Circulating Tumour Cells (CTC), Head and Neck Cancer and Radiotherapy; Future Perspectives.

Head and neck cancer is the seventh most common cancer in Australia and globally. Despite the current improved treatment modalities, there is still up to 50⁻60% local regional recurrence and or distant metastasis. High-resolution medical imaging technologies such as PET/CT and MRI do not currently detect the early spread of tumour cells, thus limiting the potential for effective minimal residual detection and early diagnosis. Circulating tumour cells (CTCs) are a rare subset of cells that escape from the primary tumour and enter into the bloodstream to form metastatic deposits or even re-establish themselves in the primary site of the cancer. These cells are more aggressive and accumulate gene alterations by somatic mutations that are the same or even greater than the primary tumour because of additional features acquired in the circulation. The potential application of CTC in clinical use is to acquire a liquid biopsy, by taking a reliable minimally invasive venous blood sample, for cell genotyping during radiotherapy treatment to monitor the decline in CTC detectability, and mutational changes in response to radiation resistance and radiation sensitivity. Currently, very little has been published on radiation therapy, CTC, and circulating cancer stem cells (CCSCs). The prognostic value of CTC in cancer management and personalised medicine for head and neck cancer radiotherapy patients requires a deeper understanding at the cellular level, along with other advanced technologies. With this goal, this review summarises the current research of head and neck cancer CTC, CCSC and the molecular targets for personalised radiotherapy response.

Delivery of expression constructs of secreted frizzled-related protein 4 and its domains by chitosan-dextran sulfate nanoparticles enhances their expression and anti-cancer effects.

In malignant mesothelioma (MM) cells, secreted frizzled-related protein 4 (SFRP4) expression is downregulated by promoter methylation. In this study, we evaluated the effect of encapsulated chitosan-dextran (CS-DS) nanoparticle formulations of SFRP4 and its cysteine-rich domain (CRD) and netrin-like domain (NLD) as means of SFRP4-GFP protein delivery and their effects in JU77 and ONE58 MM cell lines. CS-DS formulations of SFRP4, CRD, and NLD nanoparticles were prepared by a complex coacervation technique, and particle size ranged from 300 nm for empty particles to 337 nm for particles containing the proteins. Measurement of the zeta potential showed that all preparations were around 25 mV or above, suggesting stable formulation and good affinity for the DNA molecules. The CS-DS nanoparticle formulation maintained high integrity and entrapment efficiency. Gene delivery of SFRP4 and its domains showed enhanced biological effects in both JU77 and ONE58 cell lines when compared to the non-liposomal FUGENE® HD transfection reagent. In comparison to the CRD nanoparticles, both the SFRP4 and NLD nanoparticles significantly reduced the viability of MM cells, with the NLD showing the greatest effect. The CS-DS nanoparticle effects were observed at an earlier time point and with lower DNA concentrations. Morphological changes in MM cells were characterized by the formation of membrane-associated vesicles and green fluorescent protein expression specific to SFRP4 and the NLD. The findings from our proof-of-concept study provide a stepping stone for further investigations using in vivo models.

Insulin antagonises pigment epithelium-derived factor (PEDF)-induced modulation of lineage commitment of myocytes and heterotrophic ossification.

Extensive bone defects arising as a result of trauma, infection and tumour resection and other bone pathologies necessitates the identification of effective strategies in the form of tissue engineering, gene therapy and osteoinductive agents to enhance the bone repair process. PEDF is a multifunctional glycoprotein which plays an important role in regulating osteoblastic differentiation and bone formation. PEDF treatment of mice and human skeletal myocytes at physiological concentration inhibited myogenic differentiation and activated Erk1/2 MAPK- dependent osteogenic transdifferentiation of myocytes. In mice, insulin, a promoter of bone regeneration, attenuated PEDF-induced expression of osteogenic markers such as osteocalcin, alkaline phosphatase and mineralisation for bone formation in the muscle and surrounding adipose tissue. These results provide new insights into the molecular aspects of the antagonising effect of insulin on PEDF-dependent modulation of the differentiation commitment of musculoskeletal environment into osteogenesis, and suggest that PEDF may be developed as an effective clinical therapy for bone regeneration as its heterotopic ossification can be controlled via co-administration of insulin.

