Fluoroscopic Percutaneous Fixation of Periacetabular Metastatic Cancer: The Tripod Technique.

There are an increasing number of patients who present with metastatic bone disease as the survival of patients with cancer improves in recent decades. The pelvis is the second most common site for skeletal metastases. Metastatic lesions in the pelvis can be largely divided into periacetabular lesions (Enneking zone II) and non-periacetabular lesions (zones I, III, and IV). Traditionally, patients with a symptomatic zone II lesion are treated with a cemented total hip arthroplasty (THA) using variations on the traditional Harrington method. These open surgeries are accompanied by many inherent risks. Both a prolonged recovery and wide range of potential complications may delay or interrupt the adjuvant radiation and systemic therapy. It was observed that the articular surface of the hip joint was often intact and that the femoral side was frequently not involved in these patients. A novel minimally invasive technique for hip joint preservation has recently been developed. Three large-bore cannulated screws are placed percutaneously under fluoroscopy in a tripod configuration to reinforce the mechanical axis of the acetabulum. Increased stability improves pain control and permits immediate weight bearing. When the disease progresses, this construct can be easily converted to a cemented THA using the tripod screws as rebar to support an acetabular cup, as part of a staged Harrington procedure. This approach is technically demanding. A detailed guide for the tripod technique should encompass indications, preoperative preparation, operating room settings, intraoperative fluoroscopic guidance, modifications, postoperative care, and subsequent conversion to a cemented THA, if needed.

Wnt Signaling in Osteosarcoma.

Wnt molecules are a class of cysteine-rich secreted glycoproteins that participate in various developmental events during embryogenesis and adult tissue homeostasis. Since its discovery in 1982, the roles of Wnt signaling have been established in various key regulatory systems in biology. Wnt signals exert pleiotropic effects, including mitogenic stimulation, cell fate specification, and differentiation. The Wnt signaling pathway in humans has been shown to be involved in a wide variety of disorders including colon cancer, sarcoma, coronary artery disease, tetra-amelia, Mullerian duct regression, eye vascular defects, and abnormal bone mass. The canonical Wnt pathway functions by regulating the function of the transcriptional coactivator ß-catenin, whereas noncanonical pathways function independent of ß-catenin. Although the role of Wnt signaling is well established in epithelial malignancies, its role in mesenchymal tumors is more controversial. Some studies have suggested that Wnt signaling plays a pro-oncogenic role in various sarcomas by driving cell proliferation and motility; however, others have reported that Wnt signaling acts as a tumor suppressor by committing tumor cells to differentiate into a mature lineage. Wnt signaling pathway also plays an important role in regulating cancer stem cell function. In this review, we will discuss Wnt signaling pathway and its role in osteosarcoma.

Flavokawain B targets protein neddylation for enhancing the anti-prostate cancer effect of Bortezomib via Skp2 degradation.

BACKGROUND: Flavokawain B (FKB) has been identified from kava root extracts as a potent apoptosis inducer for inhibiting the growth of various cancer cell lines, including prostate cancer. However, the molecular targets of FKB in prostate cancer cells remain unknown. METHODS: An in vitro NEDD8 Initiation Conjugation Assay was used to evaluate the neddylation inhibitory activity of FKB. Molecular docking and a cellular thermal shift assay were performed to assess the direct interaction between FKB and the NEDD8 activating enzyme (NAE) complex. Protein neddylation, ubiqutination, stability and expression in cells were assessed with immunoprecipitation and Western blotting methods using specific antibodies. Deletion and site specific mutants and siRNAs were used to evaluate deep mechanisms by which FKB induces Skp2 degradation. Cell growth inhibition and apoptosis induction were measured by MTT, ELISA and Western blotting methods. RESULTS: FKB inhibits NEDD8 conjugations to both Cullin1 and Ubc12 in prostate cancer cell lines and Ubc12 neddylation in an in vitro assay. Molecular docking study and a cellular thermal shift assay reveal that FKB interacts with the regulatory subunit (i.e. APP-BP1) of the NAE. In addition, FKB causes Skp2 degradation in an ubiquitin and proteasome dependent manner. Overexpression of dominant-negative cullin1 (1-452), K720R mutant (the neddylation site) Cullin1 or the F-box deleted Skp2 that losses its binding to the Skp1/Cullin1 complex causes the resistance to FKB-induced Skp2 degradation, whereas siRNA knock-down of Cdh1, a known E3 ligase of Skp2 for targeted degradation, didn't attenuate the effect of FKB on Skp2 degradation. These results suggest that degradation of Skp2 by FKB is involved in a functional Cullin1. Furthermore, proteasome inhibitors Bortezomib and MG132 transcriptionally down-regulate the expression of Skp2, and their combinations with FKB result in enhanced inhibitory effects on the growth of prostate cancer cell lines via synergistic down-regulation of Skp2 and up-regulation of p27/Kip1 and p21/WAF1 protein expression. FKB also selectively inhibits the growth of RB deficient cells with high expression of Skp2. CONCLUSION: These findings provide a rationale for further investigating combination of FKB and Bortezomib for treatment of RB deficient, castration-resistant prostate cancer.

