Identification and characterization of novel proteolytic interactions of prostate cancer-expressed kallikrein-related peptidases, type II transmembrane serine proteases and matrix metalloproteinases

This study investigated interactions of protein-cleaving enzymes (or proteases) that promote prostate cancer progression. It provides the first evidence of a novel regulatory network of protease activity at the surface of cells. The proteases kallikrein-related peptidases 4 and 14, and matrix metalloproteinases 3 and 9 are cleaved at the cell surface by the cell surface proteases hepsin and TMPRSS2. These cleavage events potentially regulate activation of downstream targets of kallikrein 4 and 14 such as cell surface signalling via the protease-activated receptors (PARs) and cell growth-promoting factors such as hepatocyte-growth factor.

Kallikrein 4 (hK4) and prostate-specific antigen (PSA) are associated with the loss of E-cadherin and an epithelial-mesenchymal transition (EMT)-like effect in prostate cancer cells

Prostate-specific antigen (PSA) and the related kallikrein family of serine proteases are current or emerging biomarkers for prostate cancer detection and progression. Kallikrein 4 (KLK4/hK4) is of particular interest, as KLK4 mRNA has been shown to be elevated in prostate cancer. In this study, we now show that the comparative expression of hK4 protein in prostate cancer tissues, compared with benign glands, is greater than that of PSA and kallikrein 2 (KLK2/hK2), suggesting that hK4 may play an important functional role in prostate cancer progression in addition to its biomarker potential. To examine the roles that hK4, as well as PSA and hK2, play in processes associated with progression, these kallikreins were separately transfected into the PC-3 prostate cancer cell line, and the consequence of their stable transfection was investigated. PC-3 cells expressing hK4 had a decreased growth rate, but no changes in cell proliferation were observed in the cells expressing PSA or hK2. hK4 and PSA, but not hK2, induced a 2.4-fold and 1.7-fold respective increase, in cellular migration, but not invasion, through Matrigel, a synthetic extracellular matrix. We hypothesised that this increase in motility displayed by the hK4 and PSA-expressing PC-3 cells may be related to the observed change in structure in these cells from a typical rounded epithelial-like cell to a spindle-shaped, more mesenchymal-like cell, with compromised adhesion to the culture surface. Thus, the expression of E-cadherin and vimentin, both associated with an epithelial-mesenchymal transition (EMT), was investigated. E-cadherin protein was lost and mRNA levels were significantly decreased in PC-3 cells expressing hK4 and PSA (10-fold and 7-fold respectively), suggesting transcriptional repression of E-cadherin, while the expression of vimentin was increased in these cells. The loss of E-cadherin and associated increase in vimentin are indicative of EMT and provides compelling evidence that hK4, in particular, and PSA have a functional role in the progression of prostate cancer through their promotion of tumour cell migration.

An investigation of the effect of some stable nitroxide antioxidants in prostate cancer cells

The risk of prostate cancer and disease progression may potentially be increased by oxidative stress. This project examined the stability of nitroxide antioxidants and their effects on cell growth, survival and gene regulation in prostate cancer cells. The novel nitroxide, CTMIO, synthesised here at QUT, was found to have minimal toxicity and modulated the expression of a subset of oxidative stress and antioxidant-related genes distinct from those regulated by a related derivative. This study has provided a step forward in our understanding of the mechanism of action of nitroxides within cells.

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.

Identification of a novel fusion transcript between human relaxin-1 (RLN1) and human relaxin-2 (RLN2) in prostate cancer

Simultaneous expression of highly homologous RLN1 and RLN2 genes in prostate impairs their accurate delineation. We used PacBio SMRT sequencing and RNA-Seq in LNCaP cells in order to dissect the expression of RLN1 and RLN2 variants. We identified a novel fusion transcript comprising the RLN1 and RLN2 genes and found evidence of its expression in the normal and prostate cancer tissues. The RLN1-RLN2 fusion putatively encodes RLN2 isoform with the deleted secretory signal peptide. The identification of the fusion transcript provided information to determine unique RLN1-RLN2 fusion and RLN1 regions. The RLN1-RLN2 fusion was co-expressed with RLN1 in LNCaP cells, but the two gene products were inversely regulated by androgens. We showed that RLN1 is underrepresented in common PCa cell lines in comparison to normal and PCa tissue. The current study brings a highly relevant update to the relaxin field, and will encourage further studies of RLN1 and RLN2 in PCa and broader.

