Background and Objective: Reduced expression of syndecan-1 (CD138), increased proliferation index, and modifications in the expression of the molecular RANK/RANKL/OPG triad are related to an intensified potential of aggressiveness and invasion of diverse tumors and cysts. The aim was to compare the expression of Ki-67, CD138, and the molecular triad RANK, RANKL, and OPG in odontogenic keratocysts (OKC), unicystic ameloblastomas (UA), and dentigerous cysts (DC).
Methods: Immunohistochemistry for Ki-67, CD138, RANK, RANKL, and OPG was performed in 58 odontogenic cystic lesions (22 OKC, 17 DC, and 19 UA).
Results: A higher expression of Ki-67 was identified in OKC as compared to UA (
Conclusion: Higher RANKL expression together with the reduction on CD138 expression in UA could be linked to a greater invasive and destructive potential, while the increased proliferation rate observed in OKC could be related to its continuous intrabony growth. The expansion of DC does not seem to be related to such factors, justifying the different therapeutic approaches proposed for each of these entities.

BACKGROUND: Several hypotheses have been proposed regarding the mechanisms underlying meningioma-related hyperostosis. In this study, we investigated the role of osteoprotegerin (OPG), insulin-like growth factor 1 (IGF-1), endothelin 1 (ET-1), and bone morphogenetic protein (BMP) 2 and 4.
METHODS: A total of 149 patients (39 males and 110 females; mean age, 62 years) who underwent surgery were included. Depending on the relationship with the bone, meningiomas were classified as hyperostotic, osteolytic, infiltrative, or unrelated. Expression of OPG, and IGF-1, ET-1, BMP-2, and BMP-4 was evaluated by tissue microarray analysis of surgical samples.
RESULTS: Our series comprised 132 cases of grade I, 14 cases of grade II, and 3 cases of grade III meningiomas, according to the World Health Organization classification. Based on preoperative computed tomography scan, the cases were classified as follows: hyperostotic, n = 11; osteolytic, n = 11; infiltrative, n = 15; unrelated to the bone, n = 108. Four cases were excluded from the statistical analysis. Using receiver operating characteristic curve analysis, we identified a 2% cutoff for the mean value of IGF-1 that discriminated between osteolytic and osteoblastic lesions; cases with a mean IGF-1 expression of <2% were classified as osteolytic (P = 0.0046), whereas those with a mean OPG expression of <10% were classified as osteolytic (P = 0.048). No other significant relationships were found.
CONCLUSIONS: Expression of OPG and expression of IGF-1 were found to be associated with the development of hyperostosis. Preliminary findings suggest that hyperostosis can be caused by an overexpression of osteogenic molecules that influence osteoblast/osteoclast activity. Based on our results, further studies on hyperostotic bony tissue in meningiomas are needed to better understand how meningiomas influence bone overproduction.

BACKGROUND: The cytokine system RANKL (receptor activator of NF-κB ligand), its receptor RANK and the antagonist OPG (osteoprotegerin) play a critical role in bone turnover. Our investigation was conducted to describe the gene expression at primary tumour site in prostate cancer patients and correlate the results with Gleason Score and PSA level.
METHODS: Seventy-one samples were obtained from prostate cancer patients at the time of radical prostatectomy and palliative prostate resection (n = 71). Patients with benign prostate hyperplasia served as controls (n = 60). We performed real-time RT-PCR after microdissection of the samples.
RESULTS: The mRNA expression of RANK was highest in tumour tissue from patients with bone metastases (p < 0.001) as compared to BPH or locally confined tumours, also shown in clinical subgroups distinguished by Gleason Score (< 7 or ≥ 7, p = 0.028) or PSA level (< 10 or ≥ 10 µg/l, p = 0.004). RANKL and OPG mRNA expression was higher in tumour tissue from patients with metastatic compared to local disease. The RANKL/OPG ratio was low in normal prostate tissue and high tumours with bone metastases (p < 0.05). Expression of all three cytokines was high in BPH tissue but did not exceed as much as in the tumour tissue.
CONCLUSION: We demonstrated that RANK, RANKL and OPG are directly expressed by prostate cancer cells at the primary tumour site and showed a clear correlation with Gleason Score, serum PSA level and advanced disease. In BPH, mRNA expression is also detectable, but RANK expression does not exceed as much as compared to tumour tissue.