Epidemiology, Therapeutic Strategies, Outcomes, and Complications of Pathologic Fractures.

Much can be learned about the epidemiology of metastatic disease of bone through large databases. Secondary data analyses add substantial knowledge of the incidence, prevalence, cost, complications, risk factors, and treatment variability by using modern statistical methods in a large patient cohort. Investigators must be aware of the intentions of these data sources as well as the limitations in any conclusions drawn. Large databases are primarily beneficial in generating hypotheses and will likely continue to be an important source of information. For the general orthopaedist, surgical management of metastatic skeletal disease can be a challenging problem with many potential risks. Complications are often encountered and can be influenced by the patient's medical conditions, stage of disease, and the selected surgical procedure. Patients diagnosed with skeletal metastases are often at higher risk of having perioperative complications, such as infection and thromboembolism, than is the general population. A case-based approach highlights potential risks with examples of common scenarios that can arise.

Mechanical analysis of the vascularized fibular graft prosthetic composite (VFGPC) for internal hemipelvectomy reconstruction.

INTRODUCTION: The vascularized fibular graft prosthetic composite (VFGPC) is used for reconstruction after internal hemipelvectomy. The purpose of this study was to create a mathematical model that calculates the mechanical effects of the vascularized fibular graft on the VFGPC. METHODS: The effects of the VFG positioning were calculated based on three-dimensional static analyzes to determine the direction, magnitude, and distribution of the forces through the prosthesis and VFG. The shear stress (SS) and cyclic loads to failure (CLF) were calculated. By varying the location of the VFG on the sacrum the zone of acceptable placement was calculated. RESULTS: Utilization of the VFG decreased the forces through the implant by 15-35% and decreased SS 20-54%, depending on stance. The CLF improved by 94%. The zone of acceptable placement for the VFG was found to be between 0° and 15° of the vertical axis in the sagittal plane and 0° and 30° of the posterior axis in coronal plane. CONCLUSION: Determining the position of the VFG pre-operatively allows for the creation of a customized cutting jig can be utilized to create graft allowing for accurate fibular osteotomies, minimization of ischemia time, and decreased intra-operative handling of the graft.

Induction of G2M Arrest by Flavokawain A, a Kava Chalcone, Increases the Responsiveness of HER2-Overexpressing Breast Cancer Cells to Herceptin.

HER2/neu positive breast tumors predict a high mortality and comprise 25%-30% of breast cancer. We have shown that Flavokawain A (FKA) preferentially reduces the viabilities of HER2-overexpressing breast cancer cell lines (i.e., SKBR3 and MCF7/HER2) versus those with less HER2 expression (i.e., MCF7 and MDA-MB-468). FKA at cytotoxic concentrations to breast cancer cell lines also has a minimal effect on the growth of non-malignant breast epithelial MCF10A cells. FKA induces G2M arrest in cell cycle progression of HER2-overexpressing breast cancer cell lines through inhibition of Cdc2 and Cdc25C phosphorylation and downregulation of expression of Myt1 and Wee1 leading to increased Cdc2 kinase activities. In addition, FKA induces apoptosis in SKBR3 cells by increasing the protein expression of Bim and BAX and decreasing expression of Bcl2, BclX/L, XIAP, and survivin. FKA also downregulates the protein expression of HER-2 and inhibits AKT phosphorylation. Herceptin plus FKA treatment leads to an enhanced growth inhibitory effect on HER-2 overexpressing breast cancer cell lines through downregulation of Myt1, Wee1, Skp2, survivin, and XIAP. Our results suggest FKA as a promising and novel apoptosis inducer and G2 blocking agent that, in combination with Herceptin, enhances for the treatment of HER2-overexpressing breast cancer.

