Integrin-linked kinase as a target for ERG-mediated invasive properties in prostate cancer models

Approximately half of prostate cancers (PCa) carry TMPRSS2-ERG translocations; however, the clinical impact of this genomic alteration remains enigmatic. Expression of v-ets erythroblastosis virus E26 oncogene like (avian) gene (ERG) promotes prostatic epithelial dysplasia in transgenic mice and acquisition of epithelial-to-mesenchymal transition (EMT) characteristics in human prostatic epithelial cells (PrECs). To explore whether ERG-induced EMT in PrECs was associated with therapeutically targetable transformation characteristics, we established stable populations of BPH-1, PNT1B and RWPE-1 immortalized human PrEC lines that constitutively express flag-tagged ERG3 (fERG). All fERG-expressing populations exhibited characteristics of in vitro and in vivo transformation. Microarray analysis revealed >2000 commonly dysregulated genes in the fERG-PrEC lines. Functional analysis revealed evidence that fERG cells underwent EMT and acquired invasive characteristics. The fERG-induced EMT transcript signature was exemplified by suppressed expression of E-cadherin and keratins 5, 8, 14 and 18; elevated expression of N-cadherin, N-cadherin 2 and vimentin, and of the EMT transcriptional regulators Snail, Zeb1 and Zeb2, and lymphoid enhancer-binding factor-1 (LEF-1). In BPH-1 and RWPE-1-fERG cells, fERG expression is correlated with increased expression of integrin-linked kinase (ILK) and its downstream effectors Snail and LEF-1. Interfering RNA suppression of ERG decreased expression of ILK, Snail and LEF-1, whereas small interfering RNA suppression of ILK did not alter fERG expression. Interfering RNA suppression of ERG or ILK impaired fERG-PrEC Matrigel invasion. Treating fERG-BPH-1 cells with the small molecule ILK inhibitor, QLT-0267, resulted in dose-dependent suppression of Snail and LEF-1 expression, Matrigel invasion and reversion of anchorage-independent growth. These results suggest that ILK is a therapeutically targetable mediator of ERG-induced EMT and transformation in PCa.

Speckle-type POZ protein mutations interrupt tumor suppressor function of speckle-type POZ protein in prostate cancer by affecting androgen receptor degradation

Large scale exome sequencing studies have revealed regions of the genome, which contribute to the castrate resistant prostate cancer (CRPC) phenotype. [1],[2],[3] Such studies have identified mutations in genes, which may have diagnostic/prognostic potential, or which may be targeted therapeutically. Two of these genes include the androgen receptor (AR) and speckle-type POZ protein (SPOP) genes. However, the findings from these exome sequencing studies can only be translated therapeutically once the functional consequences of these mutations have been determined. Here, we highlight the recent study by An et al. [4] which investigated the functional effects of mutations in the SPOP gene that were identified in the aforementioned exome sequencing studies, particularly in the context of SPOP-mediated degradation of the AR.

Fine-mapping the HOXB region detects common variants tagging a rare coding allele : evidence for synthetic association in prostate cancer

The HOXB13 gene has been implicated in prostate cancer (PrCa) susceptibility. We performed a high resolution fine-mapping analysis to comprehensively evaluate the association between common genetic variation across the HOXB genetic locus at 17q21 and PrCa risk. This involved genotyping 700 SNPs using a custom Illumina iSelect array (iCOGS) followed by imputation of 3195 SNPs in 20,440 PrCa cases and 21,469 controls in The PRACTICAL consortium. We identified a cluster of highly correlated common variants situated within or closely upstream of HOXB13 that were significantly associated with PrCa risk, described by rs117576373 (OR 1.30, P = 2.62×10(-14)). Additional genotyping, conditional regression and haplotype analyses indicated that the newly identified common variants tag a rare, partially correlated coding variant in the HOXB13 gene (G84E, rs138213197), which has been identified recently as a moderate penetrance PrCa susceptibility allele. The potential for GWAS associations detected through common SNPs to be driven by rare causal variants with higher relative risks has long been proposed; however, to our knowledge this is the first experimental evidence for this phenomenon of synthetic association contributing to cancer susceptibility.