The canonical Wnt/β-catenin pathway is constitutively activated in more than 90% of colorectal cancer (CRC) cases in which β-catenin contributes to CRC cell growth and survival. In contrast to the Wnt/β-catenin pathway, the non-canonical Wnt pathway can antagonize functions of the canonical Wnt/β-catenin pathway. Wnt5a is a key factor in the non-canonical Wnt pathway, and it plays diverse roles in different types of cancers. It was shown that reintroducing Wnt5a into CRC cells resulted in inhibited cell proliferation and impaired cell motility. However, contradictory results were reported describing increased Wnt5a expression being associated with a poor prognosis of CRC patients. Recently, it was shown that the diverse roles of Wnt5a are due to two distinct roles of Wnt5a isoforms. However, the exact roles and functions of the Wnt5a isoforms in CRC remain largely unclear. The present study for the first time showed the ambiguous role of Wnt5a in CRC was due to the encoding of distinct roles of the various Wnt5a mRNA isoforms. A relatively high expression level of the Wnt5a-short (S) isoform transcript and a low expression level of the Wnt5a-long (L) isoform transcript were detected in CRC cell lines and specimens. In addition, high expression levels of the Wnt5a-S mRNA isoform and low expression levels of the Wnt5a-L mRNA isoform were significantly positively correlated with tumor depth of CRC patients. Furthermore, knockdown of the endogenous expression of the Wnt5a-S mRNA isoform in HCT116 cells drastically inhibited their growth ability by inducing apoptosis through induction of FASLG expression and reduction of TNFRSF11B expression. Moreover, reactivation of methylation inactivation of the Wnt5a-L mRNA isoform by treatment with 5-azacytidine (5-Aza) enhanced the siWnt5a-S isoform's ability to induce apoptosis. Finally, we showed that the simultaneous reactivation of Wnt5a-L mRNA isoform and knockdown of Wnt5a-S mRNA isoform expression enhanced siWnt5a-S isoform-induced apoptosis and siWnt5a-L isoform-regulated suppression of β-catenin expression in vitro. High expression levels of the Wnt5a-S mRNA isoform and low expression levels of the Wnt5a-L mRNA isoform were significantly positively correlated with high mRNA levels of β-catenin detection in vivo. Altogether, our study showed that, for the first time, different Wnt5a mRNA isoforms play distinct roles in CRC and can be used as novel prognostic markers for CRC in the future.

Receptor activator of nuclear factor-kappa B (RANK)-RANK ligand (RANKL) signaling promotes mammary tumor development in experimental models. Circulating concentrations of soluble RANKL (sRANKL) may influence breast cancer risk via activation of RANK signaling; this may be modulated by osteoprotegerin (OPG), the decoy receptor for RANKL. sRANKL and breast cancer risk by hormone receptor subtype has not previously been investigated. A case-control study was nested in the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. This study included 1,976 incident invasive breast cancer cases [estrogen receptor positive (ER+),

PURPOSE: The receptor activator of nuclear factor kappa B (RANK)/RANK ligand (RANKL)/osteoprotegerin (OPG) axis may contribute to the development of bone metastases (BM). We studied gene expression in this pathway in primary breast cancer (BC) to determine correlations with clinical characteristics and outcomes in the neoadjuvant I-SPY1 study.
METHODS: We evaluated RANK/RANKL/OPG expression using expression microarrays in I-SPY1 (n = 149). Associations with clinical features were determined using t test and ANOVA. Associations between biomarker high versus low groups (dichotomized at an optimal cutpoint) and recurrence-free survival (RFS) were evaluated using the log-rank test and in a multivariate Cox proportional hazard model. A pooled external neoadjuvant cohort with gene expression data (GSE25066) (Hatzis et al. in JAMA 305(18):1873-1881, 30) (n = 425) was used for validation. Associations with site-specific relapse were evaluated using the t-test and multivariate logistic regression adjusting for hormone receptor (HR) status.
RESULTS: RANK was significantly higher in HR negative versus HR positive (p = 0.027), in basal versus non-basal disease (p = 0.004), and in those achieving pathologic complete response (p = 0.038); the associations with HR negative and basal BC were also significant in GSE25066. In both datasets, higher RANK associated with significantly worse RFS (I-SPY1: p = 0.045, GSE25066: p = 0.044). However, this association did not remain significant after adjusting for HR status. In I-SPY1 patients with recurrence, higher RANK correlated with BM versus non-BM (p = 0.045), even after adjusting for HR status (p = 0.035).
CONCLUSIONS: RANK is increased in HR negative and basal BC, and correlates with worse RFS and risk of BM. The RANK pathway is a potential therapeutic target in BC.

The present study was carried out to investigate the clinical significance of TMPRSS3 and TNFRSF11B in breast cancer. Thus, the expression levels of TMPRSS3 and TNFRSF11B and the correlation with prognosis in patients with breast cancer were analyzed in silico using gene microarray and hierarchical clustering analysis. Then, the differential expression in breast cancer vs. normal breast tissue was explored in the Oncomine platform and verified in our independent samples using IHC technique. Our results indicated that TMPRSS3 was upregulated and TNFRSF11B was downregulated in breast cancer tissues compared with the levels in the human normal breast tissues. TMPRSS3 and TNFRSF11B were confirmed to be correlated with distant organ metastasis of breast cancer. Moreover, upregulation of TMPRSS3 accompanied by downregulation of TNFRSF11B was found to be associated with a shorter median overall survival and indicated a poor prognosis. In conclusion, TMPRSS3 and TNFRSF11B may have potential prognostic value to be used as tumor biomarkers in breast cancer patients.