Expression Patterns of the Wnt Pathway Inhibitors Dickkopf3 and Secreted Frizzled-Related Proteins 1 and 4 in Endometrial Endometrioid Adenocarcinoma: An NRG Oncology/Gynecologic Oncology Group Study.

OBJECTIVE: The aim of the study was to determine the differential expression patterns of the wingless-type (Wnt) pathway inhibitors Dkk3 (Dickkopf 3), SFRP1 (secreted frizzled-related protein 1), and SFRP4 in normal müllerian tissue and endometrial endometrioid adenocarcinoma specimens. METHODS: Messenger RNA (mRNA) and protein levels of the Wnt pathway inhibitors Dkk3, SFRP1, and SFRP4 were evaluated by real-time reverse transcription-polymerase chain reaction and Western blot analysis. A total of 87 human tissue specimens were obtained from 60 women who participated in Gynecologic Oncology Group protocol 210. Twenty-seven normal müllerian tissues, 32 early-stage, and 28 advanced-stage endometrial endometrioid cancer specimens were analyzed. RESULTS: Median age for this cohort was 60 years, with median body mass index of 32 kg/m. There was a difference in Dkk3 protein expression between normal müllerian tissues and primary endometrial endometrioid adenocarcinoma samples (P = 0.05). There was down-regulation of Dkk3, SFRP1, and SFRP4 mRNA expression in patients with high-grade disease (P = 0.08, 0.06, and 0.05, respectfully). Furthermore, a decrease in SFRP1 and SFPR4 mRNA expression was noted in patients with a diagnosis of locoregional and distant disease recurrence. Lastly, a trend toward decreased progression-free survival in patients with low Dkk3, SFRP1, and SFRP4 mRNA expression levels was noted. CONCLUSIONS: Wnt pathway inhibitor (Dkk3, sFRP1, and/or sFRP4) expression was down-regulated in patients with high-grade disease and was associated with locoregional and distant disease recurrence. Despite sample size (power) limitations, these results support previous preclinical studies and may suggest a therapeutic role for Wnt signaling in endometrial cancer.

Transcriptional Profiling Identifies the Signaling Axes of IGF and Transforming Growth Factor-b as Involved in the Pathogenesis of Osteosarcoma.

BACKGROUND: Osteosarcoma is the most common primary bone tumor in adolescents associated with skeletal development. The molecular pathogenesis of osteosarcoma has not been completely determined, although many molecular alterations have been found in human osteosarcomas and cell lines. QUESTIONS/PURPOSES: We questioned whether (1) we could identify gene expression in osteosarcoma specimens that differs from normal osteoblasts and mesenchymal stem cells and (2) this would provide clues to the molecular pathogenesis of osteosarcoma? METHODS: The whole-genome transcriptional profiles of osteosarcomas, including two primary biopsy specimens, two cell lines, two xenografts derived from patient specimens, and one from normal osteoblasts and from mesenchymal stem cells, respectively, were quantitatively measured using serial analysis of gene expression. A statistical enrichment was performed, which selects the common genes altered in each of the osteosarcomas compared with each of the normal counterparts independently. RESULTS: Sixty (92%) of 65 total genes that were at least twofold downregulated in osteosarcoma compared with osteoblasts and mesenchymal stem cells, could be classified in four categories: (1) seven genes in the insulin­like growth factor (IGF) signaling axis, including three of the IGF-binding proteins (IGFBP) and three of the IGFBPrelated proteins (IGFBPrP); (2) eight genes in the transforming growth factor-b (TGF-b)/bone morphogenetic protein (BMP) signaling cascade; (3) 39 genes encoding cytoskeleton and extracellular matrix proteins that are regulated by TGF-b/BMPs; and (4) six genes involved in cell cycle regulation, including tumor suppressors TP63 and p21. CONCLUSIONS: Based on these transcriptional profiles, a coordinated theme of clustered gene deregulation in osteosarcoma has emerged. Cell proliferation driven by the IGF axes during bone growth is unrestrained owing to downregulation of IGFBPs and cell cycle regulators. Tumor cells may be maintained in an undifferentiated state secondary to impaired TGF-b/BMP signaling. This wellpreserved pattern suggests that the alterations in the signaling axes of IGF-1 and TGF-b, in concert with cell cycle regulators, may be an important pathogenic basis of osteosarcoma. CLINIC RELEVANCE: This study provides a possible molecular basis of pathogenesis of osteosarcoma. This may help to develop new therapeutic targets and strategy for this disease. Preclinical and subsequently clinical testing of inhibitors of the IGF-1 and TGF pathways would be warranted.

