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Analysis of DNA methylation is a valuable tool to understand disease progression and is increasingly being used to create diagnostic and prognostic clinical biomarkers. While conversion of cytosine to 5-methylcytosine (5mC) commonly results in transcriptional repression, further conversion to 5-hydroxymethylcytosine (5hmC) is associated with transcriptional activation. Here we perform the first study integrating whole-genome 5hmC with DNA, 5mC, and transcriptome sequencing in clinical samples of benign, localized, and advanced prostate cancer. 5hmC is shown to mark activation of cancer drivers and downstream targets. Furthermore, 5hmC sequencing revealed profoundly altered cell states throughout the disease course, characterized by increased proliferation, oncogenic signaling, dedifferentiation, and lineage plasticity to neuroendocrine and gastrointestinal lineages. Finally, 5hmC sequencing of cell-free DNA from patients with metastatic disease proved useful as a prognostic biomarker able to identify an aggressive subtype of prostate cancer using the genes TOP2A and EZH2, previously only detectable by transcriptomic analysis of solid tumor biopsies. Overall, these findings reveal that 5hmC marks epigenomic activation in prostate cancer and identify hallmarks of prostate cancer progression with potential as biomarkers of aggressive disease. Significance: In prostate cancer, 5-hydroxymethylcytosine delineates oncogene activation and stage-specific cell states and can be analyzed in liquid biopsies to detect cancer phenotypes. See related article by Wu and Attard, p. 3880

We report a covalent chemistry-based hepatocellular carcinoma (HCC)-specific extracellular vesicle (EV) purification system for early detection of HCC by performing digital scoring on the purified EVs. Earlier detection of HCC creates more opportunities for curative therapeutic interventions. EVs are present in circulation at relatively early stages of disease, providing potential opportunities for HCC early detection. We develop an HCC EV purification system (i.e., EV Click Chips) by synergistically integrating covalent chemistry-mediated EV capture/release, multimarker antibody cocktails, nanostructured substrates, and microfluidic chaotic mixers. We then explore the translational potential of EV Click Chips using 158 plasma samples of HCC patients and control cohorts. The purified HCC EVs are subjected to reverse-transcription droplet digital PCR for quantification of 10 HCC-specific mRNA markers and computation of digital scoring. The HCC EV-derived molecular signatures exhibit great potential for noninvasive early detection of HCC from at-risk cirrhotic patients with an area under receiver operator characteristic curve of 0.93 (95% CI, 0.86 to 1.00; sensitivity = 94.4%, specificity = 88.5%)., Extracellular vesicles (EVs) are present in circulation at relatively early stages of disease, providing potential opportunities for early cancer diagnosis. Here, the authors report a covalent chemistry-based hepatocellular carcinoma (HCC)-specific EV purification system for early detection of HCC by performing digital scoring on the purified EVs.

Simple Summary Extracellular vesicles (EVs) are heterogenous in size, cargo, and mechanism of biogenesis. While the mechanism of formation of small EVs, such as exosomes, has been widely investigated, little is known about the pathobiology of large EVs. We identify here a microRNA that alters cellular vesicologenesis increasing shedding of large EVs and slightly reducing shedding of small EVs. We also demonstrate that this microRNA, miR1227, targets SEC23A to promote this phenotype. Importantly, large EVs are released by cells undergoing a mesenchymal-amoeboid transition that functionally translates into a more metastatic phenotype. Abstract Cancer cells shed a heterogenous mixture of extracellular vesicles (EVs), differing in both size and composition, which likely influence physiological processes in different manners. However, how cells differentially control the shedding of these EV populations is poorly understood. Here, we show that miR-1227, which is enriched in prostate cancer EVs, compared to the cell of origin, but not in EVs derived from prostate benign epithelial cells, induces the shedding of large EVs (such as large oncosomes), while inhibiting the shedding of small EVs (such as exosomes). RNA sequencing from cells stably expressing miR-1227, a modified RISCTRAP assay that stabilizes and purifies mRNA-miR-1227 complexes for RNA sequencing, and in silico target prediction tools were used to identify miR-1227 targets that may mediate this alteration in EV shedding. The COPII vesicle protein SEC23A emerged and was validated by qPCR, WBlot, and luciferase assays as a direct target of miR-1227. The inhibition of SEC23A was sufficient to induce the shedding of large EVs. These results identify a novel mechanism of EV shedding, by which the inhibition of SEC23A by miR-1227 induces a shift in EV shedding, favoring the shedding of large EV over small EV.