Breast cancer is the most common female cancer, affecting approximately one in eight women during their lifetime in North America and Europe. Receptor Activator of NF-kB Ligand (RANKL), its receptor RANK and the natural antagonist osteoprotegerin (OPG) are essential regulators of bone resorption. We have initially shown that RANKL/RANK are essential for hormone-driven mammary epithelial proliferation in pregnancy and RANKL/RANK have been implicated in mammary stem cell biology. Using genetic mouse-models, we and others identified the RANKL/RANK system as a key regulator of sex hormone, BRCA1-mutation, and oncogene-driven breast cancer and we proposed that RANKL/RANK might be involved in the initiation of breast tumors. We now report that in postmenopausal women without known genetic predisposition, high RANKL and progesterone serum levels stratify a subpopulation of women at high risk of developing breast cancer 12-24 months before diagnosis (5.33-fold risk, 95%CI 1.5-25.4; P=0.02). In women with established breast cancer, we demonstrate that RANKL/OPG ratios change dependent on the presence of circulating tumor cells (CTCs). Finally, we show in a prospective human breast cancer cohort that alterations in RANKL/OPG ratios are significantly associated with breast cancer manifestation. These data indicate that the RANKL/RANK/OPG system is deregulated in post-menopausal women at high risk for breast cancer and in women with circulating tumor cells. Thus, serum levels of RANKL/OPG are potentially indicative of predisposition and progression of breast cancer in humans. Advancement of our findings towards clinical application awaits prior validation in independent patient cohorts.

The present study aimed to systematically examine the molecular mechanisms of papillary thyroid cancer (PTC), and identify potential biomarkers and drugs for the treatment of PTC. Two microarray data sets (GSE3467 and GSE3678), containing 16 PTC samples and 16 paired normal samples, were downloaded from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) were identified using the Linear Models for Microarray Analysis package. Subsequently, the common DEGs were screened for functional and pathway enrichment analysis using the Database for Annotation Visualization and Integrated Discovery. The representative interaction subnetwork was further derived using Molecular Complex Detection software. In addition, the potential drugs for the hub DEGs in the subnetwork were screened from DrugBank and the potential drug‑like ligands, which interacted with genes, were selected using MTiOpenScreen. A total of 167 common DEGs, including 77 upregulated and 90 downregulated DEGs, were screened. The common DEGs were associated with the functions of plasma membrane, extracellular matrix, response to steroid hormone stimulus and cell adhesion, and the pathways of tyrosine metabolism and cell adhesion molecules were significantly enriched. A total of eight common DEGs (MET, SERPINA1, LGALS3, FN1, TNFRSF11B, LAMB3 and COL13A1) were involved in the subnetwork. The two drugs, lanoteplase and ocriplasmin, and four drugs, β‑mercaptoethanol, recombinant α 1‑antitrypsin, PPL‑100 and API, were found for FN1 and SERPINA1, respectively. The common DEGs identified may be potential biomarkers for PCT. FN1 and SERPINA1 may be involved in PTC by regulating epithelial‑to‑mesenchymal transition and responding to steroid hormone stimuli, respectively. Ocriplasmin, β‑mercaptoethanol and recombinant α 1‑antitrypsin may be potential drugs for the treatment of PTC.

We recently reported the downregulation of osteoprotegerin expression in primary colorectal carcinoma and its significant association with aggressive oncogenic behavior, which suggest that this process contributes to colorectal carcinoma development and progression. In this study, we used immunohistochemical staining to evaluate osteoprotegerin expression in 81 colorectal liver metastasis tissue samples and investigated its possible association with the clinicopathological characteristics and outcomes of patients with colorectal liver metastasis. These tissues exhibited significantly reduced expression of osteoprotegerin compared to primary colorectal carcinomas and normal colorectal mucosa. This reduced expression was significantly associated with the extent of colorectal liver metastasis, including multiplicity of metastatic tumors, involvement of the bilateral hepatic lobes, and higher histological grade. In addition, reduced osteoprotegerin expression was an independent significant predictor of recurrent liver metastasis and prognostic factor for reduced patient survival. These findings suggest that osteoprotegerin expression may be a novel predictor of recurrent liver metastasis and a prognostic biomarker in patients with colorectal liver metastasis. Patients harboring colorectal liver metastasis with reduced osteoprotegerin expression should be carefully monitored after hepatic resection for colorectal liver metastasis to enable early detection of potentially resectable metastatic recurrences.

Oncogenic events combined with a favourable environment are the two main factors in the oncological process. The tumour microenvironment is composed of a complex, interconnected network of protagonists, including soluble factors such as cytokines, extracellular matrix components, interacting with fibroblasts, endothelial cells, immune cells and various specific cell types depending on the location of the cancer cells (e.g. pulmonary epithelium, osteoblasts). This diversity defines specific "niches" (e.g. vascular, immune, bone niches) involved in tumour growth and the metastatic process. These actors communicate together by direct intercellular communications and/or in an autocrine/paracrine/endocrine manner involving cytokines and growth factors. Among these glycoproteins, RANKL (receptor activator nuclear factor-κB ligand) and its receptor RANK (receptor activator nuclear factor), members of the TNF and TNFR superfamilies, have stimulated the interest of the scientific community. RANK is frequently expressed by cancer cells in contrast with RANKL which is frequently detected in the tumour microenvironment and together they participate in every step in cancer development. Their activities are markedly regulated by osteoprotegerin (OPG, a soluble decoy receptor) and its ligands, and by LGR4, a membrane receptor able to bind RANKL. The aim of the present review is to provide an overview of the functional implication of the RANK/RANKL system in cancer development, and to underline the most recent clinical studies.

