Somatic copy number alterations by whole‐exome sequencing implicates YWHAZ and PTK2 in castration‐resistant prostate cancer

Castration‐resistant prostate cancer (CRPC) is the most aggressive form of prostate cancer (PCa) and remains a significant therapeutic challenge. The key to the development of novel therapeutic targets for CRPC is to decipher the molecular alterations underlying this lethal disease. The aim of our study was to identify therapeutic targets for CRPC by assessing somatic copy number alterations (SCNAs) by whole‐exome sequencing on five CRPC/normal paired formalin‐fixed paraffin‐embedded (FFPE) samples, using the SOLiD4 next‐generation sequencing (NGS) platform. Data were validated using fluorescence in situ hybridization (FISH) on a PCa progression cohort. PTK2 and YWHAZ amplification, mRNA and protein expression were determined in selected PCa cell lines. Effects of PTK2 inhibition using TAE226 inhibitor and YWHAZ knock‐down on cell proliferation and migration were tested in PC3 cells in vitro. In a larger validation cohort, the amplification frequency of YWHAZ was 3% in localized PCa and 48% in CRPC, whereas PTK2 was amplified in 1% of localized PCa and 35% in CRPC. YWHAZ knock‐down and PTK2 inhibition significantly affected cell proliferation and migration in the PC3 cells. Our findings suggest that inhibition of YWHAZ and PTK2 could delay the progression of the disease in CRPC patients harbouring amplification of the latter genes. Furthermore, our validated whole‐exome sequencing data show that FFPE tissue could be a promising alternative for SCNA screening using next‐generation sequencing technologies. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

前列腺癌非编码RNA1在雄激素非依赖去势抵抗型前列腺癌细胞中的作用

目的 探讨长链非编码RNA前列腺癌非编码RNA1(PRNCR1)在雄激素非依赖的前列腺癌细胞中的作用.方法 应用实时定量PCR(qRT-PCR)检测雄激素依赖的前列腺癌细胞LNCaP及雄激素非依赖的前列腺癌细胞C4-2中PRNCR1的表达情况,通过RNA干扰技术沉默C4-2细胞中的PRNCR1,Western blot技术检测C4-2细胞中雄激素受体(AR)的表达变化,流式细胞术检测细胞凋亡的变化,MTT实验检测沉默PRNCR1表达对细胞增殖的影响,Transwell侵袭实验检测细胞侵袭能力的变化.结果 PRNCR1在雄激素非依赖的细胞系C4-2中高表达,干扰其表达可以明显降低前列腺癌细胞中AR的表达,抑制前列腺癌细胞的细胞增殖及细胞的侵袭能力,并促进细胞的凋亡.结论 PRNCR1介导AR在前列腺癌去势抵抗中发挥着重要作用.     

The androgen receptor co-activator CBP is up-regulated following androgen withdrawal and is highly expressed in advanced prostate cancer

The androgen receptor co-activator CREB (cAMP-response element binding protein)-binding protein (CBP) enhances androgen receptor activity after stimulation by androgenic hormones and androgen receptor antagonists. The aim of the present study was to investigate the regulation of CBP expression by steroid and peptide hormones in prostate cancer. For this purpose, LNCaP cells were treated with the synthetic androgen methyltrienolone (R1881), epidermal growth factor, insulin-like growth factor-I or interleukin-6 (IL-6). CBP protein and mRNA expression were studied by western blotting and real-time PCR, respectively. CBP expression was also investigated in tissue specimens obtained from 26 patients with therapy-resistant carcinoma of the prostate. In LNCaP cells, CBP protein was down-regulated by R1881 or IL-6. The non-steroidal anti-androgen bicalutamide antagonized the effects of R1881 and the Janus kinase inhibitor AG 490 reversed the effects of IL-6. In contrast, neither R1881 nor IL-6 caused any effect on CBP expression in the PC-3 cell line. In LNCaP cells, the inhibition of CBP expression by R1881 or IL-6 was also observed at the mRNA level. CBP protein was detected in all 26 specimens by immunohistochemistry. The results suggest that up-regulation of CBP during androgen ablation may be relevant to the failure of endocrine therapy in patients with prostate carcinoma.     