Neuropilin-2 expression is inhibited by secreted Wnt antagonists and its down-regulation is associated with reduced tumor growth and metastasis in osteosarcoma.

BACKGROUND: Neuropilin 2 (NRP2) isa multi-functional co-receptor to many receptors, including VEGF receptor, c-Met and others. NRP2 has recently been implicated in tumor angiogenesis, growth, and metastasis of many other cancers. However, its role in osteosarcoma remains poorly understood. RESULTS: NRP2 was overexpressed in osteosarcoma cell lines and tissues, and associated with poor survival of osteosarcoma patients. Knockdown of NRP2 expression by short-hairpin (Sh) RNA resulted in reduced tumor growth, metastasis, and blood vessel formation of osteosarcoma. Knockdown of NRP2 expression by ShRNA also inhibited the recruitment of HUVEC cells to osteosarcoma cells. Inhibition of Wnt signaling by overexpression of secreted Wnt antagonists soluble LRP5, Frzb, and WIF1 markedly down-regulated mRNA and protein expression of NRP2 in osteosarcoma cell lines. CONCLUSIONS: Regulation of NRP2 receptor expression may represent a novel approach for treatment of osteosarcoma through retarding osteosarcoma growth, metastasis and blood vessel formation. In addition, down-regulation of NRP2 expression can be achieved by expression of secreted Wnt antagonists.

Surgical techniques for spinopelvic reconstruction following total sacrectomy: a systematic review.

PURPOSE: To identify all available reconstruction methods for a total sacrectomy. Secondarily, we aimed to evaluate outcomes based on different interventions. METHODS: We searched PubMed to identify sacral resections for tumors requiring internal fixation for stabilization. Demographic information, fixation techniques and postoperative outcomes were abstracted. RESULTS: Twenty-three publications (43 patients) met inclusion criteria from an initial search of 856 (κ 0.93). Mean age was 37 years and follow-up was 33 months. Fixation methods included a combination of spinopelvic fixation (SPF), posterior pelvic ring fixation (PPRF), and/or anterior spinal column fixation (ASCF). For the purposes of analysis, patients were segregated based on whether they received ASCF. Postoperative complications including wound/instrument infections, GI or vascular complications were reported at a higher rate in the non-ASCF group (1.63 complications/patient vs. 0.7 complications/patient). Instrument failure was seen in 5 (16.1 %) out of the 31 patients with reported outcomes. Specifically, 1 out of 8 patients (12.5 %) with ASCF compared with 4 out of 23 patients (17.4 %) without ASCF had hardware failure. At final follow-up, 35 of 39 patients were ambulating. CONCLUSION: While surgical treatment of primary sacral tumors remains a challenge, there have been advances in reconstruction techniques following total sacrectomy. SPF has shifted from intrapelvic rod and hook constructs to pedicle and iliac screw-rod systems for improved rigidity. PPRF and ASCF have adapted for deficiencies in the posterior ring and anterior column. A trend toward a lower rate of hardware failure emerged in the group utilizing anterior spinal column support. Despite a more involved reconstruction with ASCF, surgical complications such as infection rates and blood loss were lower compared to the group without ASCF. While we cannot definitively say one system is superior to the other, based on the data gleaned from this systematic review, it is our opinion that incorporation of ASCF in reconstructing the spinopelvic junction may lead to improved outcomes. However, most importantly, we recommend that the treating surgeon operate on patients requiring a total sacrectomy based on his or her level of comfort, as these cases can be extremely challenging even among experts.

