Acoustic Enrichment of Heterogeneous Circulating Tumor Cells and Clusters from Metastatic Prostate Cancer Patients.

BACKGROUND: There are important unmet clinical needs to develop cell enrichment technologies to enable unbiased label-free isolation of both single cell and clusters of circulating tumor cells (CTCs) manifesting heterogeneous lineage specificity. Here, we report a pilot study based on the microfluidic acoustophoresis enrichment of CTCs using the CellSearch CTC assay as a reference modality. METHODS: Acoustophoresis uses an ultrasonic standing wave field to separate cells based on biomechanical properties (size, density, and compressibility), resulting in inherently label-free and epitope-independent cell enrichment. Following red blood cell lysis and paraformaldehyde fixation, 6 mL of whole blood from 12 patients with metastatic prostate cancer and 20 healthy controls were processed with acoustophoresis and subsequent image cytometry. RESULTS: Acoustophoresis enabled enrichment and characterization of phenotypic CTCs (EpCAM+, Cytokeratin+, DAPI+, CD45-/CD66b-) in all patients with metastatic prostate cancer and detected CTC-clusters composed of only CTCs or heterogeneous aggregates of CTCs clustered with various types of white blood cells in 9 out of 12 patients. By contrast, CellSearch did not detect any CTC clusters, but detected comparable numbers of phenotypic CTCs as acoustophoresis, with trends of finding a higher number of CTCs using acoustophoresis. CONCLUSION: Our preliminary data indicate that acoustophoresis provides excellent possibilities to detect and characterize CTC clusters as a putative marker of metastatic disease and outcomes. Moreover, acoustophoresis enables the sensitive label-free enrichment of cells with epithelial phenotypes in blood and offers opportunities to detect and characterize CTCs undergoing epithelial-to-mesenchymal transitioning and lineage plasticity.

Ki67 and prostate specific antigen are prognostic in metastatic hormone naïve prostate cancer.

BACKGROUND: For metastatic hormone naïve prostate cancer patients, androgen deprivation therapy (ADT) with escalation therapy including docetaxel and/or androgen targeting drugs is the standard therapy. However, de-escalation is preferable to avoid unnecessary side effects, especially from docetaxel, but markers to identify these patients are lacking. The purpose of the present study was to investigate the potential of PSA and Ki67 immunoreactive scores as prognostic and treatment-predictive markers. MATERIAL AND METHODS: Prostate biopsies from 92 patients with metastatic hormone naïve PC (PSA > 80 ng/mL or clinical metastases) were immunohistochemically evaluated for PSA and Ki67. Gene expression analysis was performed with Clariom D microarrays to identify the phenotypic profile associated with the immunohistochemistry scores of biopsies. Cox regression analysis for progression free survival after ADT adjustment for age, ISUP, and serum PSA and Kaplan-Meier analyses were performed to assess prognostic values of Ki67, PSA, and the Ki67/PSA ratio. RESULTS: The immunohistochemical score for PSA was the strongest prognostic factor for progression-free and overall survival after ADT. Consequently, the ratio between Ki67 and PSA displayed a stronger prognostic value than Ki67 itself. Further, mRNA expression data analysis showed an association between high Ki67/PSA ratio, cell-cycle regulation, and DNA damage repair. In an exploratory sub-analysis of 12 patients treated with early docetaxel as addition to ADT and matched controls, a high Ki67/PSA ratio showed potential to identify those who benefit from docetaxel. CONCLUSION: PSA and Ki67 immunoreactive scores are prognostic in the metastatic hormone-sensitive setting, with PSA being superior. The combination of Ki67 and PSA did not give additional prognostic value. The results suggest immunohistochemical scoring of PSA to have potential to improve identification of patients responding well to ADT alone.

Androgens, aging, and prostate health.