Peripheral CD4+ T‐cell tolerance is induced in vivo by rare antigen‐bearing B cells in follicular,marginal zone,and B‐1 subsets

B cells are efficient APCs when they internalize antigen via BCR‐mediated uptake. Adoptively transferred antigen‐presenting B cells can induce T‐cell tolerance to foreign and self antigens; however, it is unknown whether endogenous B cells presenting self‐peptides interact with naïve T cells and contribute to peripheral T‐cell self‐tolerance. Moreover, the relative abilities of mature B‐cell subsets to induce T‐cell tolerance have not been examined. To address these questions, we created a new mouse model wherein a very small fraction of B cells expresses an antigen transgene that cannot be transferred to other APCs. We limited antigen expression to follicular, marginal zone, or B‐1 B‐cell subsets and found that small numbers of each subset interacted with naïve antigen‐specific T cells. Although antigen expressed by B‐1 B cells induced the most T‐cell division, divided T cells subsequently disappeared from secondary lymphoid tissues. Independent of which B‐cell subset presented antigen, the remaining T cells were rendered hypo‐responsive, and this effect was not associated with Foxp3 expression. Our data show that physiologically relevant proportions of B cells can mediate peripheral T‐cell tolerance, and suggest that the mechanisms of tolerance induction might differ among follicular, marginal zone, and B‐1 B‐cell subsets.     

Protective effect of γ‐tocopherol‐enriched diet on N‐methyl‐N‐nitrosourea‐induced epithelial dysplasia in rat ventral prostate

Despite recent advances in understanding the biological basis of prostate cancer (PCa), the management of this disease remains a challenge. Chemoprotective agents have been used to protect against or eradicate prostate malignancies. Here, we investigated the protective effect of γ‐tocopherol on N‐methyl‐N‐nitrosourea (MNU)‐induced epithelial dysplasia in the rat ventral prostate (VP). Thirty‐two male Wistar rats were divided into four groups (n = 8): control (CT): healthy control animals fed a standard diet; control+γ‐tocopherol (CT+γT): healthy control animals without intervention fed a γ‐tocopherol‐enriched diet (20 mg/kg); N‐methyl‐N‐nitrosourea (MNU): rats that received a single dose of MNU (30 mg/kg) plus testosterone propionate (100 mg/kg) and were fed a standard diet; and MNU+γ‐tocopherol (MNU+γT): rats that received the same treatment of MNU plus testosterone and were fed with a γ‐tocopherol‐enriched diet (20 mg/kg). After 4 months, the VPs were excised to evaluate morphology, cell proliferation and apoptosis, as well as cyclooxygenase‐2 (Cox‐2), glutathione‐S‐transferase‐pi (GST‐pi) and androgen receptor (AR) protein expression, and matrix metalloproteinase‐9 (MMP‐9) activity. An increase in the incidence of epithelial dysplasias, such as stratified epithelial hyperplasia and squamous metaplasia, in the MNU group was accompanied by augmented cell proliferation, GST‐pi and Cox‐2 immunoexpression and pro‐MMP‐9 activity. Stromal thickening and inflammatory foci were also observed. The administration of a γ‐tocopherol‐enriched diet significantly attenuated the adverse effects of MNU in the VP. The incidence of epithelial dysplasia decreased, along with the cell proliferation index, GST‐pi and Cox‐2 immunoexpression. The gelatinolytic activity of pro‐MMP‐9 returned to the levels observed for the CT group. These results suggest that γ‐tocopherol acts as a protective agent against MNU‐induced prostatic disorders in the rat ventral prostate.     

Spatial aspects of oncogenic signalling determine the response to combination therapy in slice explants from Kras‐driven lung tumours

A key question in precision medicine is how functional heterogeneity in solid tumours informs therapeutic sensitivity. We demonstrate that spatial characteristics of oncogenic signalling and therapy response can be modelled in precision‐cut slices from Kras‐driven non‐small‐cell lung cancer with varying histopathologies. Unexpectedly, profiling of in situ tumours demonstrated that signalling stratifies mostly according to histopathology, showing enhanced AKT and SRC activity in adenosquamous carcinoma, and mitogen‐activated protein kinase (MAPK) activity in adenocarcinoma. In addition, high intertumour and intratumour variability was detected, particularly of MAPK and mammalian target of rapamycin (mTOR) complex 1 activity. Using short‐term treatment of slice explants, we showed that cytotoxic responses to combination MAPK and phosphoinositide 3‐kinase–mTOR inhibition correlate with the spatially defined activities of both pathways. Thus, whereas genetic drivers determine histopathology spectra, histopathology‐associated and spatially variable signalling activities determine drug sensitivity. Our study is in support of spatial aspects of signalling heterogeneity being considered in clinical diagnostic settings, particularly to guide the selection of drug combinations. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

