Active Surveillance in Prostate Cancer: Current and Potentially Emerging Biomarkers for Patient Selection Criteria.

BACKGROUND: Prostate cancer (PCa) represents one of the most frequent malignancies and the fifth leading cause of cancer death in adult men worldwide. PCa mortality rates have been declining in several Western countries; one of the possible reasons may be related to the application of prostate-specific antigen early detection policies. These early detection protocols increase PCa-specific patient survival; however, a high percentage of these cases corresponds to low-risk PCa that grows very slowly and is unlikely to metastasize to threaten survival. Many low-risk PCa patients receive aggressive therapies, such as radical prostatectomy and radiotherapy, that are costly for patients and/or health systems and generate side effects that affect the quality of life. An alternative to surgery and radiotherapy treatments for low-risk PCa is active surveillance (AS), a strategy based on close disease monitoring and intervention only if the disease progresses. However, proper identification of low-risk PCa patients at the time of diagnosis is essential for the effectiveness AS. The selection of AS candidates remains challenging; thus, effective prognostic biomarkers are needed. SUMMARY: This review article addresses the characteristics of the current and emerging PCa prognostic biomarkers, including tests available for tissue, blood, and urine analyses, for the appropriate selection of PCa patients for AS. In addition, and based on published literature, we performed a selection of potential new biomarkers that can distinguish low-risk PCa. KEY MESSAGES: The literature search yielded four tissue-based tests, two blood-based tests, and six urine-based tests that can be used to determine PCa risk classification. However, most available tests are expensive; thus, cost-effective analyses are needed in order to obtain the approval of government agencies and to be financed by the health systems. Available prognostic urine tests have shown great progress over the last years, and they have the advantage of being minimally invasive; therefore, they may become a routine disease progression test for patients under AS. In addition, new research conducted in the last decade has shown promising biomarkers, including mRNA, miRNA, long noncoding RNA, and metabolites, that could improve existing tests or allow the development of new tools for AS patient selection.

Iron accumulation typifies renal cell carcinoma tumorigenesis but abates with pathological progression, sarcomatoid dedifferentiation, and metastasis.

Iron is a potent catalyst of oxidative stress and cellular proliferation implicated in renal cell carcinoma (RCC) tumorigenesis, yet it also drives ferroptosis that suppresses cancer progression and represents a novel therapeutic target for advanced RCC. The von Hippel Lindau (VHL)/hypoxia-inducible factor-α (HIF-α) axis is a major regulator of cellular iron, and its inactivation underlying most clear cell (cc) RCC tumors introduces both iron dependency and ferroptosis susceptibility. Despite the central role for iron in VHL/HIF-α signaling and ferroptosis, RCC iron levels and their dynamics during RCC initiation/progression are poorly defined. Here, we conducted a large-scale investigation into the incidence and prognostic significance of total tissue iron in ccRCC and non-ccRCC patient primary tumor cancer cells, tumor microenvironment (TME), metastases and non-neoplastic kidneys. Prussian Blue staining was performed to detect non-heme iron accumulation in over 1600 needle-core sections across multiple tissue microarrays. We found that RCC had significantly higher iron staining scores compared with other solid cancers and, on average, >40 times higher than adjacent renal epithelium. RCC cell iron levels correlated positively with TME iron levels and inversely with RCC levels of the main iron uptake protein, transferrin receptor 1 (TfR1/TFRC/CD71). Intriguingly, RCC iron levels, including in the TME, decreased significantly with pathologic (size/stage/grade) progression, sarcomatoid dedifferentiation, and metastasis, particularly among patients with ccRCC, despite increasing TfR1 levels, consistent with an increasingly iron-deficient tumor state. Opposite to tumor iron changes, adjacent renal epithelial iron increased significantly with RCC/ccRCC progression, sarcomatoid dedifferentiation, and metastasis. Lower tumor iron and higher renal epithelial iron each predicted significantly shorter ccRCC patient metastasis-free survival. In conclusion, iron accumulation typifies RCC tumors but declines toward a relative iron-deficient tumor state during progression to metastasis, despite precisely opposite dynamics in adjacent renal epithelium. These findings raise questions regarding the historically presumed selective advantage for high iron during all phases of cancer evolution, suggesting instead distinct tissue-specific roles during RCC carcinogenesis and early tumorigenesis versus later progression. Future study is warranted to determine how the relative iron deficiency of advanced RCC contributes to ferroptosis resistance and/or introduces a heightened susceptibility to iron deprivation that might be therapeutically exploitable.