Due to late onset hypogonadism (LOH), there is an increased usage of testosterone replacement therapy (TRT) in the aging male population. Since prostate is a target organ for androgens and anti-androgenic strategies are used to treat and palliate benign prostate hyperplasia (BPH) and prostate cancer (PC), the prevalence of both increases with age, the possible influence of TRT on prostate health becomes highly relevant. The present review summarizes existing data on the associations between endogenous hormone concentrations and prostate growth and concludes that circulating concentrations of androgens do not appear to be associated with the risks of development of BPH or initiation or progression of PC. The explanation for these findings relates to an apparent insensitivity of prostatic tissue to changes of testosterone concentrations within the physiological range.

RGS2 is prognostic for development of castration resistance and cancer-specific survival in castration-resistant prostate cancer.

BACKGROUND: Regulator of G-protein signaling 2 (RGS2) is a multifaceted protein with a prognostic value in hormone-naïve prostate cancer (PC). It has previously been associated with the development of castration resistance. However, RGS2 expression in clinical specimens of castration-resistant prostate cancer (CRPC) and its clinical relevance has not been explored. In the present study, RGS2 was assessed in CRPC and in relation to the development of castration resistance. METHODS: In the present study, RGS2 expression was evaluated with immunohistochemistry in patient materials of hormone-naïve and castration-resistant primary tumors, also in matched specimens before and after 3 months of androgen deprivation therapy (ADT). Cox regression and Kaplan-Meier curves were used to evaluate the clinical significance of RGS2 expression. RGS2 expression in association to castration-resistant growth was assessed experimentally in an orthotopic xenograft mouse model of CRPC. In vitro, hormone depletion of LNCaP and enzalutamide treatment of LNCaP, 22Rv1, and VCaP was performed to evaluate the association between RGS2 and the androgen receptor (AR). Stable RGS2 knockdown was used to evaluate the impact of RGS2 in association to PC cell growth under hormone-reduced conditions. Gene and protein expression were evaluated with quantitative polymerase chain reaction and Western blot analysis, respectively. RESULTS: RGS2 expression is increased in CRPC and enriched under ADT. Furthermore, a high RGS2 level is prognostic for poor cancer-specific survival for CRPC patients and significantly reduced failure-free survival (FFS) after an initiated ADT. Additionally, the prognostic value of RGS2 outperforms prostate-specific antigen (PSA) in terms of FFS. The present study furthermore suggests that RGS2 expression is reflective of AR activity. Moreover, low RGS2-expressing cells display hampered growth under hormone-reduced conditions, in line with the poor prognosis associated with high RGS2 expression. CONCLUSIONS: High levels of RGS2 are associated with aggressive forms of castration-resistant PC. The results demonstrate that a high level of RGS2 is associated with poor prognosis in association with castration-resistant PC growth. RGS2 alone, or in association with PSA, has the potential to identify patients that require additional treatment at an early stage during ADT.

Castration is a prerequisite for the inhibitory effect of metronomic chemotherapy on the growth of experimental castration-resistant prostate cancer.

BACKGROUND: Low-dose metronomic chemotherapy (LDMC) is an alternative for treatment of patients with late-stage prostate cancer (PC) not susceptible to regular chemotherapy due to its severe side effects. The exact working mechanisms of LDMC have not been established, although anti-angiogenic effects have been identified. In PC, several studies show clinical effects from LDMC but the mode of action and the role of androgen signaling for its effect are not known. In this study, we used a xenograft model to evaluate the effect of LDMC on PC growth in relation to androgen deprivation. MATERIAL AND METHODS: Subcutaneous human castration-resistant PC xenografts were treated with LDMC using cyclophosphamide (CPA). Treatment effect was compared to treatment with maximum tolerated dose (MTD) and also between intact and castrated mice. Microvessel density (MVD), and factors important for angiogenesis were analyzed with immunohistochemistry and real-time-PCR. RESULTS: Tumors treated with LDMC were 50% smaller than untreated controls. Tumors in non-castrated mice were not affected by LDMC, but in an androgen receptor (AR) negative tumor model, tumor inhibiting effect were seen in both intact and castrated animals, indicating mechanism via AR. MTD resulted in similar growth inhibition as LDMC in castrated mice, but resulted in severe weight loss. Despite that LDMC induced TSP1 mRNA expression, and the hypoxic area in the tumors was slightly increased, no decrease in MVD was detected. CONCLUSIONS: This study shows that a low-dose metronomic scheduling of CPA was as efficient as MTD treatment, and resulted in fewer side effects. It also demonstrates that a functional androgen signaling axis inhibits this effect despite the castration-resistance of the tumor cells. The anti-angiogenic nature of the effect of LDMC could not be confirmed and further studies to elucidate the working mechanism for treatment response are needed.