Biallelic tumour suppressor loss and DNA repair defects in de novo small‐cell prostate carcinoma

Small‐cell prostate carcinoma (SCPC) is an aggressive malignancy that is managed similarly to small‐cell lung cancer. SCPC can evolve from prostate adenocarcinoma in response to androgen deprivation therapy, but, in rare cases, is present at initial cancer diagnosis. The molecular aetiology of de novo SCPC is incompletely understood, owing to the scarcity of tumour tissue and the short life‐expectancy of patients. Through a retrospective search of our regional oncology pharmacy database, we identified 18 patients diagnosed with de novo SCPC between 2004 and 2017. Ten patients had pure SCPC pathology, and the remainder had some admixed adenocarcinoma foci, but all were treated with first‐line platinum‐based chemotherapy. The median overall survival was 28 months. We performed targeted DNA sequencing, whole exome sequencing and mRNA profiling on formalin‐fixed paraffin‐embedded archival tumour tissue. We observed frequent biallelic deletion and/or mutation of the tumour suppressor genes TP53, RB1, and PTEN, similarly to what was found in treatment‐related SCPC. Indeed, at the RNA level, pure de novo SCPC closely resembled treatment‐related SCPC. However, five patients had biallelic loss of DNA repair genes, including BRCA1, BRCA2, ATM, and MSH2/6, potentially underlying the high genomic instability of this rare disease variant. Two patients with pure de novo SCPC harboured ETS gene rearrangements involving androgen‐driven promoters, consistent with the evolution of de novo SCPC from an androgen‐driven ancestor. Overall, our results reveal a highly aggressive molecular landscape that underlies this unusual pathological variant, and suggest opportunities for targeted therapy strategies in a disease with few treatment options. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Loss of caveolin‐1 in prostate cancer stroma correlates with reduced relapse‐free survival and is functionally relevant to tumour progression

Levels of caveolin‐1 (Cav‐1) in tumour epithelial cells increase during prostate cancer progression. Conversely, Cav‐1 expression in the stroma can decline in advanced and metastatic prostate cancer. In a large cohort of 724 prostate cancers, we observed significantly decreased levels of stromal Cav‐1 in concordance with increased Gleason score (p = 0.012). Importantly, reduced expression of Cav‐1 in the stroma correlated with reduced relapse‐free survival (p = 0.009), suggesting a role for stromal Cav‐1 in inhibiting advanced disease. Silencing of Cav‐1 by shRNA in WPMY‐1 prostate fibroblasts resulted in up‐regulation of Akt phosphorylation, and significantly altered expression of genes involved in angiogenesis, invasion, and metastasis, including a > 2.5‐fold increase in TGF‐β1 and γ‐synuclein (SNCG) gene expression. Moreover, silencing of Cav‐1 induced migration of prostate cancer cells when stromal cells were used as attractants. Pharmacological inhibition of Akt caused down‐regulation of TGF‐β1 and SNCG, suggesting that loss of Cav‐1 in the stroma can influence Akt‐mediated signalling in the tumour microenvironment. Cav‐1‐depleted stromal cells exhibited increased levels of intracellular cholesterol, a precursor for androgen biosynthesis, steroidogenic enzymes, and testosterone. These findings suggest that loss of Cav‐1 in the tumour microenvironment contributes to the metastatic behaviour of tumour cells by a mechanism that involves up‐regulation of TGF‐β1 and SNCG through Akt activation. They also suggest that intracrine production of androgens, a process relevant to castration resistance, may occur in the stroma. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Generation of pralatrexate resistant T‐cell lymphoma lines reveals two patterns of acquired drug resistance that is overcome with epigenetic modifiers