Elevated level of inhibin-α subunit is pro-tumourigenic and pro-metastatic and associated with extracapsular spread in advanced prostate cancer

The biological function of inhibin-a subunit (INHa) in prostate cancer (PCa) is currently unclear. A recent study associated elevated levels of INHa in PCa patients with a higher risk of recurrence. This prompted us to use clinical specimens and functional studies to investigate the pro-tumourigenic and pro-metastatic function of INHa. We conducted a cross-sectional study to determine a link between INHa expression and a number of clinicopathological parameters including Gleason score, surgical margin, extracapsular spread, lymph node status and vascular endothelial growth factor receptor-3 expression, which are well-established prognostic factors of PCa. In addition, using two human PCa cell lines (LNCaP and PC3) representing androgen-dependent and -independent PCa respectively, we investigated the biological function of elevated levels of INHa in advanced cancer. Elevated expression of INHa in primary PCa tissues showed a higher risk of PCa patients being positive for clinicopathological parameters outlined above. Overexpressing INHa in LNCaP and PC3 cells demonstrated two different and cell-type-specific responses. INHa-positive LNCaP demonstrated reduced tumour growth whereas INHa-positive PC3 cells demonstrated increased tumour growth and metastasis through the process of lymphangiogenesis. This study is the first to demonstrate a pro-tumourigenic and pro-metastatic function for INHa associated with androgen-independent stage of metastatic prostate disease. Our results also suggest that INHa expression in the primary prostate tumour can be used as a predictive factor for prognosis of PCa.

Potential role of EPB41L3 (Protein 4.1B/Dal-1) as a target for treatment of advanced prostate cancer

Background: Loss of erythrocyte membrane protein band 4.1-like 3 (EPB41L3; aliases: protein 4.1B, differentially expressed in adenocarcinoma of the lung-1 (Dal-1)) expression has been implicated in tumor progression. Objective: To evaluate literature describing the role of EPB41L3 in tumorigenesis and metastasis, and to consider whether targeting this gene would be useful in the treatment of prostate cancer. Methods: A literature review of studies describing EPB41L3 and its aliases was conducted. Online databases (NCBI, SwissProt) were also interrogated to collect further data. Results/conclusion: A growing body of evidence supports a role for loss of EPB41L3 in tumor progression, including in prostate cancer. Therapeutic strategies that could be harnessed to upregulate EPB41L3 gene expression in prostate cancer cells are currently being developed.

Stem-like cells with luminal progenitor phenotype survive castration in human prostate cancer

Castration is the standard therapy for advanced prostate cancer (PC). Although this treatment is initially effective, tumors invariably relapse as incurable, castration-resistant PC (CRPC). Adaptation of androgen-dependent PC cells to an androgen-depleted environment or selection of pre-existing,CRPC cells have been proposed as mechanisms of CRPC development. Stem cell (SC)-like PC cells have been implicated not only as tumor initiating/maintaining in PC but also as tumor-reinitiating cells in CRPC. Recently, castration-resistant cells expressing the NK3 homeobox 1 (Nkx3-1) (CARNs), the other luminal markers cytokeratin 18 (CK18) and androgen receptor (AR), and possessing SC properties, have been found in castrated mouse prostate and proposed as the cell-of-origin of CRPC. However, the human counterpart of CARNs has not been identified yet. Here, we demonstrate that in the human PC xenograft BM18, preexisting SC-like and neuroendocrine (NE) PC cells are selected by castration and survive as totally quiescent. SClike BM18 cells, displaying the SC markers aldehyde dehydrogenase 1A1 or NANOG, coexpress the luminal markers NKX3-1, CK18, and a low level of AR (ARlow) but not basal or NE markers. These CR luminal SC-like cells, but not NE cells, reinitiate BM18 tumor growth after androgen replacement. The ARlow seems to mediate directly both castration survival and tumor reinitiation. This study identifies for the first time in human PC SC-/CARN-like cells that may represent the cell-of-origin of tumor reinitiation as CRPC. This finding will be fundamental for refining the hierarchy among human PC cancer cells and may have important clinical implications.