The Wnt regulator SFRP4 inhibits mesothelioma cell proliferation, migration, and antagonizes Wnt3a via its netrin-like domain.

Secreted frizzled related proteins (SFRPs) are a family of Wnt regulators which are frequently downregulated in cancers. In malignant mesothelioma (MM), downregulation of SFRP4 has been reported as a mechanism which contributes to aberrant activation of oncogenic Wnt signaling. Here we investigated the biological consequences of SFRP4 in two mesothelioma cell models where this protein is downregulated. We used recombinant SFRP4 and transient overexpression to study changes in proliferation, migration and downstream signaling. We found that recombinant SFRP4 inhibited both proliferation and migration of MM cells as well as abrogating the stimulatory effect of recombinant Wnt3a. Morphologically SFRP4 induced a cytotoxic effect distinct from apoptosis and consistent with mitotic catastrophe. Overexpression of SFRP4 in these cell lines displayed similar effects as endogenous protein on cell viability, migration and nuclear morphology. We also used expression constructs to examine the role of the SFRP4 cysteine rich domain (CRD) and a netrin-like domain (NLD) in these effects. Interestingly, we found it was the NLD which mediated the biological effects of SFRP4 in these cells. Our results indicate that SFRP4 inhibits mesothelioma proliferation, migration and activates alternative cell death pathways. The finding that the NLD is responsible for these has broader implications for this protein family. Overall this study suggests that the Wnt pathway may prove a promising target for therapy in mesothelioma.

sFRP4 signalling of apoptosis and angiostasis uses nitric oxide-cGMP-permeability axis of endothelium.

Nitric oxide (NO) plays a critical role in endothelial functions such as cellular migration, vascular permeability and angiogenesis. Angiogenesis, the formation of new blood vessels from "pre-existing" ones is a carefully regulated process and essential during reproduction, development and wound healing. Previously our lab group reported that Secreted Frizzled-Related Protein 4 (sFRP4) could inhibit angiogenesis in both in vitro and in vivo conditions. sFRP4 belongs to a family of secreted glycoproteins that function as antagonists of the canonical Wnt signalling pathway. Although the pro-apoptotic role of sFRP4 is well discussed in literature, little is known in regards to its anti-angiogenic property. The objective of this study was to elucidate sFRP4 implications in NO biology of the endothelium. Results demonstrate that sFRP4 causes endothelial dysfunction by suppressing NO-cGMP signaling and elevating corresponding ROS levels. The imbalance between NO and ROS levels results in apoptosis and subsequent leakiness of endothelium as confirmed in vivo (Texas red/Annxin - CAM assay) and in vitro (Monolayer permeability assay) conditions. Furthermore utilizing peptides synthesized from the CRD domain of sFRP4, our results showed that while these peptides were able to cause endothelial dysfunctions, they did not cause apoptosis of the endothelial cells. Thereby confirming that sFRP4 can mediate its anti-angiogenic effect independent of its pro-apoptotic property. In conclusion, the current study reports that sFRP4-mediated anti-angiogenesis occurs as a result of impaired NO-cGMP signaling which in turn allow for elevation of redox levels and promotion of apoptosis of endothelial cells.

microRNAs in breast cancer: regulatory roles governing the hallmarks of cancer.

A large number of etiological factors and the complexity of breast cancers present challenges for prevention and treatment. Recently, the emergence of microRNAs (miRNAs) as cancer biomarkers has added an extra dimension to the 'molecular signatures' of breast cancer. Bioinformatic analyses indicate that each miRNA can regulate hundreds of target genes and could serve functionally as 'oncogenes' or 'tumour suppressor' genes, and co-ordinate multiple cellular processes relevant to cancer progression. A number of studies have shown that miRNAs play important roles in breast tumorigenesis, metastasis, proliferation and differentiation of breast cancer cells. This review provides a comprehensive overview of miRNAs with established functional relevance in breast cancer, their established target genes and resulting cellular phenotype. The role and application of circulating miRNAs in breast cancer is also discussed. Furthermore, we summarize the role of miRNAs in the hallmarks of breast cancer, as well as the possibility of using miRNAs as potential biomarkers for detection of breast cancer.

