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Purpose Bisphosphonates (BPs) are antiresorptive drugs that provide important effects on bone turnover. The key to the high efficiency of BPs is their affinity for bone tissue, and their chemical structure provides their molecular mechanism of action. BPs are widely used to treat a variety of diseases that cause excess bone resorption, such as bone metastasis, hypercalcaemia due to malignancy and Paget’s disease. The goal of this study was to assess whether the bisphosphonate (Pamifos®) present in bone cement has any effect on bone turnover. In this paper, we present changes in cytokine levels in the serum of rats treated surgically. Methods Research was performed on 40 adult male Wistar rats. The rats were divided into four groups: two control groups (A, B) and two experimental groups (C, D). Bone in rats in the experimental groups was implanted with BP-enriched cement, whereas bone in control-groups rats was implanted with clean cement (without BPs). Results We found a higher concentration of tumour necrosis factor alpha (TNF-α) three weeks after surgery in rats implanted with BP-enriched cement compared with rats implanted with clean cement. After six weeks of treatment, TNF-α levels decreased significantly in rats treated with BP-enriched cement, whereas the control group experienced an increase in TNF-α. The concentration of osteoprotegerin ligand (OPG) was higher in rats with BP implants. We found high levels of receptor activator of nuclear factor kappa-B ligand (RANKL) in rats after implantation of cement without BP in both groups. Conclusions We conclude that use of bisphosphonate (Pamifos®), which is present in bone cement, has an effect on bone turnover in that BPs stimulate an increase in OPG and a decrease in RANKL in the bone microenvironment and thus may be an important component of mechanisms that reduce bone resorption. Therefore, the use of BP-enriched cement implants appears to be justified.

Breast cancer frequently metastasises to the skeleton, interfering with the normal bone remodelling process and inducing bone degradation. Bone degradation is caused by osteoclasts, the normal bone-resorbing cells. Osteoclast-mediated bone degradation subsequently leads to the release of bone-derived factors that promote skeletal tumour growth. Osteoclasts themselves stimulate tumour growth. This Review describes the molecular mechanisms through which osteoclasts and breast cancer cells collaborate with each other, triggering the formation of osteolytic bone metastasis., Highlights • RANK-RANKL-OPG pathway regulates osteoclast formation, function and survival. • Cancer-mediated osteolysis is caused by osteoclast stimulation and osteoblast inhibition. • Bone matrix-embedded growth factors are released upon bone degradation. • Growth factors released from resorbed bone stimulate tumour growth.

Adipocytes are a significant component of the bone marrow microenvironment. Although bone marrow adipocytes were first identified more than 100 years ago, it is only in recent years that an understanding of their complex physiological role is emerging. Bone marrow adipocytes act as local regulators of skeletal biology and homeostasis, with recent studies suggesting that marrow adipose tissue is metabolically active, and can function as an endocrine organ. As such, bone marrow adipocytes have the potential to interact with tumour cells, influencing both tumour growth and bone disease. This review discusses the current evidence for the role of bone marrow adipocytes in tumour growth within the bone marrow microenvironment and the development of the associated bone disease., Highlights • Bone marrow adipocytes are a metabolically active source of lipids and adipokines. • Marrow adipocytes increase with age, but their role in bone metastasis is ill-defined. • Marrow adipocytes have tumour-promoting and -suppressive effects in bone metastasis.

This review summarizes the role of extracellular calcium, as found present in the bone tissue, in the process of bone metastasis.

The bone marrow microenvironment is characterized by its multicellular nature, and perhaps less obviously by the high mobility of multiple transient and stationary cell lineages present in this environment. The trafficking of hematopoietic and mesenchymal cells between the bone marrow and blood compartments is regulated by a number of bone marrow-derived factors. It is suspected that transformed metastatic cells “hijack” these processes to engraft into the skeleton and eventually cause the skeletal complications associated with metastatic disease. In this short review, experimental and association data supporting the contribution of a less recognized cell type of the bone marrow – the nerves of the sympathetic nervous system – to early events of the breast cancer bone metastatic process, are summarized.

Background: Due to the severity of the primary disease in patients with lung cancer, quality of life (QoL) is often overlooked. Factors that form QoL should be taken in consideration when planning the appropriate treatment and determining therapy targets, because of the increasing frequency of bone metastasis leading to high levels of pain. Purpose of this study is to assess quality of life in patients with lung cancer, before and after treatment combined with zoledronic acid. Methods and materials: QoL was assessed in 80 patients (49 males-31 females), of which 45 developed bone metastasis. Prior and post treatment (with co administration of zoledronic acid) seven reliable scales: Pittsburgh Sleep Quality index (PSQI), Epworth Sleeping Scale (ess), Dyspnea Scale (ds), Fatigue Severity Scale (FSS), Brief Pain Inventory (BPI), Fact-G scale for sleep quality and EQ-5D for general health condition. Results: Statistically positive correlations were verified between PSQI-DS, PSQI-FSS, BPI-ESS, DS-FSS, DS-BPI and BPI-FSS (p

Prostate and breast cancers frequently metastasize to bone. The physiological bone homeostasis is perturbed once cancer cells proliferate at the bone metastatic site. Tumors are complex structures consisting of cancer cells and numerous stroma cells. In this study, we show that osteolytic cancer cells (PC-3 and MDA-MB231) induce transcriptome changes in the bone/bone marrow microenvironment (stroma). This stroma transcriptome differs from the previously reported stroma transcriptome of osteoinductive cancer cells (VCaP). While the biological process "angiogenesis/vasculogenesis" is enriched in both transcriptomes, the "vascular/axon guidance" process is a unique process that characterizes the osteolytic stroma. In osteolytic bone metastasis, angiogenesis is denoted by vessel morphology and marker expression specific for arteries/arterioles. Interestingly, intra-tumoral neurite-like structures were in proximity to arteries. Additionally, we found that increased numbers of mesenchymal stem cells and vascular smooth muscle cells, expressing osteolytic cytokines and inhibitors of bone formation, contribute to the osteolytic bone phenotype. Osteoinductive and osteolytic cancer cells induce different types of vessels, representing functionally different hematopoietic stem cell niches. This finding suggests different growth requirements of osteolytic and osteoinductive cancer cells and the need for a differential anti-angiogenic strategy to inhibit tumor growth in osteolytic and osteoblastic bone metastasis.

OBJECTIVE: This study aimed to evaluate the impact of cancer treatment on bone mineral density (BMD) in the lumbar spine (LS) and femur in the postmenopausal women with cervical or endometrial cancer without bone metastasis compared to normal control postmenopausal women. METHODS: We retrospectively evaluated the BMD data in the LS, femur neck (FN) and trochanter (FT) by dual-energy X-ray absorptiometry and laboratory data of bone turnover markers at baseline and after one year in 130 patients with cervical cancer, 68 patients with endometrial cancer, and 225 healthy controls. RESULTS: There were no significant differences in the T-scores of basal BMD in LS and femur between patients with endometrial cancer and controls, and only T-score of basal BMD at the fourth lumbar vertebra (L4) was significantly lower in patients with cervical cancer compared to controls. One year later, T-scores of BMD at all LS sites and FN in patients with cervical cancer and T-scores of BMD at L3, L4, FN, and FT in those with endometrial cancer after cancer treatment were significantly lower compared to controls. Lower proportions of normal BMD at all skeletal sites except L2 in patients with endometrial cancer and those at L1, L4, and FN in patients with cervical cancer were observed compared to controls after cancer treatment. CONCLUSIONS: Our results suggest that cancer treatment increase bone loss in postmenopausal women with cervical and endometrial cancer.