Circulating Tumor Cells as a Marker for Progression-free Survival in Metastatic Castration-naïve Prostate Cancer.

BACKGROUND: Analysis of circulating tumor cells (CTC) is a promising prognostic marker in castration-resistant prostate cancer (CRPC). The aim of this study was to investigate CTC detection and phenotyping as prognostic biomarkers for response to primary androgen deprivation therapy (ADT) of metastatic prostate cancer (PC). METHODS: PC patients presenting with a prostate specific antigen (PSA) >80 ng/ml and/or metastatic disease, intended for ADT were enrolled in the study. CTCs were analysed for expression of PSA prostate specific membrane antigen (PSMA) and epidermal growth factor receptor (EGFR) before and three months after ADT and related to progression. RESULTS: At inclusion, 46 out of 53 patients (87%) were CTC-positive with a sensitivity and specificity for distant metastases (M1) of 98% and 75%, respectively. In patients with M1-disease, EGFR-detection in CTC was an independent prognostic marker for progression-free survival, whereas PSA and alkaline phosphatase serum levels, Gleason score, or T-stage were not. EGFR-positive patients had significantly shorter time to progression (5 months) compared to EGFR-negative patients (11 months) (P < 0.05). CONCLUSIONS: In this explorative study, CTCs were detected in 98% of M1 patients and detection of EGFR in CTCs was strongly associated with poor outcome, which indicated that phenotypical analysis of CTC could be a promising prognostic marker of ADT-response in castration-naïve metastatic PC patients. Prostate 77:849-858, 2017. © 2017 Wiley Periodicals, Inc.

Tasquinimod inhibits prostate cancer growth in bone through alterations in the bone microenvironment.

BACKGROUND: Tasquinimod (ABR-215050) is an orally active quinoline-3-carboxamide analog that inhibits occurrence of experimental metastasis and delays disease progression of castration resistant prostate cancer in humans. Its mechanism of action is not fully elucidated, but previous studies show immunomodulatory and anti-angiogenic effects. The aim of the present study was to investigate the tumor inhibiting effect of tasquinimod in bone of castrated mice as well as to elucidate its working mechanism related to bone microenvironment. METHODS: Effects of tasquinimod on prostate cancer metastasis to bone was studied in an intratibial xenograft model. Animals were treated with tasquinimod and tumor establishment and growth, immunological status, as well as markers for bone remodeling were analyzed. Direct effects of tasquinimod on osteoblasts were studied in vitro. RESULTS: Establishment and growth of tumors in the bone after intratibial implantation in castrated mice was suppressed by tasquinimod treatment. The treatment effect was linked to decreased potential for immunosuppression in the pre-metastatic niche in bone (lower levels of CD206 and Arg1 expression in combination with increased iNOS expression) as well as in the tumor microenvironment (less Gr1 and CD206 staining). The shift to a pro-inflammatory, anti-tumorigenic milieu was also reflected in serum by increased levels of IFN-γ, CCL4, IL-5, LIX, IP-10, and MCP-1 as well as decreased TGF-ß. Tasquinimod treatment also affected expression of factors involved in the pre-metastatic niche in the bone microenvironment (Lox, Cdh2, Cdh11, and Cxcl12). In addition, tasquinimod treatment caused a decreased osteogenic response indicated by decreased expression of Ocn, Runx2, and Col1a2 and increased expression of osteoclast stimulating CSF2. In vitro studies on mouse osteoblasts showed impaired osteoblast mineralization upon tasquinimod treatment. CONCLUSIONS: The present study shows that tasquinimod reduces establishment and progression of tumor growth in bone likely through a combination of effects on the pre-metastatic niche, homing, immunological status, and osteogenesis. It was concluded that tasquinimod interferes with the metastatic process, presumably by inhibition of tumor establishment. Hence, our data suggest that tasquinimod might be most effective in inhibiting the occurrence of new metastatic lesions.