Number and brightness analysis of sFRP4 domains in live cells demonstrates vesicle association signal of the NLD domain and dynamic intracellular responses to Wnt3a.

The Wnts are secreted, lipidated glycoproteins that play a role in cellular processes of differentiation, proliferation, migration, survival, polarity and stem cell self-renewal. The majority of Wnts biological effects are through binding to specific frizzled (Fzd) receptor complexes leading to activation of downstream pathways. Secreted frizzled-related proteins (sFRPs) were first identified as antagonists of Wnt signalling by binding directly to Wnts. They comprise two domains, a Fzd-like cysteine rich domain (CRD) and a netrin-like domain (NLD). Subsequently sFRPs have been shown to also interact with Fzd receptors and more diverse functions have been identified, including potentiation of Wnt signalling. Many aspects of the biology of this family remain to be elucidated. We used the number and brightness (N&B) method, a technique based on fluorescence fluctuation analysis, to characterise the intracellular aggregation and trafficking of sFRP4 domains. We expressed sFRP4 and its' domains as EGFP fusions and then characterised the effect of endogenous Wnt3a by fluorescence confocal imaging. We observed vesicular trafficking of sFRP4 and that the NLD domain has a vesicular association signal. We found that sFRP4 and the CRD formed oligomeric aggregates in the perinuclear region while the NLD was distributed evenly throughout the cell with a larger proportion of aggregates. Most significantly we observed intracellular redistribution of sFRP4 in response to Wnt3a suggesting that Wnt3a can modulate intracellular localisation and secretion of sFRP4. Our results reveal a number of novel findings regarding sFRP4 which are likely to have relevance to this wider family.

Secreted frizzled related proteins: Implications in cancers.

The Wnt (wingless-type) signaling pathway plays an important role in embryonic development, tissue homeostasis, and tumor progression becaluse of its effect on cell proliferation, migration, and differentiation. Secreted frizzled-related proteins (SFRPs) are extracellular inhibitors of Wnt signaling that act by binding directly to Wnt ligands or to Frizzled receptors. In recent years, aberrant expression of SFRPs has been reported to be associated with numerous cancers. As gene expression of SFRP members is often lost through promoter hypermethylation, inhibition of methylation through the use of epigenetic modifying agents could renew the expression of SFRP members and further antagonize deleterious Wnt signaling. Several reports have described epigenetic silencing of these Wnt signaling antagonists in various human cancers, suggesting their possible role as tumor suppressors. SFRP family members thus come across as potential tools in combating Wnt-driven tumorigenesis. However, little is known about SFRP family members and their role in different cancers. This review comprehensively covers all the available information on the role of SFRP molecules in various human cancers.

Expression profile and function of Wnt signaling mechanisms in malignant mesothelioma cells.

Malignant mesothelioma (MM) is an uncommon and particularly aggressive cancer associated with asbestos exposure, which currently presents an intractable clinical challenge. Wnt signaling has been reported to play a role in the neoplastic properties of mesothelioma cells but has not been investigated in detail in this cancer. We surveyed expression of Wnts, their receptors, and other key molecules in this pathway in well established in vitro mesothelioma models in comparison with primary mesothelial cultures. We also tested the biological response of MM cell lines to exogenous Wnt and secreted regulators, as well as targeting ß-catenin. We detected frequent expression of Wnt3 and Wnt5a, as well as Fzd 2, 4 and 6. The mRNA of Wnt4, Fzd3, sFRP4, APC and axin2 were downregulated in MM relative to mesothelial cells while LEF1 was overexpressed in MM. Functionally, we observed that Wnt3a stimulated MM proliferation while sFRP4 was inhibitory. Furthermore, directly targeting ß-catenin expression could sensitise MM cells to cytotoxic drugs. These results provide evidence for altered expression of a number of Wnt/Fzd signaling molecules in MM. Modulation of Wnt signaling in MM may prove a means of targeting proliferation and drug resistance in this cancer.