Bone metastasis is an important prognostic factor in renal cell carcinoma (RCC). The calcium-sensing receptor (CaSR) has been associated with bone metastasis in several different malignancies. We analyzed the impact of CaSR in bone metastasis in RCC in vitro and in vivo. The RCC cell line 786-O was stably transfected with the CaSR gene and treated with calcium alone or in combination with the CaSR antagonist NPS2143. Afterwards migration, adhesion, proliferation and prominent signaling molecules were analyzed. Calcium treated CaSR-transfected 768-O cells showed an increased adhesion to endothelial cells and the extracellular matrix components fibronectin and collagen I, but not to collagen IV. The chemotactic cell migration and proliferation was also induced by calcium. The activity of SHC, AKT, ERK, P90RSK and JNK were enhanced after calcium treatment of CaSR-transfected cells. These effects were abolished by NPS2143. Development of bone metastasis was evaluated in vivo in a mouse model. Intracardiac injection of CaSR-transfected 768-O cells showed an increased rate of bone metastasis. The results indicate CaSR as an important component in the mechanism of bone metastasis in RCC. Therefore, targeting CaSR might be beneficial in patients with bone metastatic RCC with a high CaSR expression.

// Wei-Yu Chen 1, 2, * , Shih-Yang Liu 3, * , Yung-Sheng Chang 4 , Juan Juan Yin 5 , Hsiu-lien Yeh 6 , Tarek H. Mouhieddine 7 , Ola Hadadeh 7 , Wassim Abou-Kheir 7 , Yen-Nien Liu 4 1 Department of Pathology, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan 2 Department of Pathology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan 3 Department of Acupuncture and Manipulation, College of International Education, Tianjin University of Traditional Chinese Medicine, Tianjin, China 4 Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan 5 Cell and Cancer Biology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA. 6 Institute of Information System and Applications, National Tsing Hua University, HsinChu, Taiwan 7 Department of Anatomy, Cell Biology and Physiological Sciences, Faculty of Medicine, American University of Beirut, Beirut, Lebanon * These authors contributed equally to this work Correspondence to: Wassim Abou-Kheir, e-mail: wa12@aub.edu.lb Yen-Nien Liu, e-mail: liuy@tmu.edu.tw Keywords: Prostate cancer, bone metastasis, miR-34a, TCF7, BIRC5 Received: August 15, 2014 Accepted: November 02, 2014 Published: November 25, 2014 ABSTRACT Aberrant activation of Ras and WNT signaling are key events that have been shown to be up-regulated in prostate cancer that has metastasized to the bone. However, the regulatory mechanism of combinatorial Ras and WNT signaling in advanced prostate cancer is still unclear. TCF7 , a WNT signaling-related gene, has been implicated as a critical factor in bone metastasis, and here we show that TCF7 is a direct target of miR-34a. In samples of prostate cancer patients, miR-34a levels are inversely correlated with TCF7 expression and a WNT dependent gene signature. Ectopic miR-34a expression inhibited bone metastasis and reduced cancer cell proliferation in a Ras-dependent xenograft model. We demonstrate that miR-34a can directly interfere with the gene expression of the anti-proliferative BIRC5, by targeting BIRC5 3’UTR. Importantly, BIRC5 overexpression was sufficient to reconstitute anti-apoptotic signaling in cells expressing high levels of miR-34a. In prostate cancer patients, we found that BIRC5 levels were positively correlated with a Ras signaling signature expression. Our data show that the bone metastasis and anti-apoptotic effects found in Ras signaling-activated prostate cancer cells require miR-34a deficiency, which in turn aids in cell survival by activating the WNT and anti-apoptotic signaling pathways thereby inducing TCF7 and BIRC5 expressions.

Neuroblastoma (NB), which arises from embryonic neural crest cells, is the most common extra-cranial solid tumor of childhood. Approximately half of NB patients manifest bone metastasis accompanied with bone pain, fractures and bone marrow failure, leading to disturbed quality of life and poor survival. To study the mechanism of bone metastasis of NB, we established an animal model in which intracardiac inoculation of the SK-N-AS human NB cells in nude mice developed osteolytic bone metastases with increased osteoclastogenesis. SK-N-AS cells induced the expression of receptor activator of NF-κB ligand and osteoclastogenesis in mouse bone marrow cells in the co-culture. SK-N-AS cells expressed COX-2 mRNA and produced substantial amounts of prostaglandin E2 (PGE2). In contrast, the SK-N-DZ and SK-N-FI human NB cells failed to develop bone metastases, induce osteoclastogenesis, express COX-2 mRNA and produce PGE2. Immunohistochemical examination of SK-N-AS bone metastasis and subcutaneous tumor showed strong expression of COX-2. The selective COX-2 inhibitor NS-398 inhibited PGE2 production and suppressed bone metastases with reduced osteoclastogenesis. NS-398 also inhibited subcutaneous SK-N-AS tumor development with decreased angiogenesis and vascular endothelial growth factor-A expression. Of interest, metastasis to the adrenal gland, a preferential site for NB development, was also diminished by NS-398. Our results suggest that COX2/PGE2 axis plays a critical role in the pathophysiology of osteolytic bone metastases and tumor development of the SK-NS-AS human NB. Inhibition of angiogenesis by suppressing COX-2/PGE2 may be an effective therapeutic approach for children with NB.

The L1 cell adhesion molecule (L1CAM) has been implicated in tumor progression of many types of cancers, but its role in prostate cancer and its application in targeted gene therapy have not been investigated. Herein, we demonstrated that the L1CAM was expressed in androgen-insensitive and highly metastatic human prostate cancer cell lines. The correlation between L1CAM expression and prostate cancer metastasis was also validated in serum samples of prostate cancer patients. Knockdown of L1CAM expression in prostate cancer cells by RNA interference significantly decreased their aggressive behaviors, including colony formation, migration and invasion in vitro, and tumor formation in a metastatic murine model. These anti-malignant phenotypes of L1CAM-knockdown cancer cells were accompanied by G0/G1 cell cycle arrest and suppression of matrix metalloproteinase (MMP)-2 and MMP-9 expression and nuclear factor NF-κB activation. In vivo targeting of L1CAM expression using liposome-encapsulated L1CAM siRNAs effectively inhibited prostate cancer growth in mouse bone, which was associated with decreased L1CAM expression and cell proliferation by tumor cells. These results provide the first evidence for L1CAM being a major contributor to prostate cancer metastasis and translational application of siRNA-based L1CAM-targeted therapy.

// Huai-Ching Tai 1 , An-Chen Chang 2 , Hong-Jeng Yu 1 , Chao-Yuan Huang 1 , Yu-Chieh Tsai 3 , Yu-Wei Lai 4 , Hui-Lung Sun 5 , Chih-Hsin Tang 2,6,7 and Shih-Wei Wang 8 1 Department of Urology, National Taiwan University Hospital, Taipei, Taiwan 2 Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan 3 Department of Oncology, National Taiwan University Hospital, Taipei, Taiwan 4 Division of Urology, Taipei City Hospital Renai Branch, Taipei, Taiwan 5 Department of Molecular Virology, Immunology and Mediccal Genetics, Ohio state University, Columbus, OH, USA 6 Department of Pharmacology, School of Medicine, China Medical University, Taichung, Taiwan 7 Department of Biotechnology, College of Health Science, Asia University, Taichung, Taiwan 8 Department of Medicine, Mackay Medical College, New Taipei City, Taiwan Correspondence: Shih-Wei Wang, email: // Chih-Hsin Tang, email: // Keywords : WISP-1; Osteoblasts; Prostate cancer; miR-126; VCAM-1 Received : May 29, 2014 Accepted : July 29, 2014 Published : July 30, 2014 Abstract Bone metastases of prostate cancer (PCa) may cause intractable pain. Wnt-induced secreted protein-1 (WISP-1) belongs to the CCN family (CTGF/CYR61/NOV) that plays a key role in bone formation. We found that osteoblast-conditioned medium (OBCM) stimulates migration and vascular cell adhesion molecule-1 (VCAM-1) expression in human PCa (PC3 and DU145) cells. Osteoblast transfection with WISP-1 shRNA reduced OBCM-mediated PCa migration and VCAM-1 expression. Stimulation of PCa with OBCM or WISP-1 elevated focal adhesion kinase (FAK) and p38 phosphorylation. Either FAK and p38 inhibitors or siRNA abolished osteoblast-derived WISP-1-induced migration and VCAM-1 expression. Osteoblast-derived WISP-1 inhibited miR-126 expression. Moreover, miR-216 mimic reversed the WISP-1-enhanced migration and VCAM-1 expression. This study suggests that osteoblast-derived WISP-1 promotes migration and VCAM-1 expression in human PCa cells by down-regulating miR-126 expression via αvβ1 integrin, FAK, and p38 signaling pathways. Thus, WISP-1 may be a new molecular therapeutic target in PCa bone metastasis.