Androgen-targeted therapy-induced epithelial mesenchymal plasticity and neuroendocrine transdifferentiation in prostate cancer : an opportunity for intervention

Androgens regulate biological pathways to promote proliferation, differentiation, and survival of benign and malignant prostate tissue. Androgen receptor (AR) targeted therapies exploit this dependence and are used in advanced prostate cancer to control disease progression. Contemporary treatment regimens involve sequential use of inhibitors of androgen synthesis or AR function. Although targeting the androgen axis has clear therapeutic benefit, its effectiveness is temporary, as prostate tumor cells adapt to survive and grow. The removal of androgens (androgen deprivation) has been shown to activate both epithelial-to-mesenchymal transition (EMT) and neuroendocrine transdifferentiation (NEtD) programs. EMT has established roles in promoting biological phenotypes associated with tumor progression (migration/invasion, tumor cell survival, cancer stem cell-like properties, resistance to radiation and chemotherapy) in multiple human cancer types. NEtD in prostate cancer is associated with resistance to therapy, visceral metastasis, and aggressive disease. Thus, activation of these programs via inhibition of the androgen axis provides a mechanism by which tumor cells can adapt to promote disease recurrence and progression. Brachyury, Axl, MEK, and Aurora kinase A are molecular drivers of these programs, and inhibitors are currently in clinical trials to determine therapeutic applications. Understanding tumor cell plasticity will be important in further defining the rational use of androgen-targeted therapies clinically and provides an opportunity for intervention to prolong survival of men with metastatic prostate cancer.

Establishing prostate cancer patient derived xenografts: Lessons learned from older studies

Background Understanding the progression of prostate cancer to androgen-independence/castrate resistance and development of preclinical testing models are important for developing new prostate cancer therapies. This report describes studies performed 30 years ago, which demonstrate utility and shortfalls of xenografting to preclinical modeling. Methods We subcutaneously implanted male nude mice with small prostate cancer fragments from transurethral resection of the prostate (TURP) from 29 patients. Successful xenografts were passaged into new host mice. They were characterized using histology, immunohistochemistry for marker expression, flow cytometry for ploidy status, and in some cases by electron microscopy and response to testosterone. Two xenografts were karyotyped by G-banding. Results Tissues from 3/29 donors (10%) gave rise to xenografts that were successfully serially passaged in vivo. Two, (UCRU-PR-1, which subsequently was replaced by a mouse fibrosarcoma, and UCRU-PR-2, which combined epithelial and neuroendocrine features) have been described. UCRU-PR-4 line was a poorly differentiated prostatic adenocarcinoma derived from a patient who had undergone estrogen therapy and bilateral castration after his cancer relapsed. Histologically, this comprised diffusely infiltrating small acinar cell carcinoma with more solid aggregates of poorly differentiated adenocarcinoma. The xenografted line showed histology consistent with a poorly differentiated adenocarcinoma and stained positively for prostatic acid phosphatase (PAcP), epithelial membrane antigen (EMA) and the cytokeratin cocktail, CAM5.2, with weak staining for prostate specific antigen (PSA). The line failed to grow in female nude mice. Castration of three male nude mice after xenograft establishment resulted in cessation of growth in one, growth regression in another and transient growth in another, suggesting that some cells had retained androgen sensitivity. The karyotype (from passage 1) was 43–46, XY, dic(1;12)(p11;p11), der(3)t(3:?5)(q13;q13), -5, inv(7)(p15q35) x2, +add(7)(p13), add(8)(p22), add(11)(p14), add(13)(p11), add(20)(p12), -22, +r4[cp8]. Conclusions Xenografts provide a clinically relevant model of prostate cancer, although establishing serially transplantable prostate cancer patient derived xenografts is challenging and requires rigorous characterization and high quality starting material. Xenografting from advanced prostate cancer is more likely to succeed, as xenografting from well differentiated, localized disease has not been achieved in our experience. Strong translational correlations can be demonstrated between the clinical disease state and the xenograft model

A cellular automata model to investigate immune cell–tumor cell interactions in growing tumors in two spatial dimensions

We develop a hybrid cellular automata model to describe the effect of the immune system and chemokines on a growing tumor. The hybrid cellular automata model consists of partial differential equations to model chemokine concentrations, and discrete cellular automata to model cell–cell interactions and changes. The computational implementation overlays these two components on the same spatial region. We present representative simulations of the model and show that increasing the number of immature dendritic cells (DCs) in the domain causes a decrease in the number of tumor cells. This result strongly supports the hypothesis that DCs can be used as a cancer treatment. Furthermore, we also use the hybrid cellular automata model to investigate the growth of a tumor in a number of computational “cancer patients.” Using these virtual patients, the model can explain that increasing the number of DCs in the domain causes longer “survival.” Not surprisingly, the model also reflects the fact that the parameter related to tumor division rate plays an important role in tumor metastasis.