Mechanisms of cisplatin-induced cell death in malignant mesothelioma cells: role of inhibitor of apoptosis proteins (IAPs) and caspases.

Malignant mesothelioma (MM) is an aggressive and highly chemoresistant tumour. Although cisplatin is used in frontline therapy of this disease treatment remains palliative at best. The biochemical pathways activated by cisplatin and the mechanisms of resistance in mesothelioma cells are poorly understood. Overexpression of inhibitor of apoptosis proteins (IAPs) has been described in clinical mesothelioma tumours and proposed as therapeutic targets. In this study, we examined cisplatin-induced cell death pathways and IAPs in three mesothelioma-derived cell lines. Cisplatin induced cell death in mesothelioma cell lines was characterised by biochemical mechanisms classically associated with apoptosis including: mitochondrial depolarisation, phosphatidylserine translocation and caspase activation. Surprisingly mRNA expression of IAPs in mesothelioma was not upregulated relative to primary mesothelial cells except for survivin which was higher in the most resistant cell line. In contrast, protein expression of both XIAP and survivin was upregulated in all mesothelioma cells, consistent with post-translational regulation. Knockdown of either XIAP or survivin by RNAi did not affect the sensitivity to cisplatin in any of the cell lines. Survivin RNAi did, however, inhibit proliferation in the highest expressing cell line, ONE58. The pan-caspase inhibitor z-VAD and the more selective caspase 3/7 inhibitor z-DEVD had no effect upon the sensitivity of any of the cell lines to cisplatin indicating that caspase-independent pathways predominate. The findings of the present study provide insights into cisplatin-induced mechanisms in mesothelioma cells and show that alternative pathways are operating which may provide new options for targeting this extremely resistant tumour.

Function of caspase-14 in trophoblast differentiation.

BACKGROUND: Within the human placenta, the cytotrophoblast consists of a proliferative pool of progenitor cells which differentiate to replenish the overlying continuous, multi-nucleated syncytiotrophoblast, which forms the barrier between the maternal and fetal tissues. Disruption to trophoblast differentiation and function may result in impaired fetal development and preeclampsia. Caspase-14 expression is limited to barrier forming tissues. It promotes keratinocyte differentiation by cleaving profilaggrin to stabilise keratin intermediate filaments, and indirectly providing hydration and UV protection. However its role in the trophoblast remains unexplored. METHODS: Using RNA Interference the reaction of control and differentiating trophoblastic BeWo cells to suppressed caspase-14 was examined for genes pertaining to hormonal, cell cycle and cytoskeletal pathways. RESULTS: Transcription of hCG, KLF4 and cytokeratin-18 were increased following caspase-14 suppression suggesting a role for caspase-14 in inhibiting their pathways. Furthermore, hCG, KLF4 and cytokeratin-18 protein levels were disrupted. CONCLUSION: Since expression of these molecules is normally increased with trophoblast differentiation, our results imply that caspase-14 inhibits trophoblast differentiation. This is the first functional study of this unusual member of the caspase family in the trophoblast, where it has a different function than in the epidermis. This knowledge of the molecular underpinnings of trophoblast differentiation may instruct future therapies of trophoblast disease.

Proteome of synaptosome-associated proteins in spinal cord dorsal horn after peripheral nerve injury.

Peripheral nerve injury may lead to neuroadaptive changes of cellular signals in spinal cord that are thought to contribute to central mechanisms underlying neuropathic pain. Here we used a 2-DE-based proteomic technique to determine the global expression changes of synaptosome-associated proteins in spinal cord dorsal horn after unilateral fifth spinal nerve injury (SNI). The fifth lumbar dorsal horns ipsilateral to SNI or sham surgery were harvested on day 14 post-surgery, and the total soluble and synaptosomal fractions were isolated. The proteins derived from the synaptosomal fraction were resolved by 2-DE. We identified 27 proteins that displayed different expression levels after SNI, including proteins involved in transmission and modulation of noxious information, cellular metabolism, membrane receptor trafficking, oxidative stress, apoptosis, and degeneration. Six of the 27 proteins were chosen randomly and further validated in the synaptosomal fraction by Western blot analysis. Unexpectedly, Western blot analysis showed that only one protein in the total soluble fraction exhibited a significant expression change after SNI. The data indicate that peripheral nerve injury changes not only protein expression but also protein subcellular distribution in dorsal horn cells. These changes might participate in the central mechanism that underlies the maintenance of neuropathic pain.