Wnt signaling in osteosarcoma.

Osteosarcoma (OS) is the most common primary bone malignancy diagnosed in children and adolescents with a high propensity for local invasion and distant metastasis. Despite current multidisciplinary treatments, there has not been a drastic change in overall prognosis within the last two decades. With current treatments, 60-70 % of patients with localized disease survive. Given a propensity of Wnt signaling to control multiple cellular processes, including proliferation, cell fate determination, and differentiation, it is a critical pathway in OS disease progression. At the same time, this pathway is extremely complex with vast arrays of cross-talk. Even though decades of research have linked the role of Wnt to tumorigenesis, there are still outstanding areas that remain poorly understood and even controversial. The canonical Wnt pathway functions to regulate the levels of the transcriptional co-activator ß-catenin, which ultimately controls key developmental gene expressions. Given the central role of this mediator, inhibition of Wnt/ß-catenin signaling has been investigated as a potential strategy for cancer control. In OS, several secreted protein families modulate the Wnt/ß-catenin signaling, including secreted Frizzled-related proteins (sFRPs), Wnt inhibitory protein (WIF), Dickkopf proteins (DKK-1,2,3), sclerostin, and small molecules. This chapter focuses on our current understanding of Wnt/ß-catenin signaling in OS, based on recent in vitro and in vivo data. Wnt activates noncanonical signaling pathways as well that are independent of ß-catenin which will be discussed. In addition, stem cells and their association with Wnt/ß-catenin are important factors to consider. Ultimately, the multiple canonical and noncanonical Wnt/ß-catenin agonists and antagonists need to be further explored for potential targeted therapies.

Can surgical management of bone metastases improve quality of life among women with gynecologic cancer?

BACKGROUND: The evaluation, counseling, and management of gynecologic patients with bone metastasis remain a challenge for clinicians. In order to critically evaluate the role of surgery, we retrospectively analyzed the records of 18 patients surgically treated for metastatic gynecologic tumors of bone, focusing on quality of life, local tumor control, and survival. METHODS: Eighteen patients underwent surgical procedures for the treatment of bone metastases secondary to gynecologic cancer between September 2003 and April 2012. The primary cancer sites included the uterus (n = 10), the cervix (n = 5), and an ovary (n = 3). Patients were followed for an average period of 13.8 months (range, 2 to 34 months). A visual analog pain scale (VAS) and Eastern Cooperative Oncology Group (ECOG) performance status were evaluated both pre- and postoperatively. RESULTS: The median survival time following diagnosis of bone metastasis was 10.0 months. The mean VAS score was 5.8 preoperatively compared with 2.1, 3 months after surgery. The mean pre and postoperative ECOG performance status grades were 3.1 and 2.3, respectively. CONCLUSIONS: The prognosis of gynecological cancer patients with bone metastasis is poor. Some patients had improvement in their quality of life after surgical intervention for bone metastases; however, novel integrated treatment modalities should be investigated.

SKP2 Knockout in Rb1/p53-Deficient Mouse Models of Osteosarcoma Induces Immune Infiltration and Drives a Transcriptional Program with a Favorable Prognosis.

Osteosarcoma is an aggressive bone malignancy with a poor prognosis. One putative proto-oncogene in osteosarcoma is SKP2, encoding a substrate recognition factor of the SCF E3 ubiquitin ligase. We previously demonstrated that Skp2 knockout in murine osteosarcoma improved survival and delayed tumorigenesis. Here, we performed RNA sequencing (RNA-seq) on tumors from a transgenic osteosarcoma mouse model with conditional Trp53 and Rb1 knockouts in the osteoblast lineage ("DKO": Osx1-Cre;Rb1lox/lox;p53lox/lox) and a triple-knockout model with additional Skp2 germline knockout ("TKO": Osx1-Cre;Rb1lox/lox;p53lox/lox;Skp2-/-), followed by qPCR and immunohistochemistry validation. To investigate the clinical implications of our results, we analyzed a human osteosarcoma patient cohort ("NCI-TARGET OS") with RNA-seq and clinical data. We found large differences in gene expression after SKP2 knockout. Surprisingly, we observed increased expression of genes related to immune microenvironment infiltration in TKO tumors, especially the signature genes for macrophages and to a lesser extent, T cells, B cells, and vascular cells. We also uncovered a set of relevant transcription factors that may mediate these changes. In osteosarcoma patient cohorts, high expression of genes upregulated in TKO was correlated with favorable overall survival, which was largely explained by the macrophage gene signatures. This relationship was further supported by our finding that SKP2 expression was negatively correlated with macrophage infiltration in the NCI-TARGET osteosarcoma and the TCGA Sarcoma cohorts. Overall, our findings indicate that SKP2 may mediate immune exclusion from the osteosarcoma tumor microenvironment, suggesting that SKP2 modulation in osteosarcoma may induce antitumor immune activation.