Circulating tumor cell detection in high-risk non-metastatic prostate cancer

Purpose The detection of circulating tumor cells (CTCs) provides important prognostic information in men with metastatic prostate cancer. We aim to determine the rate of detection of CTCs in patients with high-risk non-metastatic prostate cancer using the CellSearch® method. Method Samples of peripheral blood (7.5 mL) were drawn from 36 men with newly diagnosed high-risk non-metastatic prostate cancer, prior to any initiation of therapy and analyzed for CTCs using the CellSearch® method. Results The median age was 70 years, median PSA was 14.1, and the median Gleason score was 9. The median 5-year risk of progression of disease using a validated nomogram was 39 %. Five out of 36 patients (14 %, 95 % CI 5–30 %) had CTCs detected in their circulation. Four patients had only 1 CTC per 7.5 mL of blood detected. One patient had 3 CTCs per 7.5 mL of blood detected, which included a circulating tumor microemboli. Both on univariate analysis and multivariate analysis, there were no correlations found between CTC positivity and the classic prognostic factors including PSA, Gleason score, T-stage and age. Conclusion This study demonstrates that patients with high-risk, non-metastatic prostate cancer present infrequently with small number of CTCs in peripheral blood. This finding is consistent with the limited literature available in this setting. Other CTC isolation and detection technologies with improved sensitivity and specificity may enable detection of CTCs with mesenchymal phenotypes, although none as yet have been validated for clinical use. Newer assays are emerging for detection of new putative biomarkers for prostate cancer. Correlation of disease control outcomes with CTC detection will be important.

Targeting Antiapoptotic Genes Upregulated by Androgen Withdrawal Using Antisense Oligodeoxynucleotides to Enhance Androgen-and Chemo-Sensitivity in Prostate Cancer

Androgen withdrawal is the only effective form of systemic therapy for men with advanced disease, producing symptomatic and/or objective response in 80% of patients. Unfortunately, androgen independent (AI) progression and death occurs within a few years in the majority of these cases (6). Prostate cancer is highly chemoresistant, with objective response rates of 10% and no demonstrated survival benefit (28). Hormone refractory prostate cancer (HRPC) is therefore the main obstacle to improving the survival and quality of life in patients with advanced disease, and novel therapeutic strategies that target the molecular basis of androgen and chemoresistance are required.

A genetic variant of MDM4 influences regulation by multiple microRNAs in prostate cancer

The oncogene MDM4, also known as MDMX or HDMX, contributes to cancer susceptibility and progression through its capacity to negatively regulate a range of genes with tumour-suppressive functions. As part of a recent genome-wide association study it was determined that the A-allele of the rs4245739 SNP (A>C), located in the 3'-UTR of MDM4, is associated with an increased risk of prostate cancer. Computational predictions revealed that the rs4245739 SNP is located within a predicted binding site for three microRNAs (miRNAs): miR-191-5p, miR-887 and miR-3669. Herein, we show using reporter gene assays and endogenous MDM4 expression analyses that miR-191-5p and miR-887 have a specific affinity for the rs4245739 SNP C-allele in prostate cancer. These miRNAs do not affect MDM4 mRNA levels, rather they inhibit its translation in C-allele-containing PC3 cells but not in LNCaP cells homozygous for the A-allele. By analysing gene expression datasets from patient cohorts, we found that MDM4 is associated with metastasis and prostate cancer progression and that targeting this gene with miR-191-5p or miR-887 decreases in PC3 cell viability. This study is the first, to our knowledge, to demonstrate regulation of the MDM4 rs4245739 SNP C-allele by two miRNAs in prostate cancer, and thereby to identify a mechanism by which the MDM4 rs4245739 SNP A-allele may be associated with an increased risk for prostate cancer.

