Stephen J. Assinder

Transgelin: an actin-binding protein and tumour suppressor.

Transgelin is a shape change sensitive 22 kDa actin-binding protein of the calponin family. It contains a C-terminal calponin-like module (CLIK(23)) and an upstream positively charged amino acid region required for actin binding. Transgelin is ubiquitous to vascular and visceral smooth muscle and is an early marker of smooth muscle differentiation, where its expression is driven by CArG box, smooth muscle gene promoter. It is also present in fibroblasts, and some epithelium where expression is likely driven by TGF-beta1. Transgelin null mice reveal that, whilst it is not required for smooth muscle development, transgelin may be involved in calcium-independent smooth muscle contraction. Recent evidence suggests that transgelin acts as a tumour suppressor. Its expression is lost in prostate, breast and colon cancers. This is consistent with suppression of the metallo matrix protease-9 (MMP-9) by transgelin, where MMP-9 is upregulated in these common cancers.

Current Status of Biomarkers for Prostate Cancer

Prostate cancer (PCa) is a leading cause of cancer-related death of men globally. Since its introduction, there has been intense debate as to the effectiveness of the prostate specific antigen (PSA) test as a screening tool for PCa. It is now evident that the PSA test produces unacceptably high rates of false positive results and is not prognostic. Here we review the current status of molecular biomarkers that promise to be prognostic and that might inform individual patient management. It highlights current efforts to identify biomarkers obtained by minimally invasive methods and discusses current knowledge with regard to gene fusions, mRNA and microRNAs, immunology, and cancer-associated microparticles.

Dp44mT targets the AKT, TGF- β and ERK pathways via the metastasis suppressor NDRG1 in normal prostate epithelial cells and prostate cancer cells

Background:Effective treatment of prostate cancer should be based on targeting interactions between tumour cell signalling pathways and key converging downstream effectors. Here, we determined how the tumourigenic phosphoinositide 3-kinase/protein kinase B (PI3K/AKT), tumour-suppressive phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and transforming growth factor-β (TGF-β) pathways are integrated via the metastasis suppressor, N-myc downstream-regulated gene-1 (NDRG1). Moreover, we assessed how the novel anti-tumour agent, Dp44mT, may target these integrated pathways by increasing NDRG1 expression.Methods:Protein expression in Dp44mT-treated normal human prostate epithelial cells and prostate cancer cells (PC-3, DU145) was assessed by western blotting. The role of NDRG1 was examined by transfection using an NDRG1 overexpression vector or shRNA.Results:Dp44mT increased levels of tumour-suppressive PTEN, and decreased phosphorylation of ERK1/2 and SMAD2L, which are regulated by oncogenic Ras/MAPK signalling. Importantly, the effects of Dp44mT on NDRG1 and p-SMAD2L expression were more marked in prostate cancer cells than normal prostate epithelial cells. This may partly explain the anti-tumour selectivity of these agents. Silencing NDRG1 expression increased phosphorylation of tumourigenic AKT, ERK1/2 and SMAD2L and decreased PTEN levels, whereas NDRG1 overexpression induced the opposite effect. Furthermore, NDRG1 silencing significantly reduced the ability of Dp44mT to suppress p-SMAD2L and p-ERK1/2 levels.Conclusion:NDRG1 has an important role in mediating the tumour-suppressive effects of Dp44mT in prostate cancer via selective targeting of the PI3K/AKT, TGF-β and ERK pathways.

The mammalian copper transporters CTR1 and CTR2 and their roles in development and disease.

Copper is vital to cell function. The influx of reduced copper ions is controlled by two functionally homologous transmembrane solute carrier transporters CTR1 (encoded by SLC31A1) and CTR2 (encoded by SLC31A2). These copper transporters vary in their expression profiles and intracellular localisation patterns. CTR1 plays roles in the developing embryo as well as regulating homeostasis in the adult mammal. In contrast, the regulation, expression and function of CTR2 is poorly defined. Both are capable of transporting other divalent metal ions and are the primary transporters for platinum-based chemotherapeutic drugs such as cisplatin. This review summarises our current understanding of these two copper transporters and highlights their roles in cellular processes, embryonic development, differentiation, cancer, immunity and disease.