[Molecular subtypes provide possibilities for precision medicine in a advanced prostate cancer]. / Molekylära subtyper och avancerad prostatacancer.

Increased molecular knowledge makes it possible to consider not only genetic defects but also expression profiles for precision medicine in advanced prostate cancer. Several prognostic and treatment-predictive classifiers for prostate cancer have been described, such as Prolaris, OncotypeDx, Decipher, Prostatype, PAM50, PCS1-2, and MetA-C, which all build upon transcript profiles. In research studies, the MetA-C classifier has shown clear prognostic information for patients with metastatic disease, in relation to outcome after androgen receptor targeting therapies, and so has immunohistochemical evaluation of tumor cell proliferation (Ki67) and PSA expression. Unfortunately, methods within clinical routine today do not allow molecular subclassification of prostate cancer. To enable comparison of the most promising treatment-predictive biomarkers and to evaluate the health economic value of implementing such precision medicine for prostate cancer, a prospective study is being planned as a joint initiative in Sweden that aims to evaluate and validate biomarkers and to establish a study platform for adaptive biomarker-driven clinical trials (sprintr.se).

Midkine is associated with neuroendocrine differentiation in castration-resistant prostate cancer.

BACKGROUND: Castration-resistant prostate cancer (CRPC) is an incurable disease and both androgen-deprivation therapy (ADT) and neuroendocrine differentiation (NED) are closely related to CRPC transition. More knowledge concerning neuroendocrine (NE)-transformed PC cells, the NED process and its association with CRPC, is needed. Expression of growth factor midkine (MDK) is correlated with poor clinical outcomes in various human cancers, including PC. In the present study, we have evaluated MDK expression and NED in two separate tumor groups: early and advanced PC. METHODS: Immunohistochemical analysis of MDK, the neuronal marker tubulin-beta III (TUBB3) and the NE-marker chromogranin A (CGA) in a human archival material consisting of hormone naive (HN)/stage T1b (n = 29) and CRPC (n = 24) tumors. Triple immunofluorescent imaging was performed on a selection of specimens. RESULTS: MDK, TUBB3, and CGA were upregulated in CRPC compared to HN tumors. MDK was highly associated to the expression of both CGA and TUBB3, and identified MDK-positive NE-like looking cells found to co-express CGA or, more commonly, CGA together with TUBB3. CGA and TUBB3 staining displayed a partial expression overlap, an overlap almost exclusively displaying also MDK expression. CONCLUSIONS: MDK upregulation in CRPC is associated with NED (shown by its relation to CGA and TUBB3). The results suggest that MDK represents an over-bridging marker between different populations of NE-like tumor cells, possibly as part of the NED process and associated CRPC transition, something that needs to be evaluated experimentally as does the applicability of MDK as a future target.

Magnetic Resonance Imaging as a Tool for Monitoring Intratibial Growth of Experimental Prostate Cancer Metastases in Mice.

Bone metastases cause morbidity and mortality in several human cancer forms. Experimental models are used to unravel the mechanisms and identify possible treatment targets. The location inside the skeleton complicates accurate assessment. This study evaluates the performance of magnetic resonance imaging (MRI) of prostate cancer tumors growing intratibially in mice. MRI detected intratibial tumor lesions with a sensitivity and specificity of 100% and 89%, respectively, compared to histological evaluation. Location and some phenotypical features could also be readily detected with MRI. Regarding volume estimation, the correlation between MRI and histological assessment was high (p < 0.001, r = 0.936). In conclusion, this study finds MRI to be a reliable tool for in vivo, non-invasive, non-ionizing, real-time monitoring of intratibial tumor growth.