Single-Cell Analyses of a Novel Mouse Urothelial Carcinoma Model Reveal a Role of Tumor-Associated Macrophages in Response to Anti-PD-1 Therapy.

Approximately 80% of patients with advanced bladder cancer do not respond to immune checkpoint inhibitor (ICI) immunotherapy. Therefore, there is an urgent unmet need to develop clinically relevant preclinical models so that factors governing immunotherapy responses can be studied in immunocompetent mice. We developed a line of mouse triple knockout (TKO: Trp53, Pten, Rb1) urothelial carcinoma organoids transplanted into immunocompetent mice. These bladder tumors recapitulate the molecular phenotypes and heterogeneous immunotherapy responses observed in human bladder cancers. The TKO organoids were characterized in vivo and in vitro and compared to the widely used MB49 murine bladder cancer model. RNAseq analysis of the TKO tumors demonstrated a basal subtype. The TKO xenografts demonstrated the expression of urothelial markers (CK5, CK7, GATA3, and p63), whereas MB49 subcutaneous xenografts did not express urothelial markers. Anti-PD-1 immunotherapy resulted in a mixed pattern of treatment responses for individual tumors. Eight immune cell types were identified (basophils, B cells, dendritic cells, macrophages, monocytes, neutrophils, NK cells, and T cells) in ICI-treated xenografts. Responder xenografts displayed significantly increased immune cell infiltration (15.3%, 742 immune cells/4861 total cells) compared to the non-responder tumors (10.1%, 452 immune cells/4459 total cells, Fisher Exact Test p < 0.0001). Specifically, there were more T cells (1.0% vs. 0.4%, p = 0.002) and macrophages (8.6% vs. 6.4%, p = 0.0002) in responder xenografts than in non-responder xenografts. In conclusion, we have developed a novel preclinical model that exhibits a mixed pattern of response to anti-PD-1 immunotherapy. The higher percentage of macrophage tumor infiltration in responders suggests a potential role for the innate immune microenvironment in regulating ICI treatment responses.

A Preclinical Study to Repurpose Spironolactone for Enhancing Chemotherapy Response in Bladder Cancer.

Neoadjuvant chemotherapy (NAC) followed by radical cystectomy is the standard-of-care for patients with muscle-invasive bladder cancer (MIBC). Defects in nucleotide excision repair (NER) are associated with improved responses to NAC. Excision Repair Cross-Complementation group 3 (ERCC3) is a key component of NER process. No NER inhibitors are available for treating patients with bladder cancer. We have developed an ex vivo cell-based assay of 6-4 pyrimidine-pyrimidinone (6-4PP) removal as a surrogate measure of NER capacity in human bladder cancer cell lines. The protein expression of ERCC3 was examined in human MIBC specimens and cell lines. Small molecule inhibitors were screened for NER inhibition in bladder cancer cell lines. Spironolactone was identified as a potent NER inhibitor. Combined effects of spironolactone with chemo-drugs were evaluated in vitro and in vivo. The efficacy between platinum and spironolactone on cytotoxicity was determined by combination index. A correlation between NER capacity and cisplatin sensitivity was demonstrated in a series of bladder cancer cell lines. Further, siRNA-mediated knockdown of ERCC3 abrogated NER capacity and enhanced cisplatin cytotoxicity. Spironolactone inhibited ERCC3 protein expression, abrogated NER capacity, and increased platinum-induced cytotoxicity in bladder cancer cells in vivo and in patient-derived organoids. Moreover, spironolactone exhibited the potential synergism effects with other clinical chemotherapy regimens in bladder cancer cell lines. Our data support the notion of repurposing spironolactone for improving the chemotherapy response of NAC in patients with MIBC. Further clinical trials are warranted to determine the safety and efficacy of spironolactone in combination with chemotherapy.

Differential Associations of SLCO Transporters with Prostate Cancer Aggressiveness between African Americans and European Americans.