Oxytocin in health and disease

Oxytocin is a nonapeptide of the neurohypophyseal protein family that binds specifically to the oxytocin receptor to produce a multitude of central and peripheral physiological responses. Within the central nervous system oxytocin is expressed by the neurons of the hypothalamus that project into higher brain centres and the posterior pituitary gland, from where it enters the circulation by release into the portal capillaries. Centrally, it modulates, maternal, sexual, social and stress related behaviour. Peripheral actions of oxytocin are commonly associated with smooth muscle contraction, particularly within the female and male reproductive tracts. Local synthesis of oxytocin along with its receptor in these regions indicates the presence of local oxytocinergic systems. More sinister implications for oxytocin in autism, depression and several cancers have recently been identified. A greater understanding of the role of oxytocinergic mechanisms will determine the potential for targeting this regulatory peptide in the pharmacological management of these disorders.

Expression of the actin-associated protein transgelin (SM22) is decreased in prostate cancer

Transgelin is an actin-binding protein shown to be tumour-suppressive. Loss of transgelin expression in transformed cells is associated with oncogenesis. This study aimed to determine whether transgelin expression was suppressed in prostate cancer. An in silico meta-analysis with public-domain expressed-sequence-tag libraries of normal human prostate epithelium, prostatic intraepithelial neoplasia, invasive carcinoma and metastasised lesions predicted decreased transgelin expression with disease progression. Similarly, analysis of Affymetrix gene chip data and the Oncomine database indicated that transgelin was one the 2% most significant of all down-regulated genes in response to prostate cancer. Analysis by quantitative reverse transcription with the polymerase chain reaction (qRT-PCR) of patient biopsies determined transgelin expression to be significantly lower in prostate tumour tissue than in matched normal tissue. Similarly, qRT-PCR and Western blot analysis of representative prostate cancer cell lines demonstrated significantly lower levels of transgelin mRNA and protein in all but the DU145 prostate cancer cell line. Increased expression of TAGLN and increased transgelin protein in response to treatment with transforming growth factor-β suggested that reduced expression in prostate cancer was not attributable to gene promoter suppression by hypermethylation. Gene ontology function analysis highlighted the importance of transgelin in the co-deregulation of actin-binding proteins. Thus, transgelin is suppressed during prostate cancer progression and seems to be an important factor in the dysregulation of the actin cytoskeleton.

Stac3 is required for myotube formation and myogenic differentiation in vertebrate skeletal muscle

Background: Stac3, an uncharacterised gene, was identified by its high expression levels in vertebrate muscle. Results: Knockdown of stac3 inhibited zebrafish myofibrillar protein assembly and differentiation in C2C12 cells by perturbing Akt signaling and cell cycle exit. Conclusion: Stac3 is a novel regulator of myogenic differentiation. Significance: Identifying new genes controlling myogenic differentiation will allow greater understanding of processes leading to muscular diseases. Stac3 was identified as a nutritionally regulated gene from an Atlantic salmon subtractive hybridization library with highest expression in skeletal muscle. Salmon Stac3 mRNA was highly correlated with myogenin and myoD1a expression during differentiation of a salmon primary myogenic culture and was regulated by amino acid availability. In zebrafish embryos, stac3 was initially expressed in myotomal adaxial cells and in fast muscle fibers post-segmentation. Morpholino knockdown resulted in defects in myofibrillar protein assembly, particularly in slow muscle fibers, and decreased levels of the hedgehog receptor patched. The function of Stac3 was further characterized in vitro using the mammalian C2C12 myogenic cell line. Stac3 mRNA expression increased during the differentiation of the C2C12 myogenic cell line. Knockdown of Stac3 by RNAi inhibited myotube formation, and microarray analysis revealed that transcripts involved in cell cycle, focal adhesion, cytoskeleton, and the pro-myogenic factors Igfbp-5 and Igf2 were down-regulated. RNAi-treated cells had suppressed Akt signaling and exogenous insulin-like growth factor (Igf) 2 was unable to rescue the phenotype, however, Igf/Akt signaling was not blocked. Overexpression of Stac3, which results in increased levels of Igfbp-5 mRNA, did not lead to increased differentiation. In synchronized cells, Stac3 mRNA was most abundant during the G1 phase of the cell cycle. RNAi-treated cells were smaller, had higher proliferation rates and a decreased proportion of cells in G1 phase when compared with controls, suggesting a role in the G1 phase checkpoint. These results identify Stac3 as a new gene required for myogenic differentiation and myofibrillar protein assembly in vertebrates.

Proteolytic cleavage and truncation of NDRG1 in human prostate cancer cells, but not normal prostate epithelial cells.

