The role of osteoprotegerin and receptor activator of nuclear factor ᴋb in osteotropic prostate and breast cancers

Osteoprotegerin (OPG), Receptor Activator of Nuclear Factor ᴋB (RANK) and RANK ligand (RANKL), are members of the tumour necrosis factor receptor superfamily (TNFRSF), signal transducers which have pleiotropic actions. Each family member has unique structural attributes shown to couple them directly to specific signalling pathways involved in cell proliferation, differentiation and survival. Previous studies have clinically correlated OPG, RANK and RANKL expression, at both transcript and protein levels, with increasing cancer tumour burden, metastatic bone involvement and androgen status, however the mechanisms by which these molecules exert their effects remain elusive. This study aimed to establish what influence targeting OPG, RANK and RANKL expression may have on osteotrophic prostate and breast cancer cells in vitro and to subsequently explore the effect(s) Hepatocyte Growth Factor (HGF) and Bone Matrix Extract (BME) might also exert on cancer cell behaviour following manipulation of these molecules. The current study utilised 2 prostate cancer cell lines with varying androgen status, metastatic potential and bone metastasis phenotypes. Initial screening showed that the more aggressive osteolytic PC-3 cells expressed OPG, whilst weakly metastatic mixed-osseous LNCaP cells had very low expression. Whilst RANK was present in both cell lines, RANKL expression was only detected in the LNCaP cells. Reduction of OPG expression in the PC-3 cells resulted in increased cell invasion in vitro, which was further enhanced when treated with BME. No other cellular traits were affected by targeting OPG directly, however, cell migration was enhanced when the manipulated cells were exposed to the representative bone microenvironment. In contrast the addition of a recombinant form of OPG to LNCaP cells resulted in decreased cell invasion, a trend which was reversed when combined with BME. Combination of OPG and BME treatment reduced the migratory response of LNCaP cells, whilst combination of OPG and HGF were pro-migratory. The targeting of RANK in PC-3 cells affected cell proliferation and matrix adhesion in vitro though the addition of HGF or BME appeared to have no further direct influence on these manipulated cells. Targeting of the RANKL expression with a neutralising monoclonal antibody had little effect on cancer cell behaviour; however combined exposure with HGF or BME resulted in similar behaviour patterns seen under the OPG treatments. In our breast cancer cohort, RANK and RANKL expression were correlated with bone metastases and survival rates. Though OPG did not appear to be associated with grading, data also implied a role in overall survival. In the aggressive osteolytic MDA-MB-231 breast cancer cells, reduced OPG expression resulted in increased motility and invasion, traits which were little affected upon exposure to HGF or BME. In contrast the targeting of RANK expression in MDA-MB-231 cells resulted in reductions in all the cancer cell behaviours studied, but again these appeared unaffected under the influence of HGF or BME. The complexity of the bone environment underpins the vast number of soluble factors, signalling pathways and transcription regulators which can influence osteotrophic cancer cells. As indicated by the licensing of Denosumab, one therapeutic approach is not suitable for all osteotrophic cancers. Therefore further elucidation into the intricacies of these interactions is needed

Prostate transglutaminase (TGase-4) induces epithelial-to-mesenchymal transition in prostate cancer cells