A better understanding of tumor biology is important in the identification of molecules that are down-regulated in malignancy and in determining their role in tumor suppression. The aim of this study was to analyze osteoprotegerin (OPG) expression in colorectal carcinoma (CRC) and to investigate the underlying mechanism for changes in the expression of OPG. OPG expression was assessed in CRC tissue samples and cell lines. The methylation status of the OPG promoter region was determined, and the effects of demethylation on OPG expression were analyzed. The effects of recombinant OPG (rOPG) administration on cellular functions were also investigated. Clinical and prognostic implications of OPG protein expression in CRC patients were analyzed. The CRC tissues and cells showed significantly lower OPG expression. Pyrosequencing of OPG-silenced CRC cells revealed that the OPG gene promoter was highly methylated. Treatment with demethylating agent significantly elevated OPG mRNA and protein expression. rOPG significantly decreased cell viability and MMP-2 and VEGF-A production in CRC cells. Reduced OPG immunoreactivity was associated with aggressive oncogenic behavior in CRC. Also, OPG expression was found to be an independent predictor of recurrent hepatic metastasis and independent prognostic factor for worse survival rates. We demonstrated that OPG silencing in CRC occurs through epigenetic repression, and is involved in the development and progression of CRC. Our data suggest that OPG is a novel prognostic biomarker and a new therapeutic target for the treatment of patients with CRC.

BACKGROUND: Aromatase inhibitor (AI) therapy is associated with musculoskeletal (MS) toxicity, which adversely affects quality of life and therapy adherence. Our objective was to evaluate whether genetic variants may predict endocrine therapy-related MS pain and hot flashes in a prospective observational cohort study.
PATIENTS & METHODS: 254 early breast cancer patients starting AI (n = 159) or tamoxifen therapy (n = 95) were included in this genetic biomarker study. MS and vasomotor symptoms were assessed at baseline and after 3, 6 and 12 months of therapy. AI-induced MS pain was defined as an increase in arthralgia or myalgia relative to baseline. Single nucleotide polymorphisms (SNP) in candidate genes involved in oestrogen signalling or previously associated with AI-related MS pain or oestrogen levels were selected.
RESULTS: Overall, 13 SNPs in CYP19, CYP17, osteoprotegerin (OPG) and oestrogen receptor 1 exhibited an allele frequency >0.05 and were included in the analysis. Patients carrying the G allele of rs2073618 in OPG experienced significantly more AI-induced MS toxicity compared to the wildtype allele, after correction for multiple testing (P = 0.046). Furthermore, this SNP was associated with severity of pain (P = 0.018). No association was found with regard to the other SNPs, both in AI and tamoxifen-treated patients. Neither could an association with vasomotor symptoms be demonstrated.
CONCLUSION: The SNP rs2073618 in OPG is associated with an increased risk of MS symptoms and pain with AI therapy, which has not been reported previously. Validation of this finding in larger cohorts and further functional studies are required.

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.

BACKGROUND: Osteoprotegerin (OPG) is a glycoprotein that has multifaceted role and is associated with several cancer malignancies like that of bladder carcinoma, gastric carcinoma, prostate cancer, multiple myeloma and breast cancer. Also OPG has been associated with several organ pathologies. The widespread expression of OPG suggests that OPG may have multiple biological activities that are yet to be explored.
METHODS: The anchorage-independent sphere cultures of the adherent cells were instrumental in our study as it provided a deeper insight into the complexity of a 3D tumor. Cytokine profiling was performed for OPG's detection in the microenvironment. ELISA and western blotting were performed to quantify the OPG secretion and measure the protein levels respectively. OPG expression was detected in human breast cancer tissue samples by IHC. To decipher OPG's role in tumor aggressiveness both recombinant human OPG as well as OPG rich and depleted breast cancer cell conditioned media were tested. Western blotting and MTT assay were performed to detect changes in signaling pathways and proliferation that were induced in presence of OPG. Onset of aneuploidy, in presence of OPG, was measured by cell cycle analysis and western blotting. Finally, human Breast Cancer qBiomarker Copy Number PCR Array was used to detect how OPG remarkably induced gene copy numbers for oncogenic pathway regulators.
RESULTS: SUM149PT and SUM1315M02 cells secrete high levels of the cytokine OPG compared to primary human mammary epithelial cells (HMEC). High expression of OPG was also detected in human breast cancer tissue samples compared to the uninvolved tissue from the same patient. OPG induced proliferation of control HMEC spheres and triggered the onset of aneuploidy in HMEC sphere cultures. OPG induced the expression of aneuploidy related kinases Aurora-A Kinase (IAK-1), Bub1 and BubR1 probably through the receptor activator of nuclear factor kappa-B ligand (RANKL) and syndecan-1 receptors via the Erk, AKT and GSK3(3 signaling pathway. Gene copy numbers for oncogenic pathway regulators such AKT1, Aurora-A Kinase (AURKA or IAK-1), epidermal growth factor receptor (EGFR) and MYC with a reduction in the copy numbers of cyclin dependent kinase inhibitor 2A (CDKN2A), PTEN and DNA topoisomerase 2 alpha (TOP2A) were induced in presence of OPG.
CONCLUSIONS: These results highlight the role of OPG in reprogramming normal mammary epithelial cells to a tumorigenic state and suggest promising avenues for treating inflammatory breast cancer as well as highly invasive breast cancer with new therapeutic targets.