Differential modulation of cell cycle, apoptosis and PPARgamma2 gene expression by PPARgamma agonists ciglitazone and 9-hydroxyoctadecadienoic acid in monocytic cells.

We sought to compare the effects of the thiazolidinedione ciglitazone with the endogenous fatty acid PPARgamma agonists 9- and 13-hydroxyoctadecadienoic acid (9- and 13-HODE), in U937 monocytic cells. Ciglitazone and 9-HODE inhibited cell proliferation and all three agonists increased cellular content of C18:0 fatty acids. Ciglitazone and 13-HODE resulted in an increased percentage of cells in S phase and ciglitazone reduced the percentage of cells in G2/M phase of cell cycle, whilst 9-HODE increased the percentage of cells in G0/1 and reduced the fraction in S and G2/M phases. 9-HODE selectively induced apoptosis in U937 cells, and increased PPARgamma2 gene expression. Induction of apoptosis by 9-HODE was not abrogated by the presence of the PPARgamma antagonist GW9662. Synthetic (TZD) and endogenous fatty acid ligands for PPARgamma, ciglitazone and 9- and 13-HODE, possess differential, ligand specific actions in monocytic cells to regulate cell cycle progression, apoptosis and PPARgamma2 gene expression.

Placental expression of secreted frizzled related protein-4 in the rat and the impact of glucocorticoid-induced fetal and placental growth restriction.

Wnt genes regulate a diverse range of developmental processes, including placental formation. Activation of the WNT pathway results in translocation of beta-catenin (CTNNB1) into the nucleus and the subsequent activation of transcription factors that promote proliferation. The secreted frizzled related proteins (SFRPs) are thought to inhibit WNT signaling by binding to the WNT ligand or its frizzled receptor. In this study, we compared the expression patterns of one of these secreted molecules, SFRP4, in the two morphologically and functionally distinct regions of the rat placenta during the last third of pregnancy. In addition, we assessed whether placental SFRP4 expression is altered in a model of glucocorticoid-induced placental growth restriction. Temporal analyses of the rat placenta by quantitative RT-PCR, in situ hybridization, and immunohistochemistry during the final third of pregnancy demonstrated elevated levels of Sfrp4 mRNA and SFRP4 protein near term, specifically in trophoblast cells of the basal zone. This increase in expression of SFRP4 in basal zone trophoblasts was associated with a reduction in CTNNB1 nuclear translocation, consistent with inhibition of the WNT pathway. Maternal dexamethasone treatment (1 microg/ml of drinking water, Days 13-22), which has previously been shown to reduce placental growth, further increased the expression of Sfrp4 mRNA in both the basal and labyrinth zones of the placenta at Day 22. Collectively, these data demonstrate that increased expression of SFRP4 is associated with reduced growth of placental regions in normal pregnancy and after glucocorticoid-induced growth retardation. These observations, together with associated changes in CTNNB1 localization, support the hypothesis that increased placental expression of SFRP4 inhibits the WNT pathway and thereby influences placental growth via effects on cell fate signaling.

Cisplatin and TNF-alpha downregulate transcription of Bcl-xL in murine malignant mesothelioma cells.