The development of effective therapies inhibiting prostate cancer progression and metastasis may substantially impact prostate cancer mortality and potentially reduce the rates of invasive treatments by enhancing the safety of active surveillance strategies. Hepsin (HPN) is a cell surface serine protease amplified in a subset of human sarcomas (7.2%), as well as in ovarian (10%), lung adeno (5.4%), lung squamous cell (4.5%), adenoid cystic (5%), breast (2.6%), uterine (1.7%) and colon (1.4%) carcinomas. While HPN is not amplified in prostate cancer, it is one of the most prominently overexpressed genes in the majority of human prostate tumors and genetic experiments in mice indicate that Hepsin promotes prostate cancer metastasis, particularly metastasis to the bone marrow. We report here the development, analysis and animal trial of the small-molecule Hepsin inhibitor HepIn-13. Long-term exposure to HepIn-13 inhibited bone, liver and lung metastasis in a murine model of metastatic prostate cancer. These findings indicate that inhibition of Hepsin with small-molecule compounds could provide an effective tool for attenuation of prostate cancer progression and metastasis.

Approximately 80% of prostate cancers exhibit some degree of bone metastasis. The role of the bone marrow and the hematopoietic stem cell (HSC) niche in attracting metastatic cells and maintaining dormancy of disseminated tumor cells (DTCs) is an increasingly important topic towards the development of novel prostate cancer therapies. This paper reviews aspects of the HSC niche that lead to prostate cancer cell homing and dormancy in the bone marrow. This review also discusses the role of DTCs in the niche environment and discusses the role of erythropoietin in targeting DTCs within the HSC niche.

// Bongkun Choi 1,4 , Eun-Jin Lee 1,4 , Da-Hyun Song 1,4 , Sung-Chul Yoon 2 , Yeon-Ho Chung 1,4 , Youngsaeng Jang 1,4 , Sang-Min Kim 1,4 , Youngsup Song 1,3,4 , Sang-Wook Kang 1,3,4 , Seung-Yong Yoon 3,4 , and Eun-Ju Chang 1,3,4 1 Department of Biomedical Sciences, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea 2 Division of Biostatistics, Center for Medical Research and Information, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea 3 Department of Anatomy and Cell Biology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea 4 Cellular Dysfunction Research Center and BMIT, University of Ulsan College of Medicine, Seoul, Korea Correspondence: Eun-Ju Chang, email: // Keywords : breast cancer, PTX3, osteoclast, RANKL, osteolysis. Received : December 10, 2013 Accepted : January 15, 2014 Published : January 15, 2014 Abstract Pentraxin 3 (PTX3), a modulator of tumor-associated inflammation, is known to be positively correlated with tumor grade and severity of malignancies, but its exact role remains unclear. This study found that PTX3 expression was up-regulated in distant bone metastases of breast cancer compared to lung, liver, and brain metastases in 64 human breast cancer patients. Elevated expression of PTX3 was correlated with poor survival in patients with breast cancer. PTX3 expression was also up-regulated in a bone metastatic breast cancer cell line and further enhanced by pro-inflammatory cytokine TNFα. Administration of PTX3 promoted the migratory potential of breast cancer cells and the mobilization of macrophages, a precursor of osteoclasts (OCs), toward breast cancer cells. In addition, elevated expression of PTX3 by TNFα led to enhanced OC formation, implying the distinct role of PTX3 in osteolytic bone metastasis of breast cancer cells. Furthermore, PTX3 silencing using PTX3-specific siRNA prevented breast cancer cell migration, macrophage chemotaxis, and subsequent OC formation. These findings provide an important insight into the key role of PTX3 in inflammation-associated osteolytic complications of breast cancer.

Aim of the study Bone scintigraphy (BS) and fluorine-18 deoxyglucose positron emission tomography computed tomography (18F-FDG-PET/CT) are widely used for the detection of bone involvement. The optimal imaging modality for the detection of bone metastases in histological subgroups of non-small cell lung cancer (NSCLC) remains ambiguous. The aim of this study was to compare the efficacy of 18F-FDG-PET/C and 99mTc-methylene diphosphonate (99mTc-MDP) BS in the detection of bone metastases of patients in NSCLC. Specifically, we compared the diagnostic accuracies of these imaging techniques evaluating bone metastasis in histological subgroups of NSCLC. Material and methods Fifty-three patients with advanced NSCLC, who had undergone both 18F-FDG-PET/CT and BS and were eventually diagnosed as having bone metastasis, were enrolled in this retrospective study. Results The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of 18F-FDG-PET/CT and BS were 90.4%, 99.4%, 98.1%, 96.6%, 97.0% and 84.6%, 93.1%, 82.5%, 93.2, 90.8%, respectively. The κ statistics were calculated for 18F-FDG-PET/CT and BS. The κ-value was 0.67 between 18F-FDG-PET/CT and BS in all patients. On the other hand, the κ-value was 0.65 in adenocarcinoma, and 0.61 in squamous cell carcinoma between 18F-FDG-PET/CT and BS. The κ-values suggested excellent agreement between all patients and histological subgroups of NSCLC. Conclusions 18F-FDG-PET/CT was more favorable than BS in the screening of metastatic bone lesions, but the trend did not reach statistical significance in all patients and histological subgroups of NSCLC. Our results need to be validated in prospective and larger study clinical trials to further clarify this topic.

Incidence of skeletal metastases and death from prostate cancer greatly increases with age and obesity, conditions which increase marrow adiposity. Bone marrow adipocytes are metabolically active components of bone metastatic niche that modulate the function of neighboring cells; yet the mechanisms of their involvement in tumor behavior in bone have not been explored. In this study, using experimental models of intraosseous tumor growth and diet-induced obesity, we demonstrate the promoting effects of marrow fat on growth and progression of skeletal prostate tumors. We reveal that exposure to lipids supplied by marrow adipocytes induces expression of lipid chaperone FABP4, pro-inflammatory interleukin IL-1β, and oxidative stress protein HMOX-1 in metastatic tumor cells and stimulates their growth and invasiveness. We show that FABP4 is highly overexpressed in prostate skeletal tumors from obese mice and in bone metastasis samples from prostate cancer patients. In addition, we provide results suggestive of bi-directional interaction between FABP4 and PPARγ pathways that may be driving aggressive tumor cell behavior in bone. Together, our data provide evidence for functional relationship between bone marrow adiposity and metastatic prostate cancers and unravel the FABP4/IL-1β axis as a potential therapeutic target for this presently incurable disease.