More men die with prostate cancer (PCa) than from it. However, once PCa is no longer organ-confined, it is associated with significant mortality. Epithelial-to-mesenchymal transition (EMT) is one mechanism facilitating progression in cancer. Our studies of transglutaminase-4 TGase-4, a member of the TGase family, expressed in the prostate gland, have implicated it in the regulation of the invasive properties of PCa. The present study investigated the role of TGase-4 on EMT of PCa cells. Materials and Methods: A panel of PCa cell lines: CA-HPV-10, PZ-HPV-7, PC-3 and DU-145 were used. An anti- TGase-4 transgene was constructed to eliminate the expression of TGase-4 in CA-HPV-10 (positive for TGase-4). An expression construct for human TGase-4 was used to transfect PCa cells negative for TGase-4. The pattern of E-cadherin, N-cadherin and vimentin in these cells were evaluated using immunofluorescent staining. Cell motility was assessed using scratch wounding and ekectric cell-substrate impedance sensing (ECIS) assays. Results: Treatment of PZ-HPV-7 and CA-HPV- 10 cells with rhTGase-4 resulted in a significant increase in cell migration (1,407.9 Ω±6.4 Ω vs. 1,691.2 Ω±8.3 Ω in non-treated and rhTGase-4 treated cells, respectively, p<0.01). Cells strongly expressing E-cadherin showed substantial changes of E-cadherin staining in that, after treatment with TGase-4, the intercellular staining of E-cadherin was diminished. Concomitantly, there was acquisition of N-cadherin in TGase- 4-treated cells. Elimination of TGase-4 from CA-HPV-10 cells significantly decreased cell motility (128.1 Ω±107.4 Ω vs. 31.7 Ω±26.2 Ω, in CA-HPV-10 control and CA-HPV-10/TGase-4 knockout cells). Knocking- out TGase-4 from CA-HPV-10 cells also resulted in substantial loss of N-cadherin in the cells. Conclusion: TGase-4 resulted in loss of E-cadherin/acquisition of N-cadherin and cell migration indicating it is a keen regulator of EMT in prostate epithelia-derived cancer cells. In concert with its other properties involved in disease progression, the present observations suggest TGase-4 as a prospective marker of disease progression

Clinical and therapeutic implications of follistatin in solid tumours

Follistatin (FST), as a single-chain glycosylated protein, has two major isoforms, FST288 and FST315. The FST315 isoform is the predominant form whilst the FST288 variant accounts for less than 5% of the encoded mRNA. FST is differentially expressed in human tissues and aberrant expression has been observed in a variety of solid tumours, including gonadal, gastric and lung cancer, hepatocellular carcinoma, basal cell carcinoma and melanoma. Based on the current evidence, FST is an antagonist of transforming growth factor beta family members, such as activin and bone morphogenetic proteins (BMPs). FST plays a role in tumourigenesis, metastasis and angiogenesis of solid tumours through its interaction with activin and BMPs, thus resulting in pathophysiological function. In terms of diagnosis, prognosis and therapy FST has shown strong promise. Through a better understanding of its biological functions, potential clinical applications may yet emerge

Importance of osteoprotegrin and receptor activator of nuclear factor kB in breast cancer response to hepatocyte growth factor and the bone microenvironment in vitro

Osteoprotegrin (OPG), receptor activator of nuclear factor κB (RANK) and RANK ligand (RANKL) are signal transducers which have pleiotropic actions. Each tumour necrosis factor receptor superfamily member has unique structural attributes which directly couples them to signalling pathways involved in cell proliferation, differentiation and survival. Previous studies have clinically linked OPG, RANK and RANKL to increasing tumour burden, metastatic bone involvement and estrogen status. This study aimed to establish the potential implications of targeting endogenously produced OPG and RANK in the osteotropic breast cancer cell line MDA-MB‑231 in vitro. Subsequently this study also aimed to explore the potential links between these molecules with regards to hepatocyte growth factor (HGF) signalling and extracted bone proteins (BME). OPG and RANK expression was successfully suppressed using hammerhead ribozyme technology. Subsequently effects were explored in MDA-MB‑231 cell proliferation, matrix adhesion, migration and invasion in vitro function assays. Reduced OPG expression resulted in increased breast cancer cell migration and invasion. These increases, particularly invasion, appeared to however be reduced under the influence of the exogenous stimuli (HGF and BME). In contrast, suppression of RANK in MDA-MB‑231 breast cancer cells resulted in decreased cancer cell proliferation, matrix-adhesion, motility and invasion with little cumulative effect being noted after the addition of exogenous stimuli. The complexity of the bone environment underpins the vast number of soluble factors and signalling pathways which can influence osteotropic cancer behaviour and progression. Further work into elucidating all the pathways affected could potentially lead to better identification of those patients most at risk

Effect of YangZheng XiaoJi Extract, DME-25, on endothelial cells and their response to Avastin.