Receptor activator for nuclear factor κB ligand (RANKL) is a member of the tumor necrosis factor (TNF) family. The interaction between RANKL and its receptor RANK plays an important role in the development and function of diverse tissues. However, the expression and role of RANKL in cervical cancer are still unknown. In the present study, we found that RANKL and RANK were highly co-expressed in cervical cancer. HeLa and SiHa cells secreted soluble RANKL (sRANKL), expressed member RANKL (mRANKL) and RANK. Recombinant human RANKL protein had no effect on the viability of HeLa and SiHa cells. Yet, blocking RANKL with an anti-human RANKL neutralizing antibody (α-RANKL) or recombinant human osteoprotegrin (OPG) protein resulted in the downregulation of Ki-67 and B-cell lymphoma 2 (Bcl-2) expression and an increase in Fas and Fas ligand (FasL) expression, as well as a high level of viability and a low level of apoptosis in the HeLa and SiHa cells. In addition, α-RANKL led to a decrease in IL-8 secretion. Recombinant human IL-8 protein reversed the effect of α-RANKL on the expression of proliferation- and apoptosis‑related molecules, and proliferation and apoptosis in the HeLa and SiHa cells. The present study suggests that a high level of mRANKL/RANK expression in cervical cancer lesions plays an important role in the rapid growth of cervical cancer cells possibly through strengthening the dialogue between cervical cancer cells and regulation of IL-8 secretion, which may be a possible target for cervical cancer therapy.

Lung cancer is the most common cause of cancer mortality and new cases are on the increase worldwide. However, the treatment of lung cancer remains unsatisfactory. Curcumin has been shown to induce cell death in many human cancer cells, including human lung cancer cells. However, the effects of curcumin on genetic mechanisms associated with these actions remain unclear. Curcumin (2 µM) was added to NCI-H460 human lung cancer cells and the cells were incubated for 24 h. Total RNA was extracted from isolated cells for cDNA synthesis, labeling, microarray hybridization and flour‑labeled cDNA hybridized on chip. Localized concentrations of fluorescent molecules were detected and quantified using Expression Console software (Affymetrix) with default RMA parameters. GeneGo software was used for the key genes involved and their possible interaction pathways. The results showed that ~170 genes were significantly upregulated and 577 genes were significantly downregulated in curcumin‑treated cells. Specifically, the up‑ and downregulated genes included CCNE2, associated with DNA damage; ID3, associated with cell survival and 146 genes with a >2- to 3-fold change including the TP53INP1 gene, associated with DNA damage; CDC6, CDCA5, TAKMIP2, CDK14, CDK5, CDCA76, CDC25A, CDC5L and SKP2, associated with cell cycle; the CARD6, ID1 and ID2 genes, associated with cell survival and the BRMS1L, associated with cell migration and invasion. Additionally, 59 downregulated genes exhibited a >4-fold change, including the DDIT3 gene, associated with DNA damage; while 97 genes had a >3- to 4-fold change including the DDIT4 gene, associated with DNA damage; the CCPG1 gene, associated with cell cycle and 321 genes with a >2- to 3-fold including the GADD45A and CGREF1 genes, associated with DNA damage; the CCPG1 gene, associated with cell cycle, the TNFRSF10B, GAS5, TSSC1 and TNFRSF11B gene, associated with cell survival and the ARHAP29 and CADM2 genes, associated with cell migration and invasion. In conclusion, gene alterations provide information regarding the cytotoxic mechanism of curcumin at the genetic level and provide additional biomarkers or targets for the treatment of human lung cancer.

miR-21 is an oncogenic microRNA (miRNA) with an emerging role as therapeutic target in human malignancies, including multiple myeloma (MM). Here we investigated whether miR-21 is involved in MM-related bone disease (BD). We found that miR-21 expression is dramatically enhanced, while osteoprotegerin (OPG) is strongly reduced, in bone marrow stromal cells (BMSCs) adherent to MM cells. On this basis, we validated the 3'UTR of OPG mRNA as miR-21 target. Constitutive miR-21 inhibition in lentiviral-transduced BMSCs adherent to MM cells restored OPG expression and secretion. Interestingly, miR-21 inhibition reduced RANKL production by BMSCs. Overexpression of protein inhibitor of activated STAT3 (PIAS3), which is a direct and validated target of miR-21, antagonized STAT3-mediated RANKL gene activation. Finally, we demonstrate that constitutive expression of miR-21 inhibitors in BMSCs restores RANKL/OPG balance and dramatically impairs the resorbing activity of mature osteoclasts. Taken together, our data provide proof-of-concept that miR-21 overexpression within MM-microenviroment plays a crucial role in bone resorption/apposition balance, supporting the design of innovative miR-21 inhibition-based strategies for MM-related BD.