BACKGROUND: Androgen receptor signaling is crucial to prostate cancer aggressiveness. Members of the solute carrier family of the organic anion transporting peptides (SLCO) are potential regulators of androgen availability in prostate tissue. It remains unknown whether genetic variations in SLCOs contribute to the differences in prostate cancer aggressiveness in African Americans (AA) and European Americans (EA). METHODS: SNPs in 11 SLCO members were selected, with addition of 139 potentially functional SNPs and 128 ancestry informative markers. A total of 1,045 SNPs were genotyped and analyzed in 993 AAs and 1,057 EAs from the North Carolina-Louisiana Prostate Cancer Project. Expression and cellular localization of SLCOs were examined using qRT-PCR, IHC, and in situ RNA hybridization in independent sets of prostate cancer cases. RESULTS: Significant associations with prostate cancer characteristics were found for SNPs in SLCO2A1 and SLCO5A1. The associations differed by race (P interaction < 0.05). SNPs in SLCO2A1 were associated with reduced tumor aggressiveness and low Gleason score in AAs; whereas, SNPs in SLCO5A1 were associated with high clinical stage in EAs. In prostate tissue, SLCO2A1 and SLCO5A1 were the most expressed SLCOs at the mRNA level and were expressed predominantly in prostate endothelial and epithelial cells at the protein level, respectively. CONCLUSIONS: SLCO2A1 and SLCO5A1 play important but different roles in prostate cancer aggressiveness in AAs versus EAs. IMPACT: The finding calls for consideration of racial differences in biomarker studies of prostate cancer and for investigations on functions of SLCO2A1 and SLCO5A1 in prostate cancer.

Hexose Transporters in Cancer: From Multifunctionality to Diagnosis and Therapy.

Cancer cells increase their metabolic activity by enhancing glucose uptake through overexpression of hexose transporters (Gluts). Gluts also have the capacity to transport other molecules besides glucose, including fructose, mannose, and dehydroascorbic acid (DHA), the oxidized form of vitamin C. The majority of research studies in this field have focused on the role of glucose transport and metabolism in cancer, leaving a substantial gap in our knowledge of the contribution of other hexoses and DHA in cancer biology. Here, we summarize the most recent advances in understanding the role that the multifunctional transport capacity of Gluts plays in biological and clinical aspects of cancer, and how these characteristics can be exploited in the search for novel diagnostic and therapeutic strategies.

Understanding the Relationship between Environmental Arsenic and Prostate Cancer Aggressiveness among African-American and European-American Men in North Carolina.

High-level exposure to arsenic, a known carcinogen and endocrine disruptor, is associated with prostate cancer (PCa) mortality. Whether low-level exposure is associated with PCa aggressiveness remains unknown. We examined the association between urinary arsenic and PCa aggressiveness among men in North Carolina. This cross-sectional study included 463 African-American and 491 European-American men with newly diagnosed, histologically confirmed prostate adenocarcinoma. PCa aggressiveness was defined as low aggressive (Gleason score < 7, stage = cT1-cT2, and PSA < 10 ng/mL) versus intermediate/high aggressive (all other cases). Total arsenic and arsenical species (inorganic arsenic (iAsIII + iAsV), arsenobetaine, monomethyl arsenic, and dimethyl arsenic)) and specific gravity were measured in spot urine samples obtained an average of 23.7 weeks after diagnosis. Multivariable logistic regression was used to estimate the covariate-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) for PCa aggressiveness in association with arsenic tertiles/quantiles overall and by race. The highest (vs. lowest) tertile of total arsenic was associated with PCa aggressiveness ORs of 1.77 (95% CI = 1.05-2.98) among European-American men, and 0.94 (95% CI = 0.57-1.56) among African-American men (PInteraction = 0.04). In contrast, total arsenic and arsenical species were not associated with PCa aggressiveness in unstratified models. Low-level arsenic exposure may be associated with PCa aggressiveness among European-Americans, but not among African-Americans.

Identification of Plasma Glycosphingolipids as Potential Biomarkers for Prostate Cancer (PCa) Status.

Prostate cancer (PCa) is the most common male cancer and the second leading cause of cancer death in United States men. Controversy continues over the effectiveness of prostate-specific antigen (PSA) for distinguishing aggressive from indolent PCa. There is a critical need for more specific and sensitive biomarkers to detect and distinguish low- versus high-risk PCa cases. Discovery metabolomics were performed utilizing ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS) on plasma samples from 159 men with treatment naïve prostate cancer participating in the North Carolina-Louisiana PCa Project to determine if there were metabolites associated with aggressive PCa. Thirty-five identifiable plasma small molecules were associated with PCa aggressiveness, 15 of which were sphingolipids; nine common molecules were present in both African-American and European-American men. The molecules most associated with PCa aggressiveness were glycosphingolipids; levels of trihexosylceramide and tetrahexosylceramide were most closely associated with high-aggressive PCa. The Cancer Genome Atlas was queried to determine gene alterations within glycosphingolipid metabolism that are associated with PCa and other cancers. Genes that encode enzymes associated with the metabolism of glycosphingolipids were altered in 12% of PCa and >30% of lung, uterine, and ovarian cancers. These data suggest that the identified plasma (glyco)sphingolipids should be further validated for their association with aggressive PCa, suggesting that specific sphingolipids may be included in a diagnostic signature for PCa.