NDRG1 (N-myc downstream regulated gene-1) is a metastasis suppressor that is down-regulated in prostate cancer. NDRG1 phosphorylation is associated with inhibition of metastasis and Western blots indicate two bands at ~41 and ~46 kDa. Previous investigations by others suggest the higher band is due to NDRG1 phosphorylation. However, the current study using a dephosphorylation assay and the Phos-tag (phosphate-binding tag) SDS/PAGE assay, demonstrated that the 46 kDa NDRG1 protein band was not due to phosphorylation. Further experiments showed that the NDRG1 protein bands were not affected upon glycosidase treatment, despite marked effects of these enzymes on the glycosylated protein, fetuin. Analysis using RT–PCR (reverse transcriptase–PCR) demonstrated only a single amplicon, and thus, the two bands could not result from an alternatively spliced NDRG1 transcript. Western-blot analysis of prostate cancer cell lysates identified the 41 kDa band to be a truncated form of NDRG1, with MS confirming the full and truncated proteins to be NDRG1. Significantly, this truncated protein was not present in normal human PrECs (prostate epithelial cells). Western-blot analysis using anti-NDRG1 raised to its N-terminal sequence failed to detect the truncated protein, suggesting that it lacked N-terminus amino acids (residues 1–49). Sequence analysis predicted a pseudotrypsin protease cleavage site between Cys49–Gly50. Such cleavage of NDRG1 in cancer cells may result in loss of NDRG1 tumour suppressive activity.

Seasonal Changes in Mesotocin and Localization of Its Receptor in the Prostate of the Brushtail Possum (Trichosurus vulpecula)

Abstract The prostate gland in the brushtail possum grows and regresses seasonally. It has similarities to the human prostate and may therefore provide a unique model for investigating prostatic hyperplasia. Oxytocin has been implicated in the regulation of prostate growth in eutherian mammals, and the initial aim of this study was to identify and localize the marsupial equivalent, mesotocin, and its receptor in the prostate of the brushtail possum. Seasonal changes in prostatic mesotocin concentrations and receptor localization were then assessed and related to prostate growth. Mesotocin and mesotocin receptor gene transcripts with high sequence homology to eutherian oxytocin/oxytocin receptors were demonstrated, and mesotocin, neurophysin, and the receptor were all localized predominantly in the epithelial cells of the glandular acini. Western blot analysis confirmed the presence of a single immunoreactive receptor protein of ∼60 Mr−3. Prostatic mesotocin concentrations were highest immediately before the increases in prostate weight associated with the autumn and spring breeding periods. At this time, mesotocin receptors were also present in the prostatic capsule in addition to those present in the glandular tissue. Mesotocin concentrations proceeded to decrease in association with the regression of prostate size toward the end of the breeding periods. No significant differences were present in serum testosterone or dihydrotestosterone throughout the year. The identification of mesotocin and its receptor in the possum prostate and the demonstration of seasonal changes in local mesotocin concentrations preceding changes in prostate size suggests that mesotocin may play a physiological role in regulating prostate growth and regression.

A novel splice variant of the β-tropomyosin (TPM2) gene in prostate cancer

Decreased expression of high molecular weight isoforms of tropomyosin (Tm) is associated with oncogenic transformation and is evident in cancers, with isoform Tm1 seemingly an important tumor suppressor. Tm1 expression in prostate cancer has not previously been described. In this study, while demonstrating suppressed levels of Tm1 in the prostate cancer cell lines LNCaP, PC3, and DU‐145 compared to normal prostate epithelial cell primary isolates (PrEC), a novel splice variant of the TPM2 gene was identified. Quantitative RT‐PCR determined significantly greater levels of the transcript variant in all three prostate cancer cell lines than in normal prostate epithelial cells. Characterization of this novel variant demonstrated it to include exon 6b, previously thought unique to the muscle‐specific β‐Tm isoform, with an exon arrangement of 1–2–3–4–5–6a–6b–7–8–10. Inclusion of exon 6b introduces a premature stop codon directly following the 6a–6b exon boundary. Western blot analysis demonstrated the presence of a truncated protein in prostate cancer cell lines that was absent in normal prostate epithelial cells. It is hypothesized that this truncated protein will result in suppression of Tm1 polymer formation required for actin filament association. The lack of Tm polymer–actin association will result in loss of the stable actin microfilament organization and stress fiber formation, a state associated with cell transformation. Mol. Carcinog.