Breast cancer is the most common cause of cancer death among women (522,000 deaths in 2012). Imbalance between RANKL and OPG is observed in many cancers, including breast cancer. Consequently, SNPs in the genes of RANKL and OPG may be involved in breast cancer development. This study included 276 subjects. Group I (n = 100) healthy females as a control group, group II (n = 96) breast cancer patients without bone metastases, and group III (n = 80) breast cancer patients with bone metastases. RANKL rs9533156, OPG rs2073618, and OPG rs2073617 SNPs and their serum protein levels were studied for a possible association with breast cancer development. The allele frequency [(OR: 4.832 CI 2.18-10.71, P = 0.001) and genotype distribution (P = 0.001)] of OPG SNP rs2073618 showed a highly significant difference between breast cancer patients and healthy controls. The allele C is more common in breast cancer patients. The allele frequency [(OR: 0.451 CI 0.232-0.879, P = 0.018) and genotype distribution (P = 0.003)] of RANKL SNP rs9533156 differed significantly between breast cancer patients and healthy controls. The allele T is more common in breast cancer patients. The allele frequency [(OR: 0.36 CI 0.184-0.705, P = 0.002) and genotype distribution (P = 0.011)] of OPG SNP rs2073617 differed significantly between breast cancer patients and healthy controls. The allele T is more common in breast cancer patients. The C allele of OPG SNP rs2073618 may be associated with breast cancer development. No association was found between any of the SNPs and the serum protein levels of RANKL and OPG.

BACKGROUND: Gastric cancer (GC) is an aggressive malignancy whose mechanisms of development and progression are poorly understood. The identification of prognosis-related genomic loci and genes may suffer from the relatively small case numbers and a lack of systematic validation in previous studies.
METHODS: Array-based comparative genomic hybridization (aCGH) coupled with patient clinical information was applied to identify prognosis-related loci and genes with high-frequency recurrent gains in 129 GC patients. The candidate loci and genes were then validated using an independent cohort of 384 patients through branched DNA signal amplification analysis (QuantiGene assays).
RESULTS: In the 129 patients, a copy number gain of three chromosome regions-namely, 8q22 (including ESRP1 and CCNE2), 8q24 (including MYC and TNFRSF11B), and 20q11-q13 (including SRC, MMP9, and CSE1L)--conferred poor survival for patients. In addition, the correlation between the branched DNA signal amplification analysis results and the aCGH results was analyzed in 73 of these 129 patients, and MYC, TNFRSF11B, ESRP1, CSE1L, and MMP9 were found to be well correlated. Further validation using an independent cohort (n = 384) verified that only MYC and TNFRSF11B within 8q24 are related to survival. Patients with gains in both MYC and TNFRSF11B had poorer survival than those with no gains, particularly those with noncardia GC. Gains in both of these genes were also a significant independent prognostic indicator.
CONCLUSIONS: Our results revealed that copy number gains in MYC and TNFRSF11B located at 8q24 are associated with survival in GC, particularly noncardia GC.

Serum levels of sRANKL, RANK, OPG, IL-8, IL-6, IL-16, MMP-2, and calcitonin were measured by ELISA in patients with malignant, borderline, and benign bone tumors and in healthy individuals (control). Serum levels of RANK, OPG, IL-8, IL-6, and the OPG/sRANKL ratio were significantly higher, while the level of MMP-2 was significantly lower in patients with bone tumors than in controls. Serum concentration of IL-16 in osteosarcoma patients was significantly lower than in chondrosarcoma patients. No significant differences between bone sarcomas of different differentiation were detected for any of the studied markers. Calcitonin level depended on the tumor location and type.