Recapitulation of prostate tissue cell type-specific transcriptomes by an in vivo primary prostate tissue xenograft model.

Studies of the normal functions and diseases of the prostate request in vivo models that maintain the tissue architecture and the multiple-cell type compartments of human origin in order to recapitulate reliably the interactions of different cell types. Cell type-specific transcriptomes are critical to reveal the roles of each cell type in the functions and diseases of the prostate. A primary prostate tissue xenograft model was developed using fresh human prostate tissue specimens transplanted onto male mice that were castrated surgically and implanted with a device to maintain circulating testosterone levels comparable to adult human males. Endothelial cells and epithelial cells were isolated from 7 fresh human prostate tissue specimens and from primary tissue xenografts established from 9 fresh human prostate tissue specimens, using antibody-conjugated magnetic beads specific to human CD31 and human EpCAM, respectively. Transcriptomes of endothelial, epithelial and stromal cell fractions were obtained using RNA-Seq. Global and function-specific gene expression profiles were compared in inter-cell type and inter-tissue type manners. Gene expression profiles in the individual cell types isolated from xenografts were similar to those of cells isolated from fresh tissue, demonstrating the value of the primary tissue xenograft model for studies of the inter-relationships between prostatic cell types and the role of such inter-relationships in organ development, disease progression, and response to drug treatments.

A CD24-p53 axis contributes to African American prostate cancer disparities.

BACKGROUND: Using a functional analysis of prostate cancer cells, we found a CD24-dependent inactivation of mutant p53, but the clinical significance of this observation remained uncertain. Here, we validated these results with samples of human prostate cancer and explored the role of a CD24-p53 axis in racial disparities of prostate cancer. METHODS: Samples of formalin-fixed, paraffin-embedded prostate cancer from 141 European Americans (EAs) and 147 African Americans (AAs) in two independent sample cohorts were assessed for protein expression of CD24, mutant p53, mouse double minute 2 human homolog (MDM2), and cyclin dependent kinase inhibitor 2A (ARF) using immunohistochemical analyses. All samples were analyzed for TP53R175H and TP53R273H . RESULTS: CD24, mutant p53, MDM2, and ARF proteins were expressed in 55%, 24%, 39%, and 68% of prostate cancer samples, respectively. CD24 and mutant p53 were present more frequently in late-stage and metastatic prostate cancer. The presence of CD24 was associated with a greater than fourfold risk of metastasis, which included lymph node and distant metastases. H score analysis showed positive correlations of CD24 expression with mutant p53 (r = .308, P < .001) and MDM2 (r = .227, P = .004). There was a negative correlation for CD24 with ARF (r = -.280, P < .001). A racial disparity was evident for CD24 (AAs/EAs: 64% vs 47%; P = .004) but not for mutant p53 (AA/EA: 28% vs 21%; P = .152). In 32 CD24+ /mutant p53+ cases, a TP53R273H mutation was found in five cases, but no TP53R175H mutation was found. CONCLUSION: The CD24-p53 axis may contribute to aggressive and metastatic prostate cancers, especially those of AAs. This observation enhances understanding of the pathogenesis of prostate cancer and its associated racial disparities.

Association among plasma 1,25(OH)2 D, ratio of 1,25(OH)2 D to 25(OH)D, and prostate cancer aggressiveness.

BACKGROUND: African-American (AA) men tend to present with more aggressive prostate cancer (Gleason score >7) than European-American (EA) men. Vitamin D and its metabolites are implicated in prostate cancer biology with vitamin D deficiency, indicated by its metabolite levels in serum or plasma, usually observed in AA men. OBJECTIVE: To determine if 1, 25-dihydroxy vitamin D3 [1,25(OH)2 D] plasma levels in AA and EA prostate cancer patients alter the risk of having aggressive prostate cancer. DESIGN: Research subjects from the North Carolina-Louisiana Prostate Cancer Project (AA n = 435 and EA n = 532) were included. Plasma metabolites 1,25(OH)2 D and 25-hydroxyvitamin D3 [25(OH)D] were measured using liquid chromatography with tandem mass spectrophotometry. Research subjects were classified into low (Gleason sum < 7, stage T1-T2, and Prostate-specific antigen (PSA) < 9 ng/mL) or high (Gleason sum > 8 or Gleason sum = 7 with 4 + 3, or PSA > 20 ng/mL, or Gleason sum = 7 and stage T3-T4) aggressive disease. RESULTS: Research subjects in the second and third tertiles of plasma levels of 1, 25(OH)2 D had lower odds of high aggressive prostate cancer (AA [ORT2vsT1 : 0.66, 95%CI: 0.39-1.12; ORT3vsT1 : 0.83, 95%CI: 0.49-1.41] and EA [ORT2vsT1 : 0.68, 95%CI: 0.41-1.11; ORT3vsT1 : 0.67, 95%CI: 0.40-1.11]) compared with the first tertile, though confidence intervals included the null. Greater 1,25(OH)2 D/25(OH)D molar ratios were associated with lower odds of high aggressive prostate cancer more evidently in AA (ORQ4vsQ1 : 0.45, CI: 0.24-0.82) than in EA (ORQ4vsQ1 : 0.64, CI: 0.35-1.17) research subjects. CONCLUSIONS: The 1,25(OH)2 D/25(OH)D molar ratio was associated with decreased risk of high aggressive prostate cancer in AA men, and possibly in EA men. Further studies analyzing vitamin D polymorphisms, vitamin D binding protein levels, and prostatic levels of these metabolites may be useful. These studies may provide a better understanding of the vitamin D pathway and its biological role underlying health disparities in prostate cancer.