Bone metastasis is the major deleterious event in prostate cancer (PCa). TMPRSS2-ERG fusion is one of the most common chromosomic rearrangements in PCa. However, its implication in bone metastasis development is still unclear. Since bone metastasis starts with the tropism of cancer cells to bone through specific migratory and invasive processes involving osteomimetic capabilities, it is crucial to better our understanding of the influence of TMPRSS2-ERG expression in the mechanisms underlying the bone tropism properties of PCa cells. We developed bioluminescent cell lines expressing the TMPRSS2-ERG fusion in order to assess its role in tumor growth and bone metastasis appearance in a mouse model. First, we showed that the TMPRSS2-ERG fusion increases cell migration and subcutaneous tumor size. Second, using intracardiac injection experiments in mice, we showed that the expression of TMPRSS2-ERG fusion increases the number of metastases in bone. Moreover, TMPRSS2-ERG affects the pattern of metastatic spread by increasing the incidence of tumors in hind limbs and spine, which are two of the most frequent sites of human PCa metastases. Finally, transcriptome analysis highlighted a series of genes regulated by the fusion and involved in the metastatic process. Altogether, our work indicates that TMPRSS2-ERG increases bone tropism of PCa cells and metastasis development., info:eu-repo/semantics/published

Neuroendocrine tumors (NETs) metastasize to the skeleton in approximately 20% of patients. We have previously shown that the epithelial-mesenchymal transition (EMT) regulates the NET osteotropism and that CXCR4 overexpression predicts bone spreading. Here, we unravel the molecular mechanisms linking the activation of the CXCL12/CXCR4 axis to the bone colonization of NETs using cell lines representative of pancreatic (BON1, CM, QGP1), intestinal (CNDT 2.5), and bronchial origin (H727). By combining flow cytometry and ELISA, BON1, CM and QGP1 cells were defined as CXCR4high/CXCL12low, while H727 and CNDT 2.5 were CXCR4low/CXCL12high. CXCL12 was inert on cell proliferation, but significantly increased the in vitro osteotropism of CXCR4high/CXCL12low cells, as assessed by transwell assays with or without Matrigel membranes. In these cells, CXCL12 induced in vitro a marked EMT-like transcriptional shift with acquirement of a mesenchymal shape. The nuclei of CXCR4high/CXCL12low NET cells were typically enriched in non-phosphorylated CXCR4, particularly upon agonist stimulation. Silencing of CXCR4 via siRNA prevented the CXCL12-induced EMT in CXCR4high/CXCL12low NET cell lines resulting in the abrogation of both migration and transcriptional mesenchymal patterns. Our data suggest that CXCL12 conveys EMT-promoting signals in NET cells through CXCR4, which in turn regulates transcriptional, morphologic and functional modifications resulting in enhanced in vitro osteotropism of NET cells. Unique functions of CXCR4 may be segregated in relation to its subcellular localization and may acquire potential relevance in future in vivo studies.

WOS: 000391457100008, PubMed ID: 27780164, Objective: To investigate the symptoms of lung cancer in Turkey and to evaluate approaches to alleviate these symptoms. Subjects and Methods: This study included 1,245 lung cancer patients from 26 centers in Turkey. Demographic characteristics as well as information regarding the disease and treatments were obtained from medical records and patient interviews. Symptoms were evaluated using the Edmonton Symptom Assessment Scale (ESAS) and were graded on a scale between 0 and 10 points. Data were compared using the. 2, Student t, and Mann-Whitney U tests. Potential predictors of symptoms were analyzed using logistic regression analysis. Results: The most common symptom was tiredness (n = 1,002; 82.1%), followed by dyspnea (n = 845; 69.3%), appetite loss (n = 801; 65.7%), pain (n = 798; 65.4%), drowsiness (n = 742; 60.8%), anxiety (n = 704; 57.7%), depression (n = 623; 51.1%), and nausea (n = 557; 45.5%). Of the 1,245 patients, 590 (48.4%) had difficulty in initiating or maintaining sleep. The symptoms were more severe in stages III and IV. Logistic regression analysis indicated a clear association between demographic characteristics and symptom distress, as well as between symptom distress (except nausea) and well-being. Overall, 804 (65.4%) patients used analgesics, 630 (51.5%) received treatment for dyspnea, 242 (19.8%) used enteral/parenteral nutrition, 132 (10.8%) used appetite stimulants, and 129 (10.6%) used anxiolytics/antidepressants. Of the 799 patients who received analgesics, 173 (21.7%) reported that their symptoms were under control, and also those on other various treatment modalities (dyspnea: 78/627 [12.4%], appetite stimulant: 25/132 [18.9%], and anxiolytics/antidepressants: 25/129 [19.4%]) reported that their symptoms were controlled. Conclusion: In this study, the symptoms progressed and became more severe in the advanced stages of lung cancer, and palliative treatment was insufficient in most of the patients in Turkey. (C) 2016 S. Karger AG, Basel

Prostate cancer (PCa) patients with bone metastases are primarily treated with androgen deprivation therapy (ADT). Less pronounced ADT effects are seen in metastases than in primary tumors. To test if acute effects of ADT was enhanced by concurrent inhibition of pro-survival insulin-like growth factor 1 (IGF-1), rats were inoculated with Dunning R3327-G tumor cells into the tibial bone marrow cavity and established tumors were treated with castration in combination with IGF-1 receptor (IGF-1R) inhibitor NVP-AEW541, or by each treatment alone. Dunning R3327-G cells were stimulated by androgens and IGF-1 in vitro. In rat tibia, Dunning R3327-G cells induced bone remodeling, identified through increased immunoreactivity of osteoblast and osteoclast markers. Tumor cells occasionally grew outside the tibia, and proliferation and apoptotic rates a few days after treatment were evaluated by scoring BrdU- and caspase-3-positive tumor cells inside and outside the bone marrow cavity, separately. Apoptosis was significantly induced outside, but unaffected inside, the tibial bone by either castration or NVP-AEW541, and the maximum increase (2.7-fold) was obtained by the combined treatment. Proliferation was significantly reduced by NVP-AEW541, independently of growth site, although the maximum decrease (24%) was observed when NVP-AEW541 was combined with castration. Tumor cell IGF-1R immunoreactivity was evaluated in clinical PCa bone metastases (n = 61), and positive staining was observed in most cases (74%). In conclusion, IGF-1R inhibition may be evaluated in combination with ADT in patients with metastatic PCa, or in combination with therapies for the subsequent development of castration-resistant disease, although diverse responses could be anticipated depending on metastasis site. Electronic supplementary material The online version of this article (doi:10.1007/s10585-017-9848-8) contains supplementary material, which is available to authorized users.

Cancer-induced bone pain (CIBP) is common in patients with bone metastases (BM), significantly impairing quality of life. The current treatments for CIBP are limited since they are often ineffective. Local acidosis derived from glycolytic carcinoma and tumor-induced osteolysis is only barely explored cause of pain. We found that breast carcinoma cells that prefer bone as a metastatic site have very high extracellular proton efflux and expression of pumps/ion transporters associated with acid-base balance (MCT4, CA9, and V-ATPase). Further, the impairment of intratumoral acidification via V-ATPase targeting in xenografts with BM significantly reduced CIBP, as measured by incapacitance test. We hypothesize that in addition to the direct acid-induced stimulation of nociceptors in the bone, a novel mechanism mediated by the acid-induced and tumor-associated mesenchymal stroma might ultimately lead to nociceptor sensitization and hyperalgesia. Consistent with this, short-term exposure of cancer-associated fibroblasts, mesenchymal stem cells, and osteoblasts to pH 6.8 promotes the expression of inflammatory and nociceptive mediators (NGF, BDNF, IL6, IL8, IL1b and CCL5). This is also consistent with a significant correlation between breakthrough pain, measured by pain questionnaire, and combined high serum levels of BDNF and IL6 in patients with BM, and also by immunofluorescence staining showing IL8 expression that was more in mesenchymal stromal cells rather than in tumors cells, and close to LAMP-2 positive acidifying carcinoma cells in BM tissue sections. In summary, intratumoral acidification in BM might promote CIBP also by activating the tumor-associated stroma, offering a new target for palliative treatments in advanced cancer.