Acoustic enrichment of heterogenous circulating tumor cells and clusters from patients with metastatic prostate cancer.

Background: There are important unmet clinical needs to develop cell enrichment technologies to enable unbiased label-free isolation of both single cell and clusters of circulating tumor cells (CTCs) manifesting heterogeneous lineage specificity. Here, we report a pilot study based on microfluidic acoustophoresis enrichment of CTCs using the CellSearch CTC assay as a reference modality. Methods: Acoustophoresis uses an ultrasonic standing wave field to separate cells based on biomechanical properties (size, density, and compressibility) resulting in inherently label-free and epitope-independent cell enrichment. Following red blood cell lysis and paraformaldehyde fixation, 6 mL of whole blood from 12 patients with metastatic prostate cancer and 20 healthy controls were processed with acoustophoresis and subsequent image cytometry. Results: Acoustophoresis enabled enrichment and characterization of phenotypic CTCs (EpCAM+, Cytokeratin+, DAPI+, CD45-/CD66b-) in all patients with metastatic prostate cancer and detected CTC-clusters composed of only CTCs or heterogenous aggregates of CTCs clustered with various types of white blood cells in 9 out of 12 patients. By contrast, CellSearch did not detect any CTC-clusters, but detected comparable numbers of phenotypic CTCs as acoustophoresis, with trends of finding higher number of CTCs using acoustophoresis. Conclusion: Our preliminary data indicate that acoustophoresis provides excellent possibilities to detect and characterize CTC-clusters as a putative marker of metastatic disease and outcomes. Moreover, acoustophoresis enables sensitive label-free enrichment of cells with epithelial phenotype in blood and offers opportunities to detect and characterize CTCs undergoing epithelial-to-mesenchymal transitioning and lineage plasticity.

Inhibition of metastasis in a castration resistant prostate cancer model by the quinoline-3-carboxamide tasquinimod (ABR-215050).

BACKGROUND: Tasquinimod (ABR-215050) is an orally active quinoline-3-carboxamide analog that has completed phase II clinical trial in patients with castration resistant prostate cancer, showing promising inhibiting effects on the occurrence of metastasis and delayed disease progression. Its mechanism of action is not fully elucidated, but previous studies show anti-angiogenic effects and strong interaction with the S100A9 protein. METHODS: This study was performed to evaluate if tasquinimod inhibits prostate cancer metastasis, by using both orthotopic and intratibial xenograft models. Animals were treated with tasquinimod, and tumor growth characteristics as well as molecular markers for metastasis and angiogenesis were analyzed. RESULTS: The results show that formation of lung and lymph node metastases from orthotopic castration resistant prostate tumors was inhibited by tasquinimod treatment. Importantly, establishment of tumors in the bone after intratibial implantation was suppressed by tasquinimod. In addition, establishment and growth of subcutaneous tumors were affected. Both in primary tumors and serum from treated mice an upregulation of thrombospondin 1 was observed. Further, downregulation of the hypoxia driven genes VEGF, CXCR4, and LOX was detected in the primary tasquinimod-treated tumors and decreased expression of chemotactic ligand SDF-1 was demonstrated in the lungs. Thus, these molecular changes could contribute to the anti-angiogenic and anti-metastatic effects of tasquinimod. CONCLUSIONS: In conclusion, this study and clinical data show that tasquinimod interferes with the metastatic process, presumably by inhibition of tumor establishment. Therefore, tasquinimod is an interesting treatment option for patients with prostate cancer prone to metastasis.

Castration resistant prostate cancer is associated with increased blood vessel stabilization and elevated levels of VEGF and Ang-2.