Adrenal androgens rescue prostatic dihydrotestosterone production and growth of prostate cancer cells after castration.

Adrenal androgens dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS) are potential substrates for intracrine production of testosterone (T) and dihydrotestosterone (DHT), or directly to DHT, by prostate cancer (PCa) cells. Production of DHT from DHEAS and DHEA, and the role of steroid sulfatase (STS), were evaluated ex vivo using fresh human prostate tissue and in vitro using human PCa cell lines. STS was expressed in benign prostate tissue and PCa tissue. DHEAS at a physiological concentration was converted to DHT in prostate tissue and PCa cell lines, which was STS-dependent. DHEAS activation of androgen receptor (AR) and stimulation of PCa cell growth were STS-dependent. DHEA at a physiological concentration was not converted to DHT ex vivo and in vitro, but stimulated in vivo tumor growth of the human PCa cell line, VCaP, in castrated mice. The findings suggest that targeting metabolism of DHEAS and DHEA may enhance androgen deprivation therapy.

Suppressive effects of iron chelation in clear cell renal cell carcinoma and their dependency on VHL inactivation.

Increasing data implicate iron accumulation in tumorigenesis of the kidney, particularly the clear cell renal cell carcinoma (ccRCC) subtype. The von Hippel Lindau (VHL)/hypoxia inducible factor-α (HIF-α) axis is uniquely dysregulated in ccRCC and is a major regulator and regulatory target of iron metabolism, yet the role of iron in ccRCC tumorigenesis and its potential interplay with VHL inactivation remains unclear. We investigated whether ccRCC iron accumulation occurs due to increased cell dependency on iron for growth and survival as a result of VHL inactivation. Free iron levels were compared between four VHL-mutant ccRCC cell lines (786-0, A704, 769-P, RCC4) and two benign renal tubule epithelial cell lines (RPTEC, HRCEp) using the Phen Green SK fluorescent iron stain. Intracellular iron deprivation was achieved using two clinical iron chelator drugs, deferasirox (DFX) and deferoxamine (DFO), and chelator effects were measured on cell line growth, cell cycle phase, apoptosis, HIF-1α and HIF-2α protein levels and HIF-α transcriptional activity based on expression of target genes CA9, OCT4/POU5F1 and PDGFß/PDGFB. Similar assays were performed in VHL-mutant ccRCC cells with and without ectopic wild-type VHL expression. Baseline free iron levels were significantly higher in ccRCC cell lines than benign renal cell lines. DFX depleted cellular free iron more rapidly than DFO and led to greater growth suppression of ccRCC cell lines (>90% at ~30-150 µM) than benign renal cell lines (~10-50% at up to 250 µM). Similar growth responses were observed using DFO, with the exception that a prolonged treatment duration was necessary to deplete cellular iron adequately for differential growth suppression of the less susceptible A704 ccRCC cell line relative to benign renal cell lines. Apoptosis and G1-phase cell cycle arrest were identified as potential mechanisms of chelator growth suppression based on their induction in ccRCC cell lines but not benign renal cell lines. Iron chelation in ccRCC cells but not benign renal cells suppressed HIF-1α and HIF-2α protein levels and transcriptional activity, and the degree and timing of HIF-2α suppression correlated with the onset of apoptosis. Restoration of wild-type VHL function in ccRCC cells was sufficient to prevent chelator-induced apoptosis and G1 cell cycle arrest, indicating that ccRCC susceptibility to iron deprivation is VHL inactivation-dependent. In conclusion, ccRCC cells are characterized by high free iron levels and a cancer-specific dependency on iron for HIF-α overexpression, cell cycle progression and apoptotic escape. This iron dependency is introduced by VHL inactivation, revealing a novel interplay between VHL/HIF-α dysregulation and ccRCC iron metabolism. Future study is warranted to determine if iron deprivation using chelator drugs provides an effective therapeutic strategy for targeting HIF-2α and suppressing tumor progression in ccRCC patients.