// Hinrich Wieder 1,2 , Ambros J. Beer 1,7 , Konstantin Holzapfel 3 , Martin Henninger 1 , Tobias Maurer 4 , Sarah Schwarzenboeck 5 , Ernst J. Rummeny 3 , Matthias Eiber 1,* and Jens Stollfuss 3,6,* 1 Department of Nuclear Medicine, Klinikum rechts der Isar, Technische Universitat Munchen, Munich, Germany 2 Centre for Radiology and Nuclear Medicine, Grevenbroich, Germany 3 Institute for Diagnostic and Interventional Radiology, Klinikum rechts der Isar, Technische Universitat Munchen, Munich, Germany 4 Department of Urology, Klinikum rechts der Isar, Technische Universitat Munchen, Munich, Germany 5 Department of Nuclear Medicine, Universitatsmedizin Rostock, Rostock, Germany 6 Department of Radiology and Nuclear Medicine, Klinikum Memmingen, Memmingen, Germany 7 Department of Nuclear Medicine, Ulm University, Ulm, Germany * These authors have contributed equally to this manuscript Correspondence to: Hinrich Wieder, email: // Keywords : prostate cancer, recurrence, 11C-choline, PET/CT, MRI Received : November 18, 2016 Accepted : March 06, 2017 Published : March 15, 2017 Abstract Purpose: To compare the detection efficacy of 11C-choline positron emission tomography and computed tomography (PET/CT) with whole-body magnetic resonance imaging (MRI) including diffusion-weighted imaging (DWI) in patients with suspected recurrent prostate cancer. Materials and Methods: Fifty-seven patients (mean age 68, range 54-80 years) underwent 11C-choline PET/CT and MRI using T1-weighted (T1w), short-tau inversion recovery (STIR), and DWI. Two readers visually rated suspicious lesions on a 5-point scale in 20 different regions. Clinical follow-up and histopathology served as the standard of reference (SOR). Results: Fifty patients (mean PSA 29.9, range 1.0-670 ng/mL) had at least one positive lesion according to the SOR. Twenty-four patients had local recurrence (LR), 27 had lymph node (LN) involvement, and 22 had bone metastases. The overall detection rates for PET/CT and MRI on a patient basis were 94% and 88%, respectively ( p = 0.07). The PSA level (>2 ng/mL vs ≤2 ng/mL) significantly influenced the overall performance of PET/CT ( p = 0.003) and MRI ( p = 0.03). PET/CT was significantly superior to MRI in detecting LR ( p = 0.03) and bone metastasis ( p = 0.02). We found no difference with respect to the detection of LN metastasis ( p = 0.65). Conclusion: 11C-choline PET/CT was superior in the detection of local recurrence and bone metastasis on a regional basis. Whole-body MRI including DWI showed similar diagnostic accuracy only for detecting lymph node metastases. Compared with 11C-choline PET/CT, therefore, whole-body MRI including DWI cannot serve as alternative imaging modality for restaging prostate cancer.

Previous studies have shown that Atp6v1c1, a regulator of the assembly of the V0 and V1 domains of the V-ATPase complex, is up-regulated in metastatic oral tumors. Despite these studies, the function of Atp6v1c1 in tumor growth and metastasis is still unknown. Atp6v1c1's expression in metastatic oral squamous cell carcinoma indicates that Atp6v1c1 has an important function in cancer growth and metastasis. We hypothesized that elevated expression of Atp6v1c1 is essential to cancer growth and metastasis and that Atp6v1c1 promotes cancer growth and metastasis through activation of V-ATPase activity. To test this hypothesis, a Lentivirus-mediated RNAi knockdown approach was used to study the function of Atp6v1c1 in mouse 4T1 mammary tumor cell proliferation and migration in vitro and cancer growth and metastasis in vivo. Our data revealed that silencing of Atp6v1c1 in 4T1 cancer cells inhibited lysosomal acidification and severely impaired 4T1 cell growth, migration, and invasion through Matrigel in vitro. We also show that Atp6v1c1 knockdown with Lenti-c1s3, a lentivirus targeting Atp6v1c1 for shRNA mediated knockdown, can significantly inhibit 4T1 xenograft tumor growth, metastasis, and osteolytic lesions in vivo. Our study demonstrates that Atp6v1c1 may promote breast cancer growth and bone metastasis through regulation of lysosomal V-ATPase activity, indicating that Atp6v1c1 may be a viable target for breast cancer therapy and silencing of Atp6v1c1 may be an innovative therapeutic approach for the treatment and prevention of breast cancer growth and metastasis.

// Rong Qiao 1, * , Runbo Zhong 1, * , Qing Chang 2 , Jiajun Teng 1 , Jun Pei 1 , Baohui Han 1 and Tianqing Chu 1 1 Department of Pulmonary Medicine, Shanghai Chest Hospital, Shanghai Jiao Tong University, Shanghai 200030, People’s Republic of China 2 Shanghai Jiao Tong University School of Medicine, Shanghai 200025, People’s Republic of China * These authors contributed equally to this work Correspondence to: Tianqing Chu, email: ctqxkyy@163.com Baohui Han, email: xkyyhan@gmail.com Keywords: dickkopf-1, non-small cell lung cancer, bone metastasis, prognosis, stage IV Received: December 20, 2016 Accepted: May 23, 2017 Published: June 12, 2017 ABSTRACT Background: The study was designed to evaluate the association between serum dickkopf-1 (DKK1) and non-small cell lung cancer (NSCLC) bone metastases. Materials and Methods: Serum DKK1 levels were quantified in 470 NSCLC patients, 140 with osseous metastases, 178 with extraosseous metastases, and 152 with early stage in complete remission. The Receiver Operating Characteristic (ROC) curve enabled us to identify a threshold value to distinguish patients with bone metastases. Results: Serum DKK1 levels in patients with osseous metastases were significantly higher than in the other 2 groups ( P < 0.001). ROC curves showed that the optimum cutoff was 311.8 pg/ml (area under curve 0.791, 95% confidence interval 0.739–0.843, sensitivity 77.1% and specificity 71.4%). Of interest, serum DKK1 correlated with the number of bone lesions ( P = 0.042) and associated with the poor survival in NSCLC patients with osseous metastases ( P = 0.029). Conclusions: Our data shows that serum DKK1 can be used for the detection of NSCLC bone metastases. More importantly this is the first report to show that serum DKK1 is a good predictor of poor prognosis in NSCLC patients with bone metastases.

It is known that V-ATPases (vacuolar H+-ATPase) are involved in breast cancer growth and metastasis. Part of this action is similar to their role in osteoclasts, where they're involved in extracellular acidification and matrix destruction; however, the roles of their subunits in cancer cell proliferation, signaling, and other pro-tumor actions are not well established. Analysis of TCGA data shows that V-ATPase subunit Atp6v1c1 is overexpressed or amplified in 34% of human breast cancer cases, with a 2-fold decrease in survival at 12 years. Whereas other subunits, such as Atp6v1c2 and Atp6v0a3, are overexpressed or genomically amplified less often, 6% each respectively, and have less impact on survival. Experiments show that lentiviral-shRNA mediated ATP6v1c1 knockdown in 4T1 mouse mammary cancer cells significantly reduces orthotopic and intraosseous tumor growth. ATP6v1c1 knockdown also significantly reduces tumor stimulated bone resorption through osteoclastogenesis at the bone and metastasis in vivo, as well as V-ATPase activity, proliferation, and mTORC1 activation in vitro. To generalize the effects of ATP6v1c1 knockdown on proliferation and mTORC1 activation we used human cancer cell lines - MCF-7, MDA-MB-231, and MDA-MB-435s. ATP6V1C1 knockdown reduced cell proliferation and impaired mTORC1 pathway activation in cancer cells but not in the untransformed cell line C3H10T1/2. Our study reveals that V-ATPase activity may be mediated through mTORC1 and that ATP6v1c1 can be knocked down to block both V-ATPase and mTORC1 activity.

We investigated whether the treatment schedule influences physicians’ decisions to refer their patients for radiotherapy. We presented a questionnaire to 104 physicians in various specialties at three hospitals. It included three hypothetical patients with uncomplicated painful bone metastasis: patients with an expected life span of one year (case 1), 6 months (case 2), and 2 months (case 3). The physicians were asked whether they would refer their patients for radiotherapy when a radiation oncologist presented three different treatment schedules: a short (8 Gy/1 fraction/1 day)-, a medium (20 Gy/5 fractions/1 week)-, and a long (30 Gy/10 fractions/2 weeks) schedule. We used Cochran’s Q-test to compare the percentage of physicians across the three schedules and a mixed-effect logistic model to identify predictors of the selection of only the one-day schedule. Of the 104 physicians, 68 (65%) responded. Of these, 37 (54%), 27 (40%), and 26 (38%) chose to refer patients for radiotherapy when the short-, medium-, and long schedules, respectively, were proposed in case 1 (p = 0.14). These numbers were 44 (65%), 29 (43%), and 15 (22%) for case 2 (p < 0.001), and 59 (87%), 12 (18%), and 1 (1%) for case 3 (p < 0.001). Hypothetical patient and the physicians’ years of practice and perspective regarding side effects were independently predictive of the selection of only the one-day schedule. In conclusion, the treatment schedule influenced the physicians’ decisions to refer patients for radiotherapy.