ackground: Angiogenesis is a cellular process that has been identified as a key target for therapy in solid cancer. However, over the course of anti-angiogenic therapies, cancer cells acquire resistance to these therapies after an initial period of success. DME-25 is an extract from Yang Zheng Xiao Ji, a traditional Chinese medicine that has been reported to benefit patients with cancer by alleviating chemotherapy-associated symptoms and possibly inhibiting key cancer cell traits. This study aimed to explore if DME-25 on its own and in combination with avastin affected endothelial cell behaviour in vitro in the presence of hypoxic lung cancer-conditioned medium (CM). Materials and Methods: Two lung cancer cell lines, A549 and SK-MES-1, were exposed to hypoxic conditions (O2 ≤1%) for 4 h, after which CM, and RNA were collected. Transcript expression of several influential angiogenic markers in lung cancer cells were assessed following hypoxic/normoxic conditions. Lung cancer CM was added in combination with avastin and DME-25, before or after vascular endothelial growth factor (VEGF) depletion, to endothelial cells (HECV) and cell migration and microtubule formation were assessed in vitro. Results: HECV cell migration was reduced in the presence of avastin, although less efficiently in the presence of lung cancer CM. A combination of DME-25 and avastin with lung cancer CM significantly reduced HECV cell migration irrespective of culture under hypoxia or normoxia. Depletion of VEGF from the CM reduced the inhibitory capacity of avastin, however, it appeared to have little impact on the anti-angiogenic effects of DME-25. Conclusion: DME-25 inhibits tubule formation irrespectively of the factors secreted by normoxic or hypoxic lung cancer cell CM depleted of VEGF

Key factors in breast cancer dissemination and establishment at the bone: past, present and future perspectives

Bone metastases associated with breast cancer remain a clinical challenge due to their associated morbidity, limited therapeutic intervention and lack of prognostic markers. With a continually evolving understanding of bone biology and its dynamic microenvironment, many potential new targets have been proposed. In this chapter, we discuss the roles of well-established bone markers and how their targeting, in addition to tumour-targeted therapies, might help in the prevention and treatment of bone metastases. There are a vast number of bone markers, of which one of the best-known families is the bone morphogenetic proteins (BMPs). This chapter focuses on their role in breast cancer-associated bone metastases, associated signalling pathways and the possibilities for potential therapeutic intervention. In addition, this chapter provides an update on the role receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL) and osteoprotegerin (OPG) play on breast cancer development and their subsequent influence during the homing and establishment of breast cancer-associated bone metastases. Beyond the well-established bone molecules, this chapter also explores the role of other potential factors such as activated leukocyte cell adhesion molecule (ALCAM) and its potential impact on breast cancer cells’ affinity for the bone environment, which implies that ALCAM could be a promising therapeutic target

Expression of osteoprotegrin is enhanced in lung cancer tissues and promotes aggressive cellular traits in H3122 lung cancer cells

Background: Osteoprotegrin (OPG), a secreted protein and a member of the tumor necrosis factor receptor superfamily has been well-characterized and is an important regulator of bone remodeling by blocking osteoclast maturation thus preventing osteolysis. In recent years, OPG has been reported to have an association with the malignant capacity of various cancer types and cancer-associated bone metastasis, although the mechanisms of this are not clearly understood. Materials and Methods: In this study, OPG expression was analyzed in human lung cancer tissue and normal tissue based on the dataset of The Cancer Genome Atlas and Oncomine. The in vitro effect of OPG on H3122 lung cancer cells was also assessed by characterizing cell function following knock-down and forced overexpression in this cell line. Results: The expression of OPG was significantly increased in lung cancer tissues compared to the normal control group and OPG promoted the malignant phenotypes of H3122 cells in in vitro models. Conclusion: OPG may be a potential driver of lung cancer cells and therefore might have potential in therapy and diagnostics

Clinical and therapeutic implications of follistatin in solid tumours

Follistatin (FST), as a single-chain glycosylated protein, has two major isoforms, FST288 and FST315. The FST315 isoform is the predominant form whilst the FST288 variant accounts for less than 5% of the encoded mRNA. FST is differentially expressed in human tissues and aberrant expression has been observed in a variety of solid tumours, including gonadal, gastric and lung cancer, hepatocellular carcinoma, basal cell carcinoma and melanoma. Based on the current evidence, FST is an antagonist of transforming growth factor beta family members, such as activin and bone morphogenetic proteins (BMPs). FST plays a role in tumourigenesis, metastasis and angiogenesis of solid tumours through its interaction with activin and BMPs, thus resulting in pathophysiological function. In terms of diagnosis, prognosis and therapy FST has shown strong promise. Through a better understanding of its biological functions, potential clinical applications may yet emerge