BACKGROUND: Angiogenesis is important for the progression of prostate cancer and may be a target for treatment in castration resistant (CR) disease. This study was performed to investigate blood vessel stabilization and expression of the pro-angiogenic factors vascular endothelial growth factor (VEGF) and Angiopoietin-2 (Ang-2) in CR and hormone naïve (HN) prostate cancer. The effect of androgen deprivation therapy (ADT) on these parameters was also studied. METHODS: VEGF and Ang-2, as well as pericyte coverage of blood vessels were studied in HN and CR prostate tumors by immunohistochemistry. The effects of ADT on VEGF expression and microvessel density (MVD) were investigated in biopsies at diagnosis, 3 months after starting ADT and at tumor relapse. Plasma was also analyzed for VEGF and Ang-2 with ELISA. RESULTS: CR tumors had higher levels of VEGF and Ang-2 as well as increased blood vessel stabilization compared to HN tumors. Three months after initiated ADT an increase of VEGF but not MVD in the tumors was observed. In contrast, plasma levels of VEGF decreased after ADT, and increased again at time of tumor relapse. Ang-2 levels were unaffected. CONCLUSIONS: CR prostate cancer is associated with elevated levels of VEGF and Ang-2, indicating that these factors could be used as targets for anti-angiogenic treatment. Still, the observed increase in blood vessel stabilization in CR tumors could influence the outcome of anti-angiogenic treatment. Furthermore, increased VEGF expression after 3 months of ADT justifies the use of VEGF-based anti-angiogenic drugs in combination with ADT for the treatment of advanced prostate cancer.

Association between pharmaceutical modulation of oestrogen in postmenopausal women in Sweden and death due to COVID-19: a cohort study.

OBJECTIVE: Determine whether augmentation of oestrogen in postmenopausal women decreases the risk of death following COVID-19. DESIGN: Nationwide registry-based study in Sweden based on registries from the Swedish Public Health Agency (all individuals who tested positive for SARS-CoV-2); Statistics Sweden (socioeconomical variables) and the National Board of Health and Welfare (causes of death). PARTICIPANTS: Postmenopausal women between 50 and 80 years of age with verified COVID-19. INTERVENTIONS: Pharmaceutical modulation of oestrogen as defined by (1) women with previously diagnosed breast cancer and receiving endocrine therapy (decreased systemic oestrogen levels); (2) women receiving hormone replacement therapy (increased systemic oestrogen levels) and (3) a control group not fulfilling requirements for group 1 or 2 (postmenopausal oestrogen levels). Adjustments were made for potential confounders such as age, annual disposable income (richest group as the reference category), highest level of education (primary, secondary and tertiary (reference)) and the weighted Charlson Comorbidity Index (wCCI). PRIMARY OUTCOME MEASURE: Death following COVID-19. RESULTS: From a nationwide cohort consisting of 49 853 women diagnosed with COVID-19 between 4 February and 14 September 2020 in Sweden, 16 693 were between 50 and 80 years of age. We included 14 685 women in the study with 11 923 (81%) in the control group, 227 (2%) women in group 1 and 2535 (17%) women in group 2. The unadjusted ORs for death following COVID-19 were 2.35 (95% CI 1.51 to 3.65) for group 1 and 0.45 (0.34 to 0.6) for group 2. Only the adjusted OR for death remained significant for group 2 with OR 0.47 (0.34 to 0.63). Absolute risk of death was 4.6% for the control group vs 10.1% and 2.1%, for the decreased and increased oestrogen groups, respectively. The risk of death due to COVID-19 was significantly associated with: age, OR 1.15 (1.14 to 1.17); annual income, poorest 2.79 (1.96 to 3.97), poor 2.43 (91.71 to 3.46) and middle 1.64 (1.11 to 2.41); and education (primary 1.4 (1.07 to 1.81)) and wCCI 1.13 (1.1 to 1.16). CONCLUSIONS: Oestrogen supplementation in postmenopausal women is associated with a decreased risk of dying from COVID-19 in this nationwide cohort study. These findings are limited by the retrospective and non-randomised design. Further randomised intervention trials are warranted.

Osteoclasts directly influence castration-resistant prostate cancer cells.