Kallikrein-related peptidases in prostate cancer: From molecular function to clinical application

Prostate cancer is a leading contributor to male cancer-related deaths worldwide. Kallikrein-related peptidases (KLKs) are serine proteases that exhibit deregulated expression in prostate cancer, with KLK3, or prostate specific antigen (PSA), being the widely-employed clinical biomarker for prostate cancer. Other KLKs, such as KLK2, show promise as prostate cancer biomarkers and, additionally, their altered expression has been utilised for the design of KLK-targeted therapies. There is also a large body of in vitro and in vivo evidence supporting their role in cancer-related processes. Here, we review the literature on studies to date investigating the potential of other KLKs, in addition to PSA, as biomarkers and in therapeutic options, as well as their current known functional roles in cancer progression. Increased knowledge of these KLK-mediated functions, including degradation of the extracellular matrix, local invasion, cancer cell proliferation, interactions with fibroblasts, angiogenesis, migration, bone metastasis and tumour growth in vivo, may help define new roles as prognostic biomarkers and novel therapeutic targets for this cancer.

Adipocytes promote prostate cancer stem cell self-renewal through amplification of the cholecystokinin autocrine loop

Obesity has long been linked with prostate cancer progression, although the underlying mechanism is still largely unknown. Here, we report that adipocytes promote the enrichment of prostate cancer stem cells (CSCs) through a vicious cycle of autocrine amplification. In the presence of adipocytes, prostate cancer cells actively secrete the peptide hormone cholecystokinin (CCK), which not only stimulates prostate CSC self-renewal, but also induces cathepsin B (CTSB) production of the adipocytes. In return, CTSB facilitates further CCK secretion by the cancer cells. More importantly, inactivation of CCK receptor not only suppresses CTSB secretion by the adipocytes, but also synergizes the inhibitory effect of CTSB inhibitor on adipocyte-promoted prostate CSC self-renewal. In summary, we have uncovered a novel mechanism underlying the mutual interplay between adipocytes and prostate CSCs, which may help explaining the role of adipocytes in prostate cancer progression and provide opportunities for effective intervention.

The effect of lycopene on the PI3K/Akt signalling pathway in prostate cancer

Prostate cancer is common in men with very high mortality which is one of leading causes of cancer-related deaths in men. The main treatment approaches for metastasized prostate cancer are androgen deprivation and chemotherapeutic agents. Although there are initial responses to castration, the resistance to the treatment will eventually occur, leading to castration-resistant prostate cancer. The common chemotherapeutic agents for the treatment of prostate cancer are docetaxel and taxane but outcomes of using these drugs have not been satisfactory. Therefore, it is necessary to find better treatment approaches for prostate cancer and to search for compounds that are effective in prostate cancer prevention. Lycopene extracted from tomato and other fruits or plants such as Gac, watermelon, pink grapefruit, pink guava, red carrot and papaya has been shown to be effective on prostate cancer prevention and treatment. The advantage of the application of lycopene for its anti-prostate cancer activity is that lycopene can reach much higher concentration in prostate tissue than other tissues. In this review, the effect of lycopene on PI3K/Akt pathway is summarised, which could be one of major mechanisms for anti-cancer activity of lycopene.

Characterisation of novel primary tumour derived epithelial prostate cancer cell lines

Current translational and basic prostate cancer research is limited by the number of cell lines that truly reflect the spectrum of disease progression, with most commonly used cell lines being derived from metastatic lesions. There are essentially no prostate cancer cell lines derived from primary tumours or localised disease in wide use.

Modulation of protease expression in prostate cancer cells after androgen deprivation

Understanding mechanisms associated with the emergence of castration resistant prostate cancer cells (CRPC) after androgen deprivation therapy (ADT) is essential to create new therapeutic agents to counteract this aggressive form of prostate cancer (PCa). Because proteases are involved in almost all cancer associated mechanisms such as cell proliferation, invasion and metastasis, we are interested in their modulation in PCa after ADT and their involvement in CRPC.

Extracellular vesicles mediate paracrine signalling in androgen deprived prostate cancer

The androgen receptor (AR) is the main therapeutic target for advanced prostate cancer (PCa). Current treatments have focused on inhibiting the transcriptional activity of the AR, however androgens can also induce non-genomic effects by facilitating the initiation of kinase signaling cascades in PCa. Cells, including PCa, secrete extracellular vesicles (EV), which are able to mediate communication between cells and can also contribute towards these processes.