Inhibiting Eph kinase activity may not be “Eph”ective for cancer treatment

Several Eph receptor tyrosine kinases (RTKs) are commonly over-expressed in epithelial and mesenchymal cancers and are recognized as promising therapeutic targets. Although normal interaction between Eph receptors and their ephrin ligands stimulates kinase activity and is generally tumor suppressive, significant Eph over-expression allows activation of ligand- and/or kinase-independent signaling pathways that promote oncogenesis. Single-agent kinase inhibitors are widely used to target RTK-driven tumors but acquired and de novo resistance to such agents is a major limitation to effective clinical use. Accumulating evidence suggests that Ephs can be inhibited by “leaky” or low-specificity kinase inhibitors targeted at other RTKs. Such off-target effects may therefore inadvertently promote ligand- and/or kinase-independent oncogenic Eph signaling, thereby providing a new mechanism by which resistance to the RTK inhibitors can emerge. We propose that combining specific, non-leaky kinase inhibitors with tumor-suppressive stimulators of Eph signaling may provide more effective treatment options for overcoming treatment-induced resistance and clinical failure.

EphB4 localises to the nucleus of prostate cancer cells

The EphB4 receptor tyrosine kinase is over-expressed in a variety of different epithelial cancers including prostate where it has been shown to be involved in survival, migration and angiogenesis. We report here that EphB4 also resides in the nucleus of prostate cancer cell lines. We used in silico methods to identify a bipartite nuclear localisation signal (NLS) in the extracellular domain and a monopartite NLS sequence in the intracellular kinase domain of EphB4. To determine whether both putative NLS sequences were functional, fragments of the EphB4 sequence containing each NLS were cloned to create EphB4NLS-GFP fusion proteins. Localisation of both NLS-GFP proteins to the nuclei of transfected cells was observed, demonstrating that EphB4 contains two functional NLS sequences. Mutation of the key amino residues in both NLS sequences resulted in diminished nuclear accumulation. As nuclear translocation is often dependent on importins we confirmed that EphB4 and importin-α can interact. To assess if nuclear EphB4 could be implicated in gene regulatory functions potential EphB4-binding genomic loci were identified using chromatin immunoprecipitation and Lef1 was confirmed as a potential target of EphB4-mediated gene regulation. These novel findings add further complexity to the biology of this important cancer-associated receptor.

Murine, but not human, ephrin-B2 can be efficiently cleaved by the serine protease kallikrein-4: Implications for xenograft models of human prostate cancer

Background Ephrin-B2 is the sole physiologically-relevant ligand of the receptor tyrosine kinase EphB4, which is over-expressed in many epithelial cancers, including 66% of prostate cancers, and contributes to cancer cell survival, invasion and migration. Crucially, however, the cancer-promoting EphB4 signalling pathways are independent of interaction with its ligand ephrin-B2, as activation of ligand-dependent signalling causes tumour suppression. Ephrin-B2, however, is often found on the surface of endothelial cells of the tumour vasculature, where it can regulate angiogenesis to support tumour growth. Proteolytic cleavage of endothelial cell ephrin-B2 has previously been suggested as one mechanism whereby the interaction between tumour cell-expressed EphB4 and endothelial cell ephrin-B2 is regulated to support both cancer promotion and angiogenesis. Methods An in silico approach was used to search accessible surfaces of 3D protein models for cleavage sites for the key prostate cancer serine protease, KLK4, and this identified murine ephrin-B2 as a potential KLK4 substrate. Mouse ephrin-B2 was then confirmed as a KLK4 substrate by in vitro incubation of recombinant mouse ephrin-B2 with active recombinant human KLK4. Cleavage products were visualised by SDS-PAGE, silver staining and Western blot and confirmed by N-terminal sequencing. Results At low molar ratios, KLK4 cleaved murine ephrin-B2 but other prostate-specific KLK family members (KLK2 and KLK3/PSA) were less efficient, suggesting cleavage was KLK4-selective. The primary KLK4 cleavage site in murine ephrin-B2 was verified and shown to correspond to one of the in silico predicted sites between extracellular domain residues arginine 178 and asparagine 179. Surprisingly, the highly homologous human ephrin-B2 was poorly cleaved by KLK4 at these low molar ratios, likely due to the 3 amino acid differences at this primary cleavage site. Conclusion These data suggest that in in vivo mouse xenograft models, endogenous mouse ephrin-B2, but not human tumour ephrin-B2, may be a downstream target of cancer cell secreted human KLK4. This is a critical consideration when interpreting data from murine explants of human EphB4+/KLK4+ cancer cells, such as prostate cancer cells, where differential effects may be seen in mouse models as opposed to human clinical situations.