Transferrin receptor 1 upregulation in primary tumor and downregulation in benign kidney is associated with progression and mortality in renal cell carcinoma patients.

The central dysregulated pathway of clear cell (cc) renal cell carcinoma (RCC), the von Hippel Lindau/hypoxia inducible factor-α axis, is a key regulator of intracellular iron levels, however the role of iron uptake in human RCC tumorigenesis and progression remains unknown. We conducted a thorough, large-scale investigation of the expression and prognostic significance of the primary iron uptake protein, transferrin receptor 1 (TfR1/CD71/TFRC), in RCC patients. TfR1 immunohistochemistry was performed in over 1500 cores from 574 renal cell tumor patient tissues (primary tumors, matched benign kidneys, metastases) and non-neoplastic tissues from 36 different body sites. TfR1 levels in RCC tumors, particularly ccRCC, were significantly associated with adverse clinical prognostic features (anemia, lower body mass index, smoking), worse tumor pathology (size, stage, grade, multifocality, sarcomatoid dedifferentiation) and worse survival outcomes, including after adjustments for tumor pathology. Highest TfR1 tissue levels in the non-gravid body were detected in benign renal tubule epithelium. Opposite to TfR1 changes in the primary tumor, TfR1 levels in benign kidney dropped during tumor progression and were inversely associated with worse survival outcomes, independent of tumor pathology. Quantitative measurement of TfR1 subcellular localization in cell lines demonstrated mixed cytoplasmic and membranous expression with increased TfR1 in clusters in ccRCC versus benign renal cell lines. Results of this study support an important role for TfR1 in RCC progression and identify TfR1 as a novel RCC biomarker and therapeutic target.

Androgens modulate male-derived endothelial cell homeostasis using androgen receptor-dependent and receptor-independent mechanisms.

BACKGROUND: Sex-related differences in the role of androgen have been reported in cardiovascular diseases and angiogenesis. Moreover, androgen receptor (AR) has been causally involved in the homeostasis of human prostate endothelial cells. However, levels of expression, functionality and biological role of AR in male- and female-derived human endothelial cells (ECs) remain poorly characterized. The objectives of this work were (1) to characterize the functional expression of AR in male- and female-derived human umbilical vein endothelial cell (HUVEC), and (2) to specifically analyze the biological effects of DHT, and the role of AR on these effects, in male-derived HUVECs (mHUVECs). RESULTS: Immunohistochemical analyses of tissue microarrays from benign human tissues confirmed expression of AR in ECs from several androgen-regulated and non-androgen-regulated human organs. Functional expression of AR was validated in vitro in male- and female-derived HUVECs using quantitative RT-PCR, immunoblotting and AR-mediated transcriptional activity assays. Our results indicated that functional expression of AR in male- and female-derived HUVECs was heterogeneous, but not sex dependent. In parallel, we analyzed in depth the biological effects of DHT, and the role of AR on these effects, on proliferation, survival and tube formation capacity in mHUVECs. Our results indicated that DHT did not affect mHUVEC survival; however, DHT stimulated mHUVEC proliferation and suppressed mHUVEC tube formation capacity. While the effect of DHT on proliferation was mediated through AR, the effect of DHT on tube formation did not depend on the presence of a functional AR, but rather depended on the ability of mHUVECs to further metabolize DHT. CONCLUSIONS: (1) Heterogeneous expression of AR in male- and female-derived HUVEC could define the presence of functionally different subpopulations of ECs that may be affected differentially by androgens, which could explain, at least in part, the pleiotropic effects of androgen on vascular biology, and (2) DHT, and metabolites of DHT, generally thought to represent progressively more hydrophilic products along the path to elimination, may have differential roles in modulating the biology of human ECs through AR-dependent and AR-independent mechanisms, respectively.

Carotenoid intake and adipose tissue carotenoid levels in relation to prostate cancer aggressiveness among African-American and European-American men in the North Carolina-Louisiana prostate cancer project (PCaP).