Malignant mesothelioma (MM) is an aggressive and highly chemo-resistant tumour. In this study, we examined cisplatin-induced apoptosis in mouse models of this disease and investigated the role of constitutive and inducible expression of apoptosis related genes in this process. All of the four mouse MM cell lines examined expressed Bax, Bcl-xL, c-Myc, and caspase-3 but not Bcl-2. Cisplatin-induced apoptosis characterised by DNA fragmentation and cell death while caspase-3/7 was activated in 3 of 4 cell lines. Quantitation of basal gene expression showed significant differences but there was no correlation between single genes and cisplatin sensitivity. In the AC29 and AB1 models, both cisplatin and TNF-alpha downregulated Bcl-xL gene expression, indicating that this gene was a common transcriptional target in these cells. The findings of the present study provide insights into apoptotic mechanisms in mesothelioma cells and show similar patterns of gene expression to that reported in the human disease.

Gonadotropin-releasing hormone-agonist inhibits synthesis of nitric oxide and steroidogenesis by luteal cells in the pregnant rat.

We have demonstrated that continuous administration of a gonadotropin-releasing hormone agonist (GnRH-Ag) in vivo suppressed progesterone production and induced apoptosis in the corpus luteum (CL) of the pregnant rat. To investigate the mechanism(s) by which progesterone secretion is suppressed and apoptosis is induced in the luteal cells, we studied nitric oxide (NO) as a messenger molecule for GnRH action. Rats were treated individually on Day 8 of pregnancy with 5 microg/day of GnRH-Ag for 4, 8, and 24 h. GnRH-Ag decreased the production of progesterone and pregnenolone 8 and 24 h after the administration. Corresponding with the reduction in these steroid hormones, luteal NO concentrations decreased at 8 and 24 h. Western blotting and immunohistochemical studies of endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and neuronal nitric oxide synthase (nNOS) in the CL demonstrated that administration of GnRH-Ag was associated with a marked decrease in eNOS and iNOS compared with sham controls at 4 and 8 h, but nNOS did not change throughout the experimental period. We demonstrated, for the first time, the presence of nNOS protein in the CL of the pregnant rat. To determine if this suppressive action of GnRH-Ag is directly on the CL, luteal cells were treated with GnRH-Ag for 4, 8, 12, and 24 h in vitro. Progesterone and NO concentrations in the media decreased at 8 and 12 h after the treatment and recovered at 24 h. Western blots revealed that eNOS and iNOS decreased in luteal cells treated with GnRH-Ag compared with controls at 4 and 8 h. These results demonstrate that suppression of luteal NO synthesis by GnRH-Ag is direct and leads to a decrease in the luteal production and release of progesterone and pregnenolone and thus suggest that GnRH could induce luteolysis in pregnant rats via NO.

Glucocorticoids and progesterone prevent apoptosis in the lactating rat mammary gland.

Apoptosis, a form of noninflammatory cell death, plays a central role in mammary gland involution after weaning. Previous studies have shown that apoptosis in the postweaning mammary gland is substantially reduced by treatment with glucocorticoids or progesterone, but whether these steroids exert a similar antiapoptotic effect during normal lactation is not known. Therefore, the present study used an in vivo rat model to assess the effects of progesterone and glucocorticoids on apoptosis in the lactating mammary gland. Rats were untreated, sham operated, ovariectomized (OVX), and/or adrenalectomized (ADX) on d 10 of lactation. Additional groups of OVX/ADX rats were treated with either progesterone or corticosterone. Mammary gland apoptosis was determined 3 d later by 3'-end labeling of fragmented DNA and by in situ terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate biotin nick end labeling analysis (TUNEL). DNA fragmentation was relatively low in the mammary gland from untreated and sham-operated rats and was unaffected by either ADX or OVX alone. In contrast, DNA fragmentation was markedly elevated in OVX/ADX rats (P < 0.01), but this effect on mammary gland apoptosis was prevented by replacement with either corticosterone or progesterone. Consistent with these data, dying cells identified by TUNEL analysis were readily observed in the alveolar epithelium of mammary tissue from OVX/ADX rats but not in any of the other groups. These data demonstrate that during normal lactation, mammary gland apoptosis is inhibited by endogenous progesterone and glucocorticoids. Importantly, the presence of either steroid alone was sufficient to prevent apoptosis, suggesting that their antiapoptotic effects in the lactating mammary gland may be mediated via similar signaling pathways.