Purpose: Apalutamide is a next-generation androgen receptor (AR) inhibitor approved for patients with nonmetastatic castration-resistant prostate cancer (CRPC) and metastatic castration-sensitive prostate cancer. We evaluated the pharmacokinetics, safety, and antitumor activity of apalutamide combined with abiraterone acetate plus prednisone (AA-P) in patients with metastatic CRPC (mCRPC). Patients and Methods: Multicenter, open-label, phase Ib drug–drug interaction study conducted in 57 patients with mCRPC treated with 1,000 mg abiraterone acetate plus 10 mg prednisone daily beginning on cycle 1 day 1 (C1D1) and 240 mg apalutamide daily starting on C1D8 in 28-day cycles. Serial blood samples for pharmacokinetic analysis were collected on C1D7 and C2D8. Results: Systemic exposure to abiraterone, prednisone, and prednisolone decreased 14%, 61%, and 42%, respectively, when apalutamide was coadministered with AA-P. No increase in mineralocorticoid excess–related adverse events was observed. Patients without prior exposure to AR signaling inhibitors had longer median treatment duration and greater mean decrease in prostate-specific antigen (PSA) from baseline compared with those who had received prior therapy. Confirmed PSA reductions of ≥50% from baseline at any time were observed in 80% (12/15) of AR signaling inhibitor–naïve patients and 14% (6/42) of AR signaling inhibitor–treated patients. Conclusions: Treatment with apalutamide plus AA-P was well tolerated and showed evidence of antitumor activity in patients with mCRPC, including those with disease progression on AR signaling inhibitors. No clinically significant pharmacokinetic interaction was observed between abiraterone and apalutamide; however, apalutamide decreased exposure to prednisone. These data support development of 1,000 mg abiraterone acetate plus 10 mg prednisone daily with 240 mg apalutamide daily in patients with mCRPC.

Asian Americans are the only racial/ethnic group in the U.S. for whom cancer is the leading cause of death in men and women, unlike heart disease for all other groups. Asian Americans face a confluence of cancer risks, with high rates of cancers endemic to their countries of origin due to infectious and cultural reasons, as well as increasing rates of “Western” cancers that are due in part to assimilation to the American diet and lifestyle. Despite the clear mortality risk, Asian Americans are screened for cancers at lower rates than the majority of Americans. Solutions to eliminate the disparity in cancer care are complicated by language and cultural concerns of this very heterogeneous group. This review addresses the disparities in cancer screening, the historical causes, the potential contribution of racism, the importance of cultural perceptions of health care, and potential strategies to address a very complicated problem. Noting that the health care disparities faced by Asian Americans may be less conspicuous than the structural racism that has inflicted significant damage to the health of Black Americans over more than four centuries, this review is meant to raise awareness and to compel the medical establishment to recognize the urgent need to eliminate health disparities for all. Implications for Practice Cancer is the leading cause of death in Asian Americans, who face cancers endemic to their native countries, perhaps because of infectious and cultural factors, as well as those faced by all Americans, perhaps because of “Westernization” in terms of diet and lifestyle. Despite the mortality rates, Asian Americans have less cancer screening than other Americans. This review highlights the need to educate Asian Americans to improve cancer literacy and health care providers to understand the important cancer risks of the fastest-growing racial/ethnic group in the U.S. Eliminating disparities is critical to achieving an equitable society for all Americans.