While pralatrexate (PDX) has been successfully developed for the treatment of T‐cell lymphoma, the mechanistic basis for its T‐cell selectivity and acquired resistance remains elusive. In an effort to potentially identify synergistic combinations that might circumnavigate or delay acquired PDX resistance, we generated resistant cells lines over a broad concentration range. PDX‐resistant cell lines H9‐12 and H9‐200 were developed, each exhibiting an IC50 of 35 and over 1000 nM, respectively. These lines were established in vitro from parental H9 cells. Expression analysis of the proteins known to be important determinants of antifolate pharmacology revealed increase expression of dihydrofolate reductase (DHFR) due to gene amplification, and reduced folate carrier1 downregulation, as the putative mechanisms of resistance in H9‐12 and H9‐200 cells. Cross resistance was only seen with methotrexate but not with romidepsin, azacitidine (AZA), decitabine, gemcitabine, doxorubicin, or bortezomib. Resistance to PDX was reversed by pretreatment with hypomethylating agents in a concentration‐dependent fashion. Comparison of gene expression profiles of parental and resistant cell lines confirmed markedly different patterns of gene expression, and identified the dual specificity phosphatase four (DUSP4) as one of the molecular target of PDX activity. Reduced STAT5 phosphorylation following exposure to PDX was observed in the H9 but not in the H9‐12 and H9‐200 cells. These data suggest that combination with hypomethylating agents could be potent, and that DUSP4 and STAT5 could represent putative biomarkers of PDX activity.     

TIMP4 expression is regulated by miR‐200b‐3p in prostate cancer cells

In prostate cancer TIMP4 expression level fluctuates with tumor progression. The mechanism and factors influencing its expression remain unclear. The aim of the study was to test the hypothesis on regulation of TIMP4 by microRNA‐200b‐3p. The levels of TIMP4 and miR‐200b‐3p expression were determined by real time PCR in 27 prostate carcinomas and eight benign prostatic hyperplasia samples. We found that miR‐200b‐3p positively correlated with TIMP4 expression in cancer samples (r = 0.46; p < 0.02). Moreover, mean miR‐200b‐3p level and TIMP4 expression were both higher in cancer tissues compared to benign prostatic hyperplasia samples (p > 0.05). Next, to test probable mechanisms of the regulation androgen‐sensitive human prostate adenocarcinoma cells (LNCaP) were transfected with synthetic‐miR‐200b‐3p or its synthetic antagonist. Modulation of miR‐200b‐3p in LNCaP cells had an impact on TIMP4 expression confirming the observation made in analyzed clinical samples. Two targets of miR‐200b‐3p: ZEB1 and ETS1 were investigated subsequently as potential regulators of TIMP4, however, no effect of their modulation on TIMP4 expression in LNCaP cells was found. Concluding, miR‐200b‐3p mediates regulation of TIMP4 expression in prostate cancer but exact mechanism needs to be investigated.     

Neural tube defects and impaired neural progenitor cell proliferation in Gβ1‐deficient mice

Heterotrimeric G proteins are well known for their roles in signal transduction downstream of G protein–coupled receptors (GPCRs), and both Gα subunits and tightly associated Gβγ subunits regulate downstream effector molecules. Compared to Gα subunits, the physiological roles of individual Gβ and Gγ subunits are poorly understood. In this study, we generated mice deficient in the Gβ1 gene and found that Gβ1 is required for neural tube closure, neural progenitor cell proliferation, and neonatal development. About 40% Gβ1^?/? embryos developed neural tube defects (NTDs) and abnormal actin organization was observed in the basal side of neuroepithelium. In addition, Gβ1^?/? embryos without NTDs showed microencephaly and died within 2 days after birth. GPCR agonist‐induced ERK phosphorylation, cell proliferation, and cell spreading, which were all found to be regulated by Gαi and Gβγ signaling, were abnormal in Gβ1^?/? neural progenitor cells. These data indicate that Gβ1 is required for normal embryonic neurogenesis. Developmental Dynamics 239:1089–1101, 2010. © 2010 Wiley‐Liss, Inc.     

P21‐activated kinase 2 is essential in maintenance of peripheral Foxp3+ regulatory T cells

The p21‐activated kinase 2 (Pak2), an effector molecule of the Rho family GTPases Rac and Cdc42, regulates diverse functions of T cells. Previously, we showed that Pak2 is required for development and maturation of T cells in the thymus, including thymus‐derived regulatory T (Treg) cells. However, whether Pak2 is required for the functions of various subsets of peripheral T cells, such as naive CD4 and helper T‐cell subsets including Foxp3⁺ Treg cells, is unknown. To determine the role of Pak2 in CD4 T cells in the periphery, we generated inducible Pak2 knockout (KO) mice, in which Pak2 was deleted in CD4 T cells acutely by administration of tamoxifen. Temporal deletion of Pak2 greatly reduced the number of Foxp3⁺ Treg cells, while minimally affecting the homeostasis of naive CD4 T cells. Pak2 was required for proliferation and Foxp3 expression of Foxp3⁺ Treg cells upon T‐cell receptor and interleukin‐2 stimulation, differentiation of in vitro induced Treg cells, and activation of naive CD4 T cells. Together, Pak2 is essential in maintaining the peripheral Treg cell pool by providing proliferation and maintenance signals to Foxp3⁺ Treg cells.     