BACKGROUND: Associations between carotenoid intake and prostate cancer (CaP) incidence have varied across studies. This may result from combining indolent with aggressive disease in most studies. This study examined whether carotenoid intake and adipose tissue carotenoid levels were inversely associated with CaP aggressiveness. METHODS: Data on African-American (AA, n = 1,023) and European-American (EA, n = 1,079) men with incident CaP from North Carolina and Louisiana were analyzed. Dietary carotenoid intake was assessed using a detailed-food frequency questionnaire (FFQ), and abdominal adipose tissue samples were analyzed for carotenoid concentrations using high-performance liquid chromatography. Multivariable logistic regression was used in race-stratified analyses to calculate odds ratios (ORs) and 95% confidence intervals (95%CI) comparing high aggressive CaP with low/intermediate aggressive CaP. RESULTS: Carotenoid intake differed significantly between AAs and EAs, which included higher intake of lycopene among EAs and higher ß-cryptoxanthin intake among AAs. Comparing the highest and lowest tertiles, dietary lycopene was associated inversely with high aggressive CaP among EAs (OR = 0.55, 95%CI: 0.34-0.89, Ptrend = 0.02), while an inverse association was observed between dietary ß-cryptoxanthin intake and high aggressive CaP among AAs (OR = 0.56, 95%CI: 0.36-0.87, Ptrend = 0.01). Adipose tissue α-carotene and lycopene (cis + trans) concentrations were higher among EAs than AAs, and marginally significant inverse linear trends were observed for adipose α-carotene (Ptrend = 0.07) and lycopene (Ptrend = 0.11), and CaP aggressiveness among EAs only. CONCLUSIONS: These results suggest that diets high in lycopene and ß-cryptoxanthin may protect against aggressive CaP among EAs and AAs, respectively. Differences in dietary behaviors may explain the observed racial differences in associations. Prostate 76:1053-1066, 2016. © 2016 Wiley Periodicals, Inc.

Dietary, supplement, and adipose tissue tocopherol levels in relation to prostate cancer aggressiveness among African and European Americans: The North Carolina-Louisiana Prostate Cancer Project (PCaP).

BACKGROUND: Controversies remain over the safety and efficacy of vitamin E (i.e., α-tocopherol) supplementation use for the prevention of prostate cancer (CaP); however, associations of different tocopherol forms and CaP aggressiveness have yet to be examined. METHODS: This study examined whether food intake of tocopherols, vitamin E supplement use, and adipose tissue biomarkers of tocopherol were associated with CaP aggressiveness among African-American (AA, n = 1,023) and European-American (EA, n = 1,079) men diagnosed with incident CaP. Dietary tocopherols were estimated from a food frequency questionnaire, supplement use from questionnaire/inventory, and biomarkers from abdominal adipose samples measured using high-performance liquid chromatography. Odds ratios (ORs) and 95% confidence intervals (95%CIs) were estimated from logistic regression comparing high-aggressive CaP to low/intermediate aggressive CaP, adjusting for covariates. RESULTS: Dietary intakes of α-and δ-tocopherol were related inversely to CaP aggressiveness among EAs [OR (95%CI), highest versus lowest quartile: α-tocopherol, 0.34 (0.17-0.69), P(trend) = 0.006; δ-tocopherol, 0.45 (0.21-0.95) P(trend) = 0.007]. Inverse associations between dietary and supplemental α-tocopherol and CaP aggressiveness were observed among AAs, though these did not reach statistical significance [OR (95%CI), highest versus lowest quartile: dietary α-tocopherol, 0.58 (0.28-1.19), P(trend) = 0.20; supplemental α-tocopherol, 0.64 (0.31-1.21) P(trend) = 0.15]. No significant association was observed between adipose tocopherol levels and CaP aggressiveness [OR (95%CI), highest versus lowest quartiles of α-tocopherol for EAs 1.43 (0.66-3.11) and AAs 0.66 (0.27-1.62)]. CONCLUSIONS: The inverse associations observed between dietary sources of tocopherols and CaP aggressiveness suggests a beneficial role of food sources of these tocopherols in CaP aggressiveness.

Anti-angiogenic activity of PSA-derived peptides.