Targeted inhibition of SCFSKP2 confers anti-tumor activities resulting in a survival benefit in osteosarcoma.

Osteosarcoma(OS) is a highly aggressive bone cancer for which treatment has remained essentially unchanged for decades. Although OS is characterized by extensive genomic heterogeneity and instability, RB1 and TP53 have been shown to be the most commonly inactivated tumor suppressors in OS. We previously generated a mouse model with a double knockout (DKO) of Rb1 and Trp53 within cells of the osteoblastic lineage, which largely recapitulates human OS with nearly complete penetrance. SKP2 is a repression target of pRb and serves as a substrate recruiting subunit of the SCFSKP2 complex. In addition, SKP2 plays a central role in regulating the cell cycle by ubiquitinating and promoting the degradation of p27. We previously reported the DKOAA transgenic model, which harbored a knock-in mutation in p27 that impaired its binding to SKP2. Here, we generated a novel p53-Rb1-SKP2 triple-knockout model (TKO) to examine SKP2 function and its potential as a therapeutic target in OS. First, we observed that OS tumorigenesis was significantly delayed in TKO mice and their overall survival was markedly improved. In addition, the loss of SKP2 also promoted an apoptotic microenvironment and reduced the stemness of DKO tumors. Furthermore, we found that small-molecule inhibitors of SKP2 exhibited anti-tumor activities in vivo and in OS organoids as well as synergistic effects when combined with a standard chemotherapeutic agent. Taken together, our results suggest that SKP2 inhibitors may reduce the stemness plasticity of OS and should be leveraged as next-generation adjuvants in this cancer.

Flavokawain B, a kava chalcone, inhibits growth of human osteosarcoma cells through G2/M cell cycle arrest and apoptosis.

BACKGROUND: Osteosarcoma (OS) is the most common primary bone malignancy with a high propensity for local invasion and distant metastasis. Limited by the severe toxicity of conventional agents, the therapeutic bottleneck of osteosarcoma still remains unconquered. Flavokawain B (FKB), a kava extract, has been reported to have significant anti-tumor effects on several carcinoma cell lines both in vitro and in vivo. Its efficacy and low toxicity profile make FKB a promising agent for use as a novel chemotherapeutic agent. RESULTS: In the current study, we investigated the anti-proliferative and apoptotic effects of FKB against human osteosarcomas. Exposure of OS cells to FKB resulted in apoptosis, evidenced by loss of cell viability, morphological changes and the externalization of phosphatidylserine. Apoptosis induced by FKB resulted in activation of Caspase-3/7, -8 and -9 in OS cell lines, 143B and Saos-2. FKB also down-regulated inhibitory apoptotic markers, including Bcl-2 and Survivin and led to concomitant increases in apoptotic proteins, Bax, Puma and Fas. Therefore, the induction of apoptosis by FKB involved both extrinsic and intrinsic pathways. FKB also caused G2/M phase cell cycle arrest, which was observed through reductions in the levels of cyclin B1, cdc2 and cdc25c and increases in Myt1 levels. Furthermore, migration and invasion ability was decreased by FKB in a dose-dependent manner. The cytotoxicity profile showed FKB had significant lower side effects on bone marrow cells and small intestinal epithelial cells compared with Adriamycin. CONCLUSIONS: Taken together, our evidence of apoptosis and cell cycle arrest by FKB treatment with less toxicity than the standard treatments provides an innovative argument for the use of FKB as a chemotherapeutic and chemopreventive compound. In vivo experiments utilizing FKB to reduce tumorigenesis and metastatic potential will be crucial to further justify clinical application.