// Bogdan-Tiberius Preca 1, 2, 3 , Karolina Bajdak 1 , Kerstin Mock 1 , Waltraut Lehmann 1 , Vignesh Sundararajan 1 , Peter Bronsert 2, 4, 5 , Alexandra Matzge-Ogi 6, 7 , Veronique Orian-Rousseau 6 , Simone Brabletz 8 , Thomas Brabletz 8 , Jochen Maurer 1, 2, 3, * , Marc P. Stemmler 8, * 1 Department of General and Visceral Surgery, Medical Center, University of Freiburg, Faculty of Medicine, Freiburg, Germany 2 German Cancer Consortium (DKTK), Heidelberg, Germany 3 German Cancer Research Center (DKFZ), Heidelberg, Germany 4 Institute for Surgical Pathology, Medical Center – University of Freiburg, Faculty of Medicine, Freiburg, Germany 5 Tumorbank Comprehensive Cancer Center Freiburg, Medical Center, University of Freiburg, Faculty of Medicine, Freiburg, Germany 6 Karlsruhe Institute of Technology, Institute of Toxicology and Genetics, Eggenstein-Leopoldshafen, Germany 7 Amcure GmbH, Eggenstein-Leopoldshafen, Germany 8 Department of Experimental Medicine I, Nikolaus-Fiebiger Center for Molecular Medicine, Friedrich-Alexander University of Erlangen-Nurnberg, Erlangen, Germany * These authors contributed equally to this work Correspondence to: Marc P. Stemmler, email: marc.stemmler@fau.de Keywords: hyaluronic acid synthase 2 (HAS2), epithelial-mesenchymal transition, invasion, metastasis, CD44 signaling Received: August 15, 2016 Accepted: December 26, 2016 Published: January 09, 2017 ABSTRACT Cancer metastasis is the main reason for poor patient survival. Tumor cells delaminate from the primary tumor by induction of epithelial-mesenchymal transition (EMT). EMT is mediated by key transcription factors, including ZEB1, activated by tumor cell interactions with stromal cells and the extracellular matrix (ECM). ZEB1-mediated EMT and motility is accompanied by substantial cell reprogramming and the acquisition of a stemness phenotype. However, understanding of the underlying mechanism is still incomplete. We identified hyaluronic acid (HA), one major ECM proteoglycan and enriched in mammary tumors, to support EMT and enhance ZEB1 expression in cooperation with CD44s. In breast cancer cell lines HA is synthesized mainly by HAS2, which was already shown to be implicated in cancer progression. ZEB1 and HAS2 expression strongly correlates in various cancer entities and high HAS2 levels associate with an early relapse. We identified HAS2, tumor cell-derived HA and ZEB1 to form a positive feedback loop as ZEB1, elevated by HA, directly activates HAS2 expression. In an in vitro differentiation model HA-conditioned medium of breast cancer cells is enhancing osteoclast formation, an indicator of tumor cell-induced osteolysis that facilitates formation of bone metastasis. In combination with the previously identified ZEB1/ESRP1/CD44s feedback loop, we found a novel autocrine mechanism how ZEB1 is accelerating EMT.

Lethal progression of prostate cancer metastasis can be improved by developing animal models that recapitulate the clinical conditions. We report here that cytokeratin 13 (KRT13), an intermediate filament protein, plays a directive role in prostate cancer bone, brain, and soft tissue metastases. KRT13 expression was elevated in bone, brain, and soft tissue metastatic prostate cancer cell lines and in primary and metastatic clinical prostate, lung, and breast cancer specimens. When KRT13 expression was determined at a single cell level in primary tumor tissues of 44 prostate cancer cases, KRT13 level predicted bone metastasis and the overall survival of prostate cancer patients. Genetically enforced KRT13 expression in human prostate cancer cell lines drove metastases toward mouse bone, brain and soft tissues through a RANKL-independent mechanism, as KRT13 altered the expression of genes associated with EMT, stemness, neuroendocrine/neuromimicry, osteomimicry, development, and extracellular matrices, but not receptor activator NF-κB ligand (RANKL) signaling networks in prostate cancer cells. Our results suggest new inhibitors targeting RANKL-independent pathways should be developed for the treatment of prostate cancer bone and soft tissue metastases.

// Ingunn Holen 1 , Diane V. Lefley 1 , Sheila E. Francis 2 , Sarah Rennicks 1 , Steven Bradbury 1 , Robert E. Coleman 1 , Penelope Ottewell 1 1 Academic Unit of Clinical Oncology, Department of Oncology and Metabolism, Mellanby Centre for Bone Research, University of Sheffield, Sheffield S10 2RX, UK 2 Department of Infection Immunity and Cardiovascular Disease, University of Sheffield, Sheffield S10 2RX, UK Correspondence to: Penelope Ottewell, email: P.D.Ottewell@sheffield.ac.uk Keywords: breast cancer, bone metastasis, IL-1B, IL-1R, anakinra Received: June 01, 2016 Accepted: September 15, 2016 Published: September 27, 2016 ABSTRACT Background: We have recently identified interleukin 1B (IL-1B) as a potential biomarker for predicting breast cancer patients at increased risk for developing bone metastasis. In mouse models, IL-1B and its receptor (IL-1R1) are upregulated in breast cancer cells that metastasise to bone compared with cells that do not. We have now investigated the functional role of IL-1 by blocking IL-1R signalling with the clinically licensed antagonist, anakinra. Methodology: 6-week old female BALB/c mice received a subcutaneous or intra-venous injection of MDA-MB-231-IV or MCF7 cells. Anakinra (1mg/kg/day) or placebo was administered 3 days before (preventative) or 7 days later (treatment). Tumour volume, apoptosis (TUNEL, Caspase 3), proliferation (Ki67) and angiogenesis (CD34, VEGF and endothelin) were analysed. Effects on bone were measured by uCT, and TRAP, P1NP, IL-1B, TNF alpha and IL-6 ELISA. Results: Anakinra significantly reduced growth of MDA-MB-231-IV tumours in bone from 6.50+/3.00mm 2 (placebo) to 2.56+/-1.07mm 2 (treatment) and 0.63+/-0.18mm 2 (preventative). Anakinra also reduced the number of mice that developed bone metastasis from 90% (placebo) to 40% (treatment) and 10% (preventative). Anti-tumour effects were not confined to bone, subcutaneous tumour volumes reduced from 656.68mm 3 (placebo) to 160.47mm 3 (treatment) and 31.08mm 3 (preventative). Anakinra did not increase tumour cell apoptosis but reduced proliferation and angiogenesis in addition to exerting significant effects on the tumour environment reducing bone turnover markers, IL-1B and TNF alpha. Conclusions: Our novel data demonstrate a functional role of IL-1 signalling in breast tumour progression and metastasis, supporting that anakinra could be repurposed for the treatment of breast cancer bone metastasis.