BACKGROUND: PSA is a biomarker for diagnosis and management of prostate cancer. PSA is known to have anti-tumorigenic activities, however, the physiological role of PSA in prostate tumor progression is not well understood. METHODS: Five candidate peptides identified based upon computer modeling of the PSA crystal structure and hydrophobicity were synthesized at >95% purity. The peptides in a linear form, and a constrained form forced by a di-sulfide bond joining the two ends of the peptide, were investigated for anti-angiogenic activity in HUVEC. RESULTS: None of the five PSA-mimetic peptides exhibited PSA-like serine protease activity. Two of the peptides demonstrated significant anti-angiogenic activity in HUVEC based on (i) inhibition of cell migration and invasion; (ii) inhibition of tube formation in Matrigel; (iii) anti-angiogenic activity in a sprouting assay; and (iv) altered expression of pro- and anti-angiogenic growth factors. Constrained PSA-mimetic peptides had greater anti-angiogenic activity than the corresponding linearized form. Complexing of PSA with ACT eliminated PSA enzymatic activity and reduced anti-angiogenic activity. In contrast, ACT had no effect on the anti-angiogenic effects of the linear or constrained PSA-mimetic peptides. Modeling of the ACT-PSA complex demonstrated ACT sterically blocks the anti-angiogenic activity of the two bioactive peptides. CONCLUSIONS: The interaction of a hydrophilic domain on the surface of the PSA molecule with a target on the cell membrane of prostate endothelial and epithelial cells was responsible for the anti-angiogenic or anti-tumorigenic activity of PSA: enzymatic activity was not associated with anti-angiogenic effects. Furthermore, since PSA and ACT are both expressed within the human prostate tissue microenvironment, the balance of their expression may represent a mechanism for endogenous regulation of tissue angiogenesis.

Association between Plasma 25-Hydroxyvitamin D, Ancestry and Aggressive Prostate Cancer among African Americans and European Americans in PCaP.

BACKGROUND: African Americans (AAs) have lower circulating 25-hydroxyvitamin D3 [25(OH)D3] concentrations and higher prostate cancer (CaP) aggressiveness than other racial/ethnic groups. The purpose of the current study was to examine the relationship between plasma 25(OH)D3, African ancestry and CaP aggressiveness among AAs and European Americans (EAs). METHODS: Plasma 25(OH)D3 was measured using LC-MS/MS (Liquid Chromatography Tandem Mass Spectrometry) in 537 AA and 663 EA newly-diagnosed CaP patients from the North Carolina-Louisiana Prostate Cancer Project (PCaP) classified as having either 'high' or 'low' aggressive disease based on clinical stage, Gleason grade and prostate specific antigen at diagnosis. Mean plasma 25(OH)D3 concentrations were compared by proportion of African ancestry. Logistic regression was used to calculate multivariable adjusted odds ratios (OR) and 95% confidence intervals (95%CI) for high aggressive CaP by tertile of plasma 25(OH)D3. RESULTS: AAs with highest percent African ancestry (>95%) had the lowest mean plasma 25(OH)D3 concentrations. Overall, plasma 25(OH)D3 was associated positively with aggressiveness among AA men, an association that was modified by calcium intake (ORT 3vs.T1: 2.23, 95%CI: 1.26-3.95 among men with low calcium intake, and ORT 3vs.T1: 0.19, 95%CI: 0.05-0.70 among men with high calcium intake). Among EAs, the point estimates of the ORs were <1.0 for the upper tertiles with CIs that included the null. CONCLUSIONS: Among AAs, plasma 25(OH)D3 was associated positively with CaP aggressiveness among men with low calcium intake and inversely among men with high calcium intake. The clinical significance of circulating concentrations of 25(OH)D3 and interactions with calcium intake in the AA population warrants further study.

Selective targeting of bioengineered platelets to prostate cancer vasculature: new paradigm for therapeutic modalities.

Androgen deprivation therapy (ADT) provides palliation for most patients with advanced prostate cancer (CaP); however, greater than 80% subsequently fail ADT. ADT has been indicated to induce an acute but transient destabilization of the prostate vasculature in animal models and humans. Human re-hydrated lyophilized platelets (hRL-P) were investigated as a prototype for therapeutic agents designed to target selectively the tumour-associated vasculature in CaP. The ability of hRL-P to bind the perturbed endothelial cells was tested using thrombin- and ADP-activated human umbilical vein endothelial cells (HUVEC), as well as primary xenografts of human prostate tissue undergoing acute vascular involution in response to ADT. hRL-P adhered to activated HUVEC in a dose-responsive manner. Systemically administered hRL-P, and hRL-P loaded with super-paramagnetic iron oxide (SPIO) nanoparticles, selectively targeted the ADT-damaged human microvasculature in primary xenografts of human prostate tissue. This study demonstrated that hRL-P pre-loaded with chemo-therapeutics or nanoparticles could provide a new paradigm for therapeutic modalities to prevent the rebound/increase in prostate vasculature after ADT, inhibiting the transition to castration-recurrent growth.