Immunohistochemical expression of multidrug resistance proteins in mature T/NK‐cell lymphomas

Multidrug resistance (MDR) is defined as resistance of tumor cells to a wide spectrum of structurally and functionally unrelated drugs. One of the most important mechanisms in mediating MDR is that involving cellular drug efflux transporters. Drug resistance is a common and formidable obstacle to therapy in mature T/NK‐cell lymphomas and the MDR phenotype is thought to be one of the contributing mechanisms. In this study we assessed the immunohistochemical expression of P‐gp (P‐glycoprotein), MRP‐1 (multidrug resistance associated protein 1), BCRP (breast cancer resistance protein) and LRP (lung resistance protein) in 45 mature T/NK‐cell lymphomas diagnosed at our hospital. We detected P‐gp expression in 31% (13/42), MRP‐1 expression in 74% (31/42), BCRP in 78% (32/ 41) and LRP in 59% (26/44) of the cases. These findings show that our T/NK‐cell lymphoma cases display high frequency of MDR protein expression.     

B‐cell inhibition by cross‐linking CD79b is superior to B‐cell depletion with anti‐CD20 antibodies in treating murine collagen‐induced arthritis

Depletion of B cells with the anti‐CD20 antibody rituximab is an established therapy for rheumatoid arthritis. However, rituximab has only moderate efficacy, most likely due to insufficient depletion of B cells in lymphoid organs and expansion of pathogenic B cells. We found that an antibody against mouse CD79b profoundly blocks B‐cell proliferation induced via the B‐cell receptor, CD40, CD180, and chondroitin sulfate, but not via TLR4 or TLR9. Treatment with anti‐CD79b also induces death in resting and activated B cells. B‐cell inhibition is mediated by cross‐linkage of CD79b, but independent of Fc‐receptor engagement. In the model of collagen‐induced arthritis, an antibody against mouse CD20 depletes B cells very efficiently but fails to suppress the humoral immune response against collagen and the development of arthritis. In contrast, the antibody against CD79b, and a deglycosylated variant of this antibody, almost completely inhibits the increase in anti‐collagen antibodies and the development of arthritis. In mice with established arthritis only the fully glycosylated antibody against CD79b is effective. Our data show that targeting B cells via CD79b is much more effective than B‐cell depletion with anti‐CD20 antibodies for therapy of arthritis. These findings may have important implications for treatment of B‐cell–mediated autoimmune diseases.     

Pyruvate‐lactate exchange and glucose uptake in human prostate cancer cell models. A study in xenografts and suspensions by hyperpolarized [1‐13C]pyruvate MRS and [18F]FDG‐PET

Reprogramming of energy metabolism in the development of prostate cancer can be exploited for a better diagnosis and treatment of the disease. The goal of this study was to determine whether differences in glucose and pyruvate metabolism of human prostate cancer cells with dissimilar aggressivenesses can be detected using hyperpolarized [1‐¹³C]pyruvate MRS and [¹⁸F]FDG‐PET imaging, and to evaluate whether these measures correlate. For this purpose, we compared murine xenografts of human prostate cancer LNCaP cells with those of more aggressive PC3 cells. [1‐¹³C]pyruvate was hyperpolarized by dissolution dynamic nuclear polarization (dDNP) and [1‐¹³C]pyruvate to lactate conversion was followed by ¹³C MRS. Subsequently [¹⁸F]FDG uptake was investigated by static and dynamic PET measurements. Standard uptake values (SUVs) for [¹⁸F]FDG were significantly higher for xenografts of PC3 compared with those of LNCaP. However, we did not observe a difference in the average apparent rate constant k_pl of ¹³C label exchange from pyruvate to lactate between the tumor variants. A significant negative correlation was found between SUVs from [¹⁸F]FDG PET measurements and k_pl values for the xenografts of both tumor types. The k_pl rate constant may be influenced by various factors, and studies with a range of prostate cancer cells in suspension suggest that LDH inhibition by pyruvate may be one of these. Our results indicate that glucose and pyruvate metabolism in the prostate cancer cell models differs from that in other tumor models and that [¹⁸F]FDG‐PET can serve as a valuable complementary tool in dDNP studies of aggressive prostate cancer with [1‐¹³C]pyruvate.