Prognostic and Therapeutic Utility of Variably Expressed Cell Surface Receptors in Osteosarcoma.

BACKGROUND: Six cell surface receptors, human epidermal growth factor receptor-2 (Her-2), platelet-derived growth factor receptor-ß (PDGFR-ß), insulin-like growth factor-1 receptor (IGF-1R), insulin receptor (IR), c-Met, and vascular endothelial growth factor receptor-3 (VEGFR-3), previously demonstrated variable expression across varying patient-derived and standard osteosarcoma (OS) cell lines. The current study sought to validate previous expression patterns and evaluate whether these receptors offer prognostic and/or therapeutic value. METHODS: Patient-derived OS cell lines (n = 52) were labeled with antibodies to Her-2, PDGFR-ß, IGF-1R, IR, c-Met, and VEGFR-3. Expression was characterized using flow cytometry. The difference in geometric mean fluorescent intensity (geoMFIdiff = geoMFIpositive - geoMFInegative) was calculated for each receptor across all cell lines. Receptor expression was categorized as low (Q1), intermediate (Q2, Q3), or high (Q4). The event-free survival (EFS) and overall survival for the six cell surface receptors were estimated by the Kaplan-Meier method. Differences in hazard for EFS event and overall survival event for patients in each of the three expression levels in each of the six cell surface receptors were assessed using the log-rank test. RESULTS: All 6 receptors were variably expressed in the majority of cell lines. IR and PDGFR-ß expressions were found to be significant predictors for EFS amongst patients with nonmetastatic disease (p=0.02 and 0.01, respectively). The hazard ratio for EFS was significantly higher between high IR and intermediate IR expression (HR = 2.66, p=0.02), as well as between high PDGFR-ß and intermediate PDGFR-ß expression (HR = 5.68, p=0.002). Her-2, c-Met, IGF-1R, and VEGFR-3 were not found to be significant predictors for either EFS or overall survival. CONCLUSION: The six cell surface receptors demonstrated variable expression across the majority of patient-derived OS cell lines tested. Limited prognostic value was offered by IR and PDGFR-ß expression within nonmetastatic patients. The remaining receptors do not provide clear prognostic utility. Nevertheless, their consistent, albeit variable, surface expression across a large panel of patient-derived OS cell lines maintains their potential use as future therapeutic targets.

The interaction of SKP2 with p27 enhances the progression and stemness of osteosarcoma.

Osteosarcoma is a highly aggressive malignancy for which treatment has remained essentially unchanged for years. Our previous studies found that the F-box protein SKP2 is overexpressed in osteosarcoma, acting as a proto-oncogene; p27Kip1 (p27) is an inhibitor of cyclin-dependent kinases and a downstream substrate of SKP2-mediated ubiquitination. Overexpression of SKP2 and underexpression of p27 are common characteristics of cancer cells. The SCFSKP2 E3 ligase ubiquitinates Thr187-phosphorylated p27 for proteasome degradation, which can be abolished by a Thr187Ala knock-in (p27T187A KI) mutation. RB1 and TP53 are two major tumor suppressors commonly coinactivated in osteosarcoma. We generated a mouse model with a double knockout (DKO) of Rb1 and Trp53 within cells of the osteoblastic lineage, which developed osteosarcoma with full penetrance. When p27T187A KI mice were crossed on to the DKO background, p27T187A protein was found to accumulate in osteosarcoma tumor tissues. Furthermore, p27T187A promoted apoptosis in DKO tumors, slowed disease progression, and significantly prolonged overall survival. RNA sequencing analysis also linked the SCFSKP2 -p27T187A axis to potentially reduced cancer stemness. Given that RB1 and TP53 loss or coinactivation is common in human osteosarcoma, our study suggests that inhibiting the SKP2-p27 axis may represent a desirable therapeutic strategy for this cancer.

The Wnt inhibitory factor 1 restoration in prostate cancer cells was associated with reduced tumor growth, decreased capacity of cell migration and invasion and a reversal of epithelial to mesenchymal transition.