Metastasis to bone is the leading cause of death from prostate cancer. Interaction between tumor cells and bone cells can promote progression and influence tumor phenotype. It is known that prostate cancer cells support osteoclast differentiation, and degradation of bone matrix by osteoclasts releases growth factors stimulating tumor cell proliferation and invasion. In the present study osteolytic (PC-3) and osteoblastic (LNCaP-19) castration-resistant prostate cancer (CRPC) cells were co-cultured with mature osteoclasts or their precursor cells (RAW 264.7) to characterize direct effects of mature osteoclasts on CRPC cells. Osteoclasts increased proliferation and decrease apoptosis of CRPC cells as assessed with flow cytometry. RNA sequencing revealed that osteolytic CRPC cells were more responsive to osteoclast stimulation regarding gene expression, but the overall induced expression patterns were similar between the prostate cancer cell lines. Genes related to DNA repair were upregulated by osteoclasts, while genes related to endoplasmic reticulum stress-induced apoptosis and cholesterol synthesis were downregulated. The results of this study shows that osteoclasts directly influence CRPC cells, increasing proliferation, decreasing apoptosis, and affecting gene expression pathways that can affect sensitivity to DNA damage and endoplasmic reticulum function. This suggests targeting of osteoclasts to be a possible way to affect efficacy of other drugs by combination regimens in treating prostate cancer metastases.

Clinical and biological relevance of the transcriptomic-based prostate cancer metastasis subtypes MetA-C.

To improve treatment of metastatic prostate cancer, the biology of metastases needs to be understood. We recently described three subtypes of prostate cancer bone metastases (MetA-C), based on differential gene expression. The aim of this study was to verify the clinical relevance of these subtypes and to explore their biology and relations to genetic drivers. Freshly-frozen metastasis samples were obtained as hormone-naive (n = 17), short-term castrated (n = 21), or castration-resistant (n = 65) from a total of 67 patients. Previously published sequencing data from 573 metastasis samples were also analyzed. Through transcriptome profiling and sample classification based on a set of predefined MetA-C-differentiating genes, we found that most metastases were heterogeneous for the MetA-C subtypes. Overall, MetA was the most common subtype, while MetB was significantly enriched in castration-resistant samples and in liver metastases, and consistently associated with poor prognosis. By gene set enrichment analysis, the phenotype of MetA was described by high androgen response, protein secretion and adipogenesis, MetB by high cell cycle activity and DNA repair, and MetC by epithelial-to-mesenchymal transition and inflammation. The MetB subtype demonstrated single nucleotide variants of RB transcriptional corepressor 1 (RB1) and loss of 21 genes at chromosome 13, including RB1, but provided independent prognostic value to those genetic aberrations. In conclusion, a distinct set of gene transcripts can be used to classify prostate cancer metastases into the subtypes MetA-C. The MetA-C subtypes show diverse biology, organ tropism, and prognosis. The MetA-C classification may be used independently, or in combination with genetic markers, primarily to identify MetB patients in need of complementary therapy to conventional androgen receptor-targeting treatments.

A Phase 2 Trial of the Effect of Antiandrogen Therapy on COVID-19 Outcome: No Evidence of Benefit, Supported by Epidemiology and In Vitro Data.