The tumour-promoting receptor tyrosine kinase, EphB4, regulates expression of Integrin-β8 in prostate cancer cells

Background The EphB4 receptor tyrosine kinase is overexpressed in many cancers including prostate cancer. The molecular mechanisms by which this ephrin receptor influences cancer progression are complex as there are tumor-promoting ligand-independent mechanisms in place as well as ligand-dependent tumor suppressive pathways. Methods We employed transient knockdown of EPHB4 in prostate cancer cells, coupled with gene microarray analysis, to identify genes that were regulated by EPHB4 and may represent linked tumor-promoting factors. We validated target genes using qRT-PCR and employed functional assays to determine their role in prostate cancer migration and invasion. Results We discovered that over 500 genes were deregulated upon EPHB4 siRNA knockdown, with integrin β8 (ITGB8) being the top hit (29-fold down-regulated compared to negative non-silencing siRNA). Gene ontology analysis found that the process of cell adhesion was highly deregulated and two other integrin genes, ITGA3 and ITGA10, were also differentially expressed. In parallel, we also discovered that over-expression of EPHB4 led to a concomitant increase in ITGB8 expression. In silico analysis of a prostate cancer progression microarray publically available in the Oncomine database showed that both EPHB4 and ITGB8 are highly expressed in prostatic intraepithelial neoplasia, the precursor to prostate cancer. Knockdown of ITGB8 in PC-3 and 22Rv1 prostate cancer cells in vitro resulted in significant reduction of cell migration and invasion. Conclusions These results reveal that EphB4 regulates integrin β8 expression and that integrin β8 plays a hitherto unrecognized role in the motility of prostate cancer cells and thus targeting integrin β8 may be a new treatment strategy for prostate cancer.

Targeting ASCT2-mediated glutamine uptake blocks prostate cancer growth and tumour development

Glutamine is conditionally essential in cancer cells, being utilized as a carbon and nitrogen source for macromolecule production, as well as for anaplerotic reactions fuelling the tricarboxylic acid (TCA) cycle. In this study, we demonstrated that the glutamine transporter ASCT2 (SLC1A5) is highly expressed in prostate cancer patient samples. Using LNCaP and PC-3 prostate cancer cell lines, we showed that chemical or shRNA-mediated inhibition of ASCT2 function in vitro decreases glutamine uptake, cell cycle progression through E2F transcription factors, mTORC1 pathway activation and cell growth. Chemical inhibition also reduces basal oxygen consumption and fatty acid synthesis, showing that downstream metabolic function is reliant on ASCT2-mediated glutamine uptake. Furthermore, shRNA knockdown of ASCT2 in PC-3 cell xenografts significantly inhibits tumour growth and metastasis in vivo, associated with the down-regulation of E2F cell cycle pathway proteins. In conclusion, ASCT2-mediated glutamine uptake is essential for multiple pathways regulating the cell cycle and cell growth, and is therefore a putative therapeutic target in prostate cancer.

Adverse effects of androgen-deprivation therapy in prostate cancer and their management

Objective To provide an up-to-date summary of current literature on the management of adverse effects of androgen-deprivation therapy (ADT). Patients and Methods All relevant medical literature on men with prostate cancer treated with ADT from 2005 to 2014, and older relevant papers, were reviewed. Recent health advisory statements from the Australian government, societies and advocacy groups have been incorporated to the document. Results There are numerous adverse effects of ADT that require pro-active prevention and treatment. Ranging from cardiovascular disease, diabetes and osteoporosis, to depression, cognitive decline and sexual dysfunction, the range of adverse effects is wide. Baseline assessment, monitoring, prevention and consultation from a multidisciplinary team are important in minimising the harm from ADT. Conclusions This review provides a series of practical recommendations to assist with managing the adverse effects of ADT.