BACKGROUND: Aberrations in the Wnt pathway have been reported to be involved in the metastasis of prostate cancer (PCa) to bone. We investigated the effect and underlying mechanism of a naturally-occurring Wnt inhibitor, WIF1, on the growth and cellular invasiveness of a bone metastatic PCa cell line, PC3. RESULTS: The WIF1 gene promoter was hypermethylated and its expression down-regulated in the majority (7 of 8) of PCa cell lines. Restoration of WIF1 expression in PC-3 cells resulted in a decreased cell motility and invasiveness via up-regulation of epithelial markers (E-cadherin, Keratin-8 and-18), down-regulation of mesenchymal markers (N-cadherin, Fibronectin and Vimentin) and decreased activity of MMP-2 and -9. PC3 cells transfected with WIF1 consistently demonstrated reduced expression of Epithelial-to-Mesenchymal Transition (EMT) transcription factors, Slug and Twist, and a change in morphology from mesenchymal to epithelial. Moreover, WIF1 expression significantly reduced tumor growth by approximately 63% in a xenograft mouse model. This was accompanied by an increased expression of E-cadherin and Keratin-18 and a decreased expression of vimentin in tumor tissues. CONCLUSION: These data suggest that WIF1 regulates tumor invasion through EMT process and thus, may play an important role in controlling metastatic disease in PCa patients. Blocking Wnt signaling in PCa by WIF1 may represent a novel strategy in the future to reduce metastatic disease burden in PCa patients.

Bone void fillers.

For this technology overview, the tools of evidence-based medicine were used to summarize information on the effectiveness and clinical outcomes related to the usage of bone void fillers- specifically, synthetic graft materials. Comprehensive literature searches were conducted to address five key questions, which the task force that prepared the report posed as follows. Question 1 addressed the use of synthetic bone void fillers alone. Question 2 was designed to determine whether synthetic bone void fillers could successfully serve as graft extenders and eliminate the need for iliac crest bone graft. Questions 3, 4, and 5 addressed the use of allografts as a comparison with synthetic fillers because clinical results with allografts are perceived as being much closer to autografts in these areas of the spine.

WIF1, a Wnt pathway inhibitor, regulates SKP2 and c-myc expression leading to G1 arrest and growth inhibition of human invasive urinary bladder cancer cells.

Epigenetic silencing of secreted wingless-type (Wnt) antagonists through hypermethylation is associated with tobacco smoking and with invasive bladder cancer. The secreted Wnt inhibitory factor-1 (WIF1) has shown consistent growth-inhibitory effect on various cancer cell lines. Therefore, we assessed the mechanisms of action of WIF1 by either restoring WIF1 expression in invasive bladder cancer cell lines (T24 and TSU-PR1) or using a recombinant protein containing functional WIF1 domain. Both ectopic expression of WIF1 and treatment with WIF1 domain protein resulted in cell growth inhibition via G(1) arrest. The G(1) arrest induced by WIF1 is associated with down-regulation of SKP2 and c-myc and up-regulation of p21/WAF1 and p27/Kip1. Conversely, reexpression of SKP2 in WIF1-overexpressing TSU-PR1 cells attenuated the WIF1-induced G(1) arrest. Furthermore, inhibition of nuclear Wnt signaling by either dominant-negative LEF1 or short hairpin RNA of TCF4 also reduced SKP2 expression. The human SKP2 gene contains two TCF/LEF1 consensus binding sites within the promoter. Chromatin immunoprecipitation/real-time PCR analysis revealed that both WIF1 and dominant-negative LEF1 expression decreased the in vivo binding of TCF4 and beta-catenin to the SKP2 promoter. Together, our results suggest that mechanisms of WIF1-induced G(1) arrest include (a) SKP2 down-regulation leading to p27/Kip1 accumulation and (b) c-myc down-regulation releasing p21/WAF1 transcription. Additionally, we show that WIF1 inhibits in vivo bladder tumor growth in nude mice. These observations suggest a mechanism for transformation of bladder epithelium on loss of WIF1 function and provide new targets such as SKP2 for intervention in WIF1-deficient bladder cancer.