// Dorien Lobbezoo 1, 2 , Wilfred Truin 1, 3 , Adri Voogd 1 , Rudi Roumen 1, 3 , Gerard Vreugdenhil 1, 2 , Marcus Wouter Dercksen 2 , Franchette van den Berkmortel 4 , Tineke Smilde 5 , Agnes van de Wouw 6 , Roel van Kampen 1, 7 , Johanna van Riel 8 , Natascha Peters 9 , Petronella Peer 10 , Vivianne C.G. Tjan-Heijnen 1 1 GROW, School for Oncology and Developmental Biology, Maastricht University Medical Center, Maastricht, The Netherlands 2 Department of Internal Medicine, Maxima Medical Center, Veldhoven, The Netherlands 3 Department of Surgery, Maxima Medical Center, Veldhoven, The Netherlands 4 Department of Internal Medicine, Atrium-Orbis, Heerlen, The Netherlands 5 Department of Internal Medicine, Jeroen Bosch Hospital, Den Bosch, The Netherlands 6 Department of Internal Medicine, VieCuri Medical Center, Venlo, The Netherlands 7 Department of Internal Medicine, Atrium-Orbis, Sittard, The Netherlands 8 Department of Internal Medicine, St Elisabeth Hospital, Tilburg, The Netherlands 9 Department of Internal Medicine, St Jans Hospital, Weert, The Netherlands 10 Department for Health Evidence, Radboud University Medical Center, Nijmegen, The Netherlands Correspondence to: Vivianne C.G. Tjan-Heijnen, email: vcg.tjan.heijnen@mumc.nl Keywords: metastatic breast cancer, histology, invasive lobular carcinoma, invasive ductal carcinoma, treatment Received: December 06, 2015 Accepted: March 28, 2016 Published: April 19, 2016 ABSTRACT Introduction: This study describes the differences between the two largest histological breast cancer subtypes (invasive ductal carcinoma (IDC) and invasive (mixed) lobular carcinoma (ILC) with respect to patient and tumor characteristics, treatment-choices and outcome in metastatic breast cancer. Results: Patients with ILC were older at diagnosis of primary breast cancer and had more often initial bone metastasis (46.5% versus 34.8%, P = 0.01) and less often multiple metastatic sites compared to IDC (23.7% versus 30.9%, P = 0.11). Six months after diagnosis of metastatic breast cancer, 28.1% of patients with ILC and 39.8% of patients with IDC had received chemotherapy with a longer median time to first chemotherapy for those with ILC ( P = 0.001). After six months 84.8% of patients with ILC had received endocrine therapy versus 72.5% of patients with IDC ( P = 0.0001). Median overall survival was 29 months for ILC and 25 months for IDC ( P = 0.53). Materials and Methods: We included 437 patients with hormone receptor-positive IDC and 131 patients with hormone receptor-positive ILC, all diagnosed with metastatic breast cancer between 2007–2009, irrespective of date of the primary diagnosis. Patient and tumor characteristics and data on treatment and outcome were collected. Survival curves were obtained using the Kaplan-Meier method. Conclusions: Treatment strategies of hormone receptor-positive metastatic breast cancer were remarkably different for patients with ILC and IDC. Further research is required to understand tumor behavior and treatment-choices in real-life.

// Hanna Taipaleenmaki 1, 2 , Nicholas H. Farina 3 , Andre J. van Wijnen 1, 4 , Janet L. Stein 1, 3 , Eric Hesse 2, 5, * , Gary S. Stein 1, 3, * , Jane B. Lian 1, 3, * 1 Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA, USA 2 Heisenberg-Group for Molecular Skeletal Biology, Department of Trauma, Hand & Reconstructive Surgery, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 3 Department of Biochemistry & Cancer Center, University of Vermont College of Medicine, Burlington, VT, USA 4 Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA 5 Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, USA * These authors have contributed equally to this work Correspondence to: Jane B. Lian, email: jane.lian@uvm.edu Hanna Taipaleenmaki, email: h.taipaleenmaeki@uke.de Keywords: metastasis, breast cancer, osteolysis, Wnt signaling, miR-218-5p Received: May 08, 2016 Accepted: September 19, 2016 Published: October 12, 2016 ABSTRACT Wnt signaling is implicated in bone formation and activated in breast cancer cells promoting primary and metastatic tumor growth. A compelling question is whether osteogenic miRNAs that increase Wnt activity for bone formation are aberrantly expressed in breast tumor cells to support metastatic bone disease. Here we report that miR-218-5p is highly expressed in bone metastases from breast cancer patients, but is not detected in normal mammary epithelial cells. Furthermore, inhibition of miR-218-5p impaired the growth of bone metastatic MDA-MB-231 cells in the bone microenvironment in vivo. These findings indicate a positive role for miR-218-5p in bone metastasis. Bioinformatic and biochemical analyses revealed a positive correlation between aberrant miR-218-5p expression and activation of Wnt signaling in breast cancer cells. Mechanistically, miR-218-5p targets the Wnt inhibitors Sclerostin (SOST) and sFRP-2, which highly enhances Wnt signaling. In contrast, delivery of antimiR-218-5p decreased Wnt activity and the expression of metastasis-related genes, including bone sialoprotein (BSP/IBSP), osteopontin (OPN/SPP1) and CXCR-4, implicating a Wnt/miR-218-5p regulatory network in bone metastatic breast cancer. Furthermore, miR-218-5p also mediates the Wnt-dependent up-regulation of PTHrP, a key cytokine promoting cancer-induced osteolysis. Antagonizing miR-218-5p reduced the expression of PTHrP and Rankl, inhibited osteoclast differentiation in vitro and in vivo , and prevented the development of osteolytic lesions in a preclinical metastasis model. We conclude that pathological elevation of miR-218-5p in breast cancer cells activates Wnt signaling to enhance metastatic properties of breast cancer cells and cancer-induced osteolytic disease, suggesting that miR-218-5p could be an attractive therapeutic target for preventing disease progression.

Aim of the study Different carcinomas have different characteristics, which may play a crucial role in diagnosis and treatment. Our study was aimed at understanding the development pattern of bone metastasis from hepatocellular carcinoma, based on its imaging characteristics, so as to provide a more targeted treatment. Material and methods Forty two patients (123 lesions) with hepatocellular carcinoma hospitalized from June 2006 to June 2011 underwent radiotherapy for bone metastasis in our department. Clinical and imaging data were analyzed retrospectively (based on CT imaging, also with reference to MRI, ECT, or PET-CT, etc.). Results One hundred of 123 lesions were vertebral metastases; 23 were non-vertebral. The major form of bone destruction was osteolytic change. Metastasis in the vertebral body was found in 87.8%, and lesions were well distributed in various sections. Vertebral appendix metastasis accounted for 52%, where lesions could be independent of vertebral body metastasis. Formation of a soft tissue mass in bone metastasis was found in 68.6% of all patients. The center of the mass from a vertebral body metastasis was mostly located at the site of the lesion; masses from the vertebral appendix and the pelvis, on the other hand, often presented as a “peripheral mass”. Masses were not formed in lesions with pure osteoblastic changes. Conclusions The most common radiographic feature is an osteolytic lesion, either replaced by soft tissue mass, or invaded by soft tissue mass from the vicinity, which often cause compression syndrome. Vertebral appendix metastasis can exist independently from vertebral body metastasis, which should be paid more attention to avoid missed diagnosis.

Myeloid-derived suppressor cells (MDSCs), identified as Gr1(+)CD11b(+) cells in mice, expand during cancer and promote tumor growth, recurrence and burden. However, little is known about their role in bone metastases. We hypothesized that MDSCs may contribute to tumor-induced bone disease, and inoculated breast cancer cells into the left cardiac ventricle of nude mice. Disease progression was monitored weekly by X-ray and fluorescence imaging and MDSCs expansion by fluorescence-activated cell sorting. To explore the contribution of MDSCs to bone metastasis, we co-injected mice with tumor cells or PBS into the left cardiac ventricle and Gr1(+)CD11b(+) cells isolated from healthy or tumor-bearing mice into the left tibia. MDSCs didn't induce bone resorption in normal mice, but increased resorption and tumor burden significantly in tumor-bearing mice. In vitro experiments showed that Gr1(+)CD11b(+) cells isolated from normal and tumor-bearing mice differentiate into osteoclasts when cultured with RANK ligand and macrophage colony-stimulating factor, and that MDSCs from tumor-bearing mice upregulate parathyroid hormone-related protein (PTHrP) mRNA levels in cancer cells. PTHrP upregulation is likely due to the 2-fold increase in transforming growth factor β expression that we observed in MDSCs isolated from tumor-bearing mice. Importantly, using MDSCs isolated from GFP-expressing animals, we found that MDSCs differentiate into osteoclast-like cells in tumor-bearing mice as evidenced by the presence of GFP(+)TRAP(+) cells. These results demonstrate that MDSCs expand in breast cancer bone metastases and induce bone destruction. Furthermore, our data strongly suggest that MDSCs are able to differentiate into osteoclasts in vivo and that this is stimulated in the presence of tumors.