The addition of docetaxel and abiraterone to androgen deprivation therapy (ADT) heralded a new era in the first-line treatment of metastatic castration-sensitive prostate cancer (mCSPC). Following the success of these combination regimens, 3 new trials presented data on using enzalutamide or apalutamide in men with mCSPC, which showed similar success. These seminal trials collectively established the addition of docetaxel, enzalutamide, apalutamide, or abiraterone to ADT as standards in the upfront treatment of mCSPC. Notably, a subset of patients in these more recent trials were treated with a combination of docetaxel, ADT, and androgen receptor signaling inhibitors or maintenance androgen receptor signaling inhibitors after docetaxel and ADT that provided an initial glimpse into the efficacy of these triplet or maintenance strategies. We discuss the implications of these recent findings and place them in context of the current mCSPC treatment landscape.

Enzalutamide and apalutamide are potent next-generation androgen receptor (AR) antagonists used in metastatic and non-metastatic prostate cancer. Despite the increased survival benefits of these agents, resistance normally occurs and the disease transitions to its lethal form. We hypothesized that enzalutamide and apalutamide suppress 11β-hydroxysteroid dehydrogenase-2 (11β-HSD2), which normally converts cortisol to cortisone, leading to elevated cortisol concentrations and increased ratio of active to inactive glucocorticoids. We measured cortisol and cortisol/cortisone ratio (substrate/product of 11β-HSD2) in serum using mass spectrometry before and 1 month on-treatment in 3 clinical trials: 1) neoadjuvant apalutamide + leuprolide (n=25) 2) enzalutamide +/- PROSTVAC for metastatic castration-resistant prostate cancer (n=54) and 3) enzalutamide +/- PROSTVAC for non-metastatic castration-sensitive prostate cancer (n=38 patients). Progression-free survival (PFS) was determined in the metastatic CRPC study of enzalutamide +/- PROSTVAC for those with glucocorticoid changes above and below the median. A statistically significant rise in cortisol concentration and cortisol/cortisone ratio with AR antagonist treatment occurred uniformly across all 3 clinical trials. For example, a rise in cortisol/cortisone ratio occurred in 23/25 (92%) patients (p < 0.001), 36/54 (67%) patients (p < 0.001), and 30/38 (79%) patients (p = 0.051), in the 3 respective trials. In the trial of enzalutamide +/- PROSTVAC for metastatic CRPC, high cortisol/cortisone ratio in the enzalutamide arm was associated with significantly improved PSA progression-free survival and radiographic progression-free survival. However, in the enzalutamide + PROSTVAC arm, the opposite trend was observed. In conclusion, treatment with enzalutamide or apalutamide increases systemic exposure to active glucocorticoids. These findings have potential consequences for immune suppression and the efficacy of treatment combinations using next-generation AR antagonists. On-treatment, glucocorticoid changes might serve as a pharmacodynamic biomarker.

Purpose: MicroRNAs in the delta-like 1 homolog–deiodinase, iodothyronine 3 (DLK1-DIO3) cluster have been shown to be critical for embryonic development and epithelial to mesenchymal transition (EMT). DLK1-DIO3 cluster miRNAs are elevated in the serum of patients with metastatic cancer. However, the biologic functions of these miRNAs in the EMT and metastasis of cancer cells are poorly understood. We previously demonstrated the oncogenic and metastatic role of miR-409-3p/5p, a member of this cluster, in prostate cancer. In this study, we defined the role of miR-154* and miR-379, two key members of this cluster, in prostate cancer progression and bone metastasis in both cell line models and clinical specimens. Experimental Design: Genetic manipulation of miR-154* and miR-379 was performed to determine their role in tumor growth, EMT, and bone metastasis in mouse models. We determined the expression of miR-154* in prostate cancer clinical samples and bone metastasis samples using in situ hybridization and quantum dot labeling. Results: Elevated expression of miR-154* and miR-379 was observed in bone metastatic prostate cancer cell lines and tissues, and miR-379 expression correlated with progression-free survival of patients with prostate cancer. Intracardiac inoculation (to mimic systemic dissemination) of miR-154* inhibitor-treated bone metastatic ARCaPM prostate cancer cells in mice led to decreased bone metastasis and increased survival. Conclusion: miR-154* and miR-379 play important roles in prostate cancer biology by facilitating tumor growth, EMT, and bone metastasis. This finding has particular translational importance because miRNAs in the DLK1-DIO3 cluster can be attractive biomarkers and possible therapeutic targets to treat bone metastatic prostate cancer. Clin Cancer Res; 20(24); 6559–69. ©2014 AACR.