BACKGROUND: Men are more severely affected by COVID-19. Testosterone may influence SARS-CoV-2 infection and the immune response. OBJECTIVE: To clinically, epidemiologically, and experimentally evaluate the effect of antiandrogens on SARS-CoV-2 infection. DESIGNS, SETTINGS, AND PARTICIPANTS: A randomized phase 2 clinical trial (COVIDENZA) enrolled 42 hospitalized COVID-19 patients before safety evaluation. We also conducted a population-based retrospective study of 7894 SARS-CoV-2-positive prostate cancer patients and an experimental study using an air-liquid interface three-dimensional culture model of primary lung cells. INTERVENTION: In COVIDENZA, patients were randomized 2:1 to 5 d of enzalutamide or standard of care. OUTCOME MEASUREMENTS: The primary outcomes in COVIDENZA were the time to mechanical ventilation or discharge from hospital. The population-based study investigated risk of hospitalization, intensive care, and death from COVID-19 after androgen inhibition. RESULTS AND LIMITATIONS: Enzalutamide-treated patients required longer hospitalization (hazard ratio [HR] for discharge from hospital 0.43, 95% confidence interval [CI] 0.20-0.93) and the trial was terminated early. In the epidemiological study, no preventive effects were observed. The frail population of patients treated with androgen deprivation therapy (ADT) in combination with abiraterone acetate or enzalutamide had a higher risk of dying from COVID-19 (HR 2.51, 95% CI 1.52-4.16). In vitro data showed no effect of enzalutamide on virus replication. The epidemiological study has limitations that include residual confounders. CONCLUSIONS: The results do not support a therapeutic effect of enzalutamide or preventive effects of bicalutamide or ADT in COVID-19. Thus, these antiandrogens should not be used for hospitalized COVID-19 patients or as prevention for COVID-19. Further research on these therapeutics in this setting are not warranted. PATIENT SUMMARY: We studied whether inhibition of testosterone could diminish COVID-19 symptoms. We found no evidence of an effect in a clinical study or in epidemiological or experimental investigations. We conclude that androgen inhibition should not be used for prevention or treatment of COVID-19.

O-glycosylation of MUC1 mucin in prostate cancer and the effects of its expression on tumor growth in a prostate cancer xenograft model.

MUC1 mucin is up-regulated and aberrantly glycosylated in many human epithelial carcinomas. Over-expression of MUC1 has also been implicated in prostate cancer, but neither the role of MUC1 in the cancer progression nor the mucin O-glycosylation has been fully elucidated. In this study, we characterized the O-glycans on MUC1 when over-expressed in the human prostate cancer cell line C4-2B(4). We found that the main O-glycan consisted of the neutral core 2 oligosaccharide Galß3(Galß3/4GlcNAcß6)GalNAc-ol, with minor components being fucosylated and sialylated variants of the same core 2 oligosaccharide. Small amounts of the shorter core 1 O-glycans were also detected.We then used the MUC1 over-expressing cell lines to study the effects of MUC1 on prostate cancer cell behavior. The results demonstrate that over-expression of MUC1 did not affect the cells' proliferation, but led to a decreased adhesion to the extracellular matrix components fibronectin and collagen I in vitro. When inoculated in BALB/c nude mice, C4-2B(4) cells expressing MUC1 showed a tendency to form fewer tumors than wt cells and the tumors also grew more slowly, but there was a large variation between different tumors. These findings suggest that MUC1 may not have the same cancer-promoting effect in prostate cancer cells that is commonly seen in other epithelial cancers such as breast, colon, and pancreatic cancer.

The roles of RUNX2 and osteoclasts in regulating expression of steroidogenic enzymes in castration-resistant prostate cancer cells.

Intratumoral steroidogenesis is involved in development of castration-resistant prostate cancer (CRPC) as bone metastases. The osteoblast transcription factor RUNX2 influences steroidogenesis and is induced in CRPC cells by osteoblasts. This study investigates osteoclastic influence on RUNX2 in intratumoral steroidogenesis. Steroidogenic enzymes and steroid receptors were detected with immunohistochemistry in xenograft intratibial tumors from CRPC cells. In vitro, expression of RUNX2 was increased by osteoclasts in osteoblastic LNCaP-19 cells, but not in osteolytic PC-3. Silencing of RUNX2 downregulates expression of CYP11A1, CYP17A1 and HSD3B1 in LNCaP-19 cells co-cultured with osteoclasts, leading to inhibition of KLK3 expression. Osteoclasts promoted CYP11A1 and RUNX2 promoted AKR1C3, HSD17B3 and CYP19A1, but suppressed ESR2 in PC-3 cells. This study shows that osteoclasts promote RUNX2 regulated induction of key steroidogenic enzymes, influencing activation of androgen receptor in CRPC cells. The potential of RUNX2 as a target to inhibit progression of skeletal metastases of CRPC needs further investigation.