting and testosterone as immunosuppressing. Additionally, an important protease involved in viral entry, TMPRSS2, is regulated by androgens. Many observational and prospective studies are ongoing or initiating to further examine the role of sex hormones in SARS-CoV-2 infection and if modulation of them is a realistic treatment option.