Purpose: miR-409-3p/-5p is a miRNA expressed by embryonic stem cells, and its role in cancer biology and metastasis is unknown. Our pilot studies demonstrated elevated miR-409-3p/-5p expression in human prostate cancer bone metastatic cell lines; therefore, we defined the biologic impact of manipulation of miR-409-3p/-5p on prostate cancer progression and correlated the levels of its expression with clinical human prostate cancer bone metastatic specimens. Experimental Design: miRNA profiling of a prostate cancer bone metastatic epithelial-to-mesenchymal transition (EMT) cell line model was performed. A Gleason score human tissue array was probed for validation of specific miRNAs. In addition, genetic manipulation of miR-409-3p/-5p was performed to determine its role in tumor growth, EMT, and bone metastasis in mouse models. Results: Elevated expression of miR-409-3p/-5p was observed in bone metastatic prostate cancer cell lines and human prostate cancer tissues with higher Gleason scores. Elevated miR-409-3p expression levels correlated with progression-free survival of patients with prostate cancer. Orthotopic delivery of miR-409-3p/-5p in the murine prostate gland induced tumors where the tumors expressed EMT and stemness markers. Intracardiac inoculation (to mimic systemic dissemination) of miR-409-5p inhibitor–treated bone metastatic ARCaPM prostate cancer cells in mice led to decreased bone metastasis and increased survival compared with control vehicle–treated cells. Conclusion: miR-409-3p/-5p plays an important role in prostate cancer biology by facilitating tumor growth, EMT, and bone metastasis. This finding bears particular translational importance as miR-409-3p/-5p appears to be an attractive biomarker and/or possibly a therapeutic target to treat bone metastatic prostate cancer. Clin Cancer Res; 20(17); 4636–46. ©2014 AACR.

Prostate cancer cells undergo neuroendocrine differentiation during androgen deprivation and secrete neuropeptides, hence activating androgen receptor-regulated genes. Src-family protein kinases are involved in neuropeptide-induced prostate cancer growth and migration. A phase II trial of AZD0530, an oral Src-family kinase inhibitor, in patients with advanced castration resistant prostate cancer was conducted. The primary endpoint was prostate cancer-specific antigen (PSA) response rate, defined as a 30% or greater decrease. A two-stage Simon design was used. Eligibility criteria included documentation of castration resistance (including antiandrogen withdrawal), adequate end-organ function, and performance status, and not more than one prior taxane-based chemotherapy regimen. AZD0530 was given at 175 mg orally once daily continuously. Rapid accrual led to 28 patients registering in the first stage. Median age was 67 years. Sixteen patients had performance status (PS) 0, eight patients had PS 1, and four patients had PS 2. Nine patients (32%) had prior docetaxel-based chemotherapy. Five patients had transient PSA reductions not meeting PSA response criteria. Median progression-free survival time was 8 weeks. Treatment was generally well tolerated. AZD0530, a potent oral Src kinase inhibitor, is feasible and tolerable in this pretreated patient population but possessed little clinical efficacy as monotherapy. Strong preclinical evidence warrants further investigation of AZD0530 in earlier-stage prostate cancer or as combination therapy. © 2009 Wolters Kluwer Health|Lippincott Williams & Wilkins.