Cancer of unknown primary (CUP) is a common, well-recognized and heterogeneous clinical syndrome. Patients with CUP present with metastatic disease in the absence of an identifiable primary tumour despite a diagnostic work-up. Aetiologically, CUP might either harbor primaries that cannot be detected by the standardized diagnostic investigations or carry distinct genetic and epigenetic aberrations. In this article we try to clarify the clinical and pathological enigma of CUP by answering 20 important questions related to this entity. © The Author 2010. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. 21 SUPPL. 7 vii303 vii307

Cell-cell and cell-matrix interactions play a major role in tumor morphogenesis and cancer metastasis. Therefore, it is crucial to create a model with a biomimetic microenvironment that allows such interactions to fully represent the pathophysiology of a disease for an in vitro study. This is achievable by using three-dimensional (3D) models instead of conventional two-dimensional (2D) cultures with the aid of tissue engineering technology. We are now able to better address the complex intercellular interactions underlying prostate cancer (CaP) bone metastasis through such models. In this study, we assessed the interaction of CaP cells and human osteoblasts (hOBs) within a tissue engineered bone (TEB) construct. Consistent with other in vivo studies, our findings show that intercellular and CaP cell-bone matrix interactions lead to elevated levels of matrix metalloproteinases, steroidogenic enzymes and the CaP biomarker, prostate specific antigen (PSA); all associated with CaP metastasis. Hence, it highlights the physiological relevance of this model. We believe that this model will provide new insights for understanding of the previously poorly understood molecular mechanisms of bone metastasis, which will foster further translational studies, and ultimately offer a potential tool for drug screening. © 2010 Landes Bioscience.

// Daniele Fanale 1, * , Valeria Amodeo 1, * , Viviana Bazan 1, * , Lavinia Insalaco 1 , Lorena Incorvaia 1 , Nadia Barraco 1 , Marta Castiglia 1 , Sergio Rizzo 1 , Daniele Santini 2 , Antonio Giordano 3 , Sergio Castorina 4, 5, # , Antonio Russo 1, # 1 Department of Surgical, Oncological and Oral Sciences, Section of Medical Oncology, University of Palermo, Palermo, Italy 2 University Campus Bio-Medico, Department of Medical Oncology, Rome, Italy 3 Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA, USA 4 Fondazione Mediterranea “G.B. Morgagni”, Catania, Italy 5 Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy # Co-last authors, these authors contributed equally to this work * These authors contributed equally to this work Correspondence to: Antonio Russo, e-mail: antonio.russo@usa.net Keywords: bone metastasis, breast cancer, microarray analysis, miRNA expression profile, zoledronic acid Received: March 08, 2016 Accepted: March 31, 2016 Published: April 13, 2016 ABSTRACT Zoledronic acid (ZOL), belonging to third generation bisphosphonate family, is a potent inhibitor of osteoclast-mediated bone resorption, widely used to effectively prevent osteolysis in breast cancer patients who develop bone metastases. Low doses of ZOL have been shown to exhibit a direct anticancer role, by inhibiting cell adhesion, invasion, cytoskeleton remodelling and proliferation in MCF-7 breast cancer cells. In order to identify the molecular mechanisms and signaling pathways underlying the anticancer activity exerted by ZOL, we analyzed for the first time the microRNA expression profile in breast cancer cells. A large-scale microarray analysis of 377 miRNAs was performed on MCF7 cells treated with 10 μM ZOL for 24 h compared to untreated cells. Furthermore, the expression of specific ZOL-induced miRNAs was analyzed in MCF-7 and SkBr3 cells through Real-time PCR. Low-dose treatment with ZOL significantly altered expression of 54 miRNAs. Nine upregulated and twelve downregulated miRNAs have been identified after 24 h of treatment. Also, ZOL induced expression of 11 specific miRNAs and silenced expression of 22 miRNAs. MiRNA data analysis revealed the involvement of differentially expressed miRNAs in PI3K/Akt, MAPK, Wnt, TGF-β, Jak-STAT and mTOR signaling pathways, and regulation of actin cytoskeleton. Our results have been shown to be perfectly coherent with the recent findings reported in literature concerning changes in expression of some miRNAs involved in bone metastasis formation, progression, therapy resistance in breast cancer. In conclusion, this data supports the hypothesis that ZOL-induced modification of the miRNA expression profile contributes to the anticancer efficacy of this agent.

// Lucia D’Amico 1, 2 , Dimas Belisario 2 , Giorgia Migliardi 3 , Cristina Grange 4 , Benedetta Bussolati 5 , Patrizia D’Amelio 6 , Timothy Perera 7 , Ettore Dalmasso 8 , Luca Dalle Carbonare 9 , Laura Godio 10 , Paolo Comoglio 3 , Livio Trusolino 3 , Riccardo Ferracini 11 , Ilaria Roato 2 1 Department of Biomedicine, Cancer Immunology, University of Basel, Basel, Switzerland 2 CeRMS, A.O. Citta della Salute e della Scienza, Torino, Italy 3 IRCC, Institute for Cancer Research and Treatment, Candiolo, Torino, Italy 4 Department of Medical Sciences, University of Turin, Torino, Italy 5 Department of Molecular Biotechnology and Health Science, Molecular Biotechnology Center, University of Turin, Torino, Italy 6 Gerontology Section, Department of Medical Sciences, University of Torino, Torino, Italy 7 Janssen Research and Development, Beerse, Belgium 8 Urology Section, A.O. Citta della Salute e della Scienza, Torino, Italy 9 Clinic of Internal Medicine, Section D, Policlinico G.B. Rossi Verona, Verona, Italy 10 Department of Pathology, A.O. Citta della Salute e della Scienza, Torino, Italy 11 Department of Orthopaedic Oncology, CTO Hospital, Torino, Italy Correspondence to: Ilaria Roato, email: ilaria.roato@unito.it Keywords: bone metastasis, renal cancer, cancer stem cells, c-MET, CCL20 Received: May 01, 2016 Accepted: May 29, 2016 Published: June 14, 2016 ABSTRACT Cancer stem cells (CSCs) are key players in bone metastasis. In some renal tumors CSCs overexpress the HGF receptor c-MET, speculating that c-MET targeting could lead to bone metastasis inhibition. To address this hypothesis we isolated renal CD105+/CD24−CSCs, expressing c-MET receptor from a primary renal carcinoma. Then, to study their ability to metastasize to bone, we injected renal CSCs in NOD/SCID mice implanted with a human bone and we tested the effect of a c-MET inhibitor (JNJ-38877605) on bone metastasis development. JNJ-38877605 inhibited the formation of metastases at bone implant site. We showed that JNJ-38877605 inhibited the activation of osteoclasts induced by RCC stem cells and it stimulated osteoblast activity, finally resulting in a reduction of bone turnover consistent with the inhibition of bone metastases. We measured the circulating levels of osteotropic factors induced by RCC stem cells in the sera of mice treated with c-Met inhibitor, showing that IL-11 and CCL20 were reduced in mice treated with JNJ-38877605, strongly supporting the involvement of c-MET in the regulation of this process. To address the clinical relevance of c-MET upregulation during tumor progression, we analysed c-MET in renal cancer patients detecting an increased expression in the bone metastatic lesions by IHC. Then, we dosed CCL20 serum levels resulting significantly increased in patients with bone metastases compared to non-metastatic ones. Collectively, our data highlight the importance of the c-MET pathway in the pathogenesis of bone metastases induced by RCC stem cells in mice and humans.