OCT4B1 is a newly discovered spliced variant of OCT4 which is primarily expressed in pluripotent and tumor cells. Based on our previous studies, OCT4B1 is significantly overexpressed in tumors, where it endows an anti-apoptotic property to tumor cells. However, the mechanism by which OCT4B1 regulates the apoptotic pathway is not yet elucidated. Here, we investigated the effects of OCT4B1 suppression on the expression alteration of 84 genes involved in apoptotic pathway. The AGS (gastric adenocarcinoma), 5637 (bladder tumor), and U-87MG (brain tumor) cell lines were transfected with OCT4B1 or irrelevant siRNAs. The expression level of apoptotic genes was then quantified using a human apoptosis panel-PCR kit. Our data revealed an almost similar pattern of alteration in the expression profile of apoptotic genes in all three studied cell lines, following OCT4B1 suppression. In general, the expression of more than 54 apoptotic genes (64 % of arrayed genes) showed significant changes. Among these, some up-regulated (CIDEA, CIDEB, TNFRSF1A, TNFRSF21, TNFRSF11B, TNFRSF10B, and CASP7) and down-regulated (BCL2, BCL2L11, TP73, TP53, BAD, TRAF3, TRAF2, BRAF, BNIP3L, BFAR, and BAX) genes had on average more than tenfold gene expression alteration in all three examined cell lines. With some minor exceptions, suppression of OCT4B1 caused upregulation of pro-apoptotic and down-regulation of anti-apoptotic genes in transfected tumor cells. Uncovering OCT4B1 down-stream targets could further elucidate its part in tumorigenesis, and could lead to finding a new approach to combat cancer, based on targeting OCT4B1.

The aim of this study was to find the apoptosis molecular markers involved in the cell death that might be related to photodynamic therapy (PDT) mechanisms in breast cancer. The mammary tumors were induced in 25 Sprague-Dawley female rats by a single, oral gavage of 7,12-dimethylbenz(a)anthracene (DMBA; 70 mg/kg body weight). Animals were divided into four groups: G1 (normal, without DMBA), G2 (control, without PDT treatment), G3 (euthanized 48 h after PDT), and G4 (euthanized 24 h after PDT). For PDT experiments, the photosensitizer used was Photodithazine, and 100 J/cm of light at a fluence rate of 100 mW/cm was delivered to treat lesions. A sample of each animal was investigated by quantitative real-time PCR using Rat Apoptosis RT2 Profiler™ PCR Array platform. The results showed 20 genes with differential expression between PDT and control groups. A significant upregulation was observed for pro-apoptotic genes CASP4, CASP12, CIDEA, GADD45A, and FAS and downregulation of anti-apoptotic genes MAPK8IP1, TNFRSF11B, and NAIP2 in PDT-treated tumors. These results indicate that these genes are more directly involved in cell apoptosis induced by PDT.

Diabetes and cancer are both serious health problems worldwide and can lead to a significant burden on society with high incidence. Studies show that diabetes, which may be the reason of cancer metastasis, can increase cancer incidence and mortality. Bone is one of the most preferential metastatic target sites for cancers. Studies also indicate that OPG and RANKL which regulate bone reabsorption play significant roles in the process of bone tumor metastasis. In addition, metformin as a commonly used medicine for type 2 diabetes is a negative regulator of RANKL and inhibits the differentiation of osteoclasts. We present a hypothesis that metformin serves an inhibitory effect on bone metastasis of cancer via OPG/RANKL/RANK system.

OBJECTIVES: Bone disease is one of the hallmarks of multiple myeloma (MM). The role of osteoprotegerin (OPG) in the RANK/RANKL/OPG signaling system is well defined in the myeloma bone disease. Polymorphisms of the TNFRSF11B gene encoding OPG have been studied in various bone diseases. However, relationship between the levels of OPG and development of bone lesions regardless of RANKL is yet unknown. In this study, the effects of OPG gene polymorphism on the development of bone lesions in MM were investigated.
METHODS: C950T and C1181G polymorphisms of the OPG gene were studied in 52 MM patients (36 with bone lesions and 16 without bone lesions) and in another 20 control subjects using DNA sequencing.
RESULTS: 1181 G and 950 T alleles were overrepresented in MM patients having bone lesions. 950 TT/1181 GG haplotype frequency and TT/GG combined haplotype were also higher in MM patients having bone lesions compared to MM patients without bone lesions or to control.
DISCUSSION: This is the first study searching for the relationship between OPG gene variants C950T (promoter), C1181G (exon 1), and myeloma bone disease. It was concluded that the presence of polymorphic 1181 G/950 T alleles and 950 TT/1181 GG genotypes may play a role in the development of bone disease.

Bone is one of the most common metastatic sites for breast cancer. In this study, we observed a promoting effect of osteoblast-conditioned medium (OCM) on the migration of MCF-7, a noninvasive cell line of breast cancer cells. Cytokine antibody array was used to compare the cytokines of OCM with the conditioned medium of non-differentiated osteoblast cells, which consequently revealed factors related to migration, such as IL8, IL6, CSF2 (G-CSF), CSF3 (GM-CSF), and TNFRSF11B (osteoprotegerin). The expression of genes related to migration was also estimated with a PCR array, which showed that 9 genes were upregulated and 26 genes downregulated. Moreover, activated p38, ERK, and AKT pathways were found in the OCM treatment group. This finding indicated the migration ability of breast cancer cells, which move toward the bone depending on the presence of specific cytokines in its surrounding microenvironment.

Tumors escape host immune responses, in part, through the release of immunomodulatory factors and decoy receptors into their microenvironment. Several cancers express surface-bound and soluble members of the tumor necrosis factor (TNF) receptor superfamily, including TNFRSF11b/osteoprotegerin (OPG). In its physiologic role, OPG regulates bone remodeling through competition for osteoclast-activating cytokines and protects newly forming bone from T cell-mediated apoptosis. In multiple tumor types, OPG production is associated with an aggressive phenotype and increased metastasis to bone, but no study has examined OPG production in human metastatic melanoma. We demonstrate that a significant proportion of human metastatic melanomas constitutively produces OPG through a mechanism governed by membrane-bound TNF-α signaling through TNF receptor 1 (TNFR1). These observations both define a specific mechanism that regulates melanoma production of OPG and establish a new molecular target for the therapeutic regulation of OPG.

BACKGROUND: The receptor activator of NF-κB (RANK), its ligand (RANKL) and osteoprotegerin (OPG) have been reported to play a role in the pathophysiological bone turnover and in the pathogenesis of breast cancer. Based on this we investigated the role of single nucleotide polymorphisms (SNPs) within RANK, RANKL and OPG and their possible association to breast cancer risk.
METHODS: Genomic DNA was obtained from Caucasian participants consisting of 307 female breast cancer patients and 396 gender-matched healthy controls. We studied seven SNPs in the genes of OPG (rs3102735, rs2073618), RANK (rs1805034, rs35211496) and RANKL (rs9533156, rs2277438, rs1054016) using TaqMan genotyping assays. Statistical analyses were performed using the χ2-tests for 2 x 2 and 2 x 3 tables.
RESULTS: The allelic frequencies (OR: 1.508 CI: 1.127-2.018, p=0.006) and the genotype distribution (p=0.019) of the OPG SNP rs3102735 differed significantly between breast cancer patients and healthy controls. The minor allele C and the corresponding homo- and heterozygous genotypes are more common in breast cancer patients (minor allele C: 18.4% vs. 13.0%; genotype CC: 3.3% vs. 1.3%; genotype CT: 30.3% vs. 23.5%). No significantly changed risk was detected in the other investigated SNPs. Additional analysis showed significant differences when comparing patients with invasive vs. non-invasive tumors (OPG rs2073618) as well as in terms of tumor localization (RANK rs35211496) and body mass index (RANKL rs9533156 and rs1054016).
CONCLUSIONS: This is the first study reporting a significant association of the SNP rs3102735 (OPG) with the susceptibility to develop breast cancer in the Caucasian population.

INTRODUCTION: The impact on the survival of bone metastases (BM) in patients with neuroendocrine tumor (NET) is a matter of debate. BM have a key role in causing symptoms and in decreasing patients' quality of life. Although the mechanisms of the development of BM are not completely clear, it is now well understood that the Receptor Activator of Nuclear factor Kappa-B-/Ligand (RANK/RANKL)/osteoprotegerin (OPG) pathway plays a relevant role.
AIM: To characterize the RANK/RANKL/OPG pathway in patients affected with NET.
PATIENTS AND METHODS: Two cohorts of 15 patients each were enrolled in the study; one cohort was affected with NET without BM and the second cohort was affected with NET with BM. The serum RANK/RANKL/OPG pathway was assessed in both the groups.
RESULTS: Serum OPG levels and RANKL/OPG ratio were lower and higher, respectively, in NET patients harboring BM than in those without BM. During the ROC analysis, a cut-off value of 1071 pg/ml for OPG and 0.62 for RANKL/OPG ratio were able to significantly distinguish between the two groups.
CONCLUSIONS: This study indicates that RANK/RANKL/OPG pathway is imbalanced in patients with NET harboring BM. Specific alterations of this pathway could predict an early development of BM.

Neoplastic degeneration represents a rare but serious complication of Paget's disease of bone (PDB). Although osteosarcomas have been described in up to 1% of PDB cases, giant cell tumors are less frequent and mainly occur in patients with polyostotic disease. We recently characterized a large pedigree with 14 affected members of whom four developed giant cell tumors at pagetic sites. The high number of affected subjects across multiple generations allowed us to better characterize the clinical phenotype and look for possible susceptibility loci. Of interest, all the affected members had polyostotic PDB, but subjects developing giant cell tumors showed an increased disease severity with a reduced clinical response to bisphosphonate treatment and an increased prevalence of bone pain, deformities, and fractures. Together with an increased occurrence of common pagetic complications, affected patients of this pedigree also evidenced a fivefold higher prevalence of coronary artery disease with respect to either the unaffected family members or a comparative cohort of 150 unrelated PDB cases from the same geographical area. This association was further enhanced in the four cases with PDB and giant cell tumors, all of them developing coronary artery disease before 60 years of age. Despite the early onset and the severe phenotype, PDB patients from this pedigree were negative for the presence of SQSTM1 or TNFRSF11A mutations, previously associated with enhanced disease severity. Genome-wide linkage analysis identified six possible candidate regions on chromosomes 1, 5, 6, 8, 10, and 20. Because the chromosome 8 and 10 loci were next to the TNFRSF11B and OPTN genes, we extended the genetic screening to these two genes, but we failed to identify any causative mutation at both the genomic and transcription level, suggesting that a different genetic defect is associated with PDB and potentially giant cell tumor of bone in this pedigree.