Albert Yee

Versican 3′-untranslated region (3′-UTR) functions as a ceRNA in inducing the development of hepatocellular carcinoma by regulating miRNA activity

This study was designed to explore the role of versican in the development of hepatocellular carcinoma (HCC). Ectopic expression of the versican 3′‐untranslated region (3′‐UTR) was studied as a competitive endogenous RNA for regulating miRNA functions. We used this approach to modulate the expression of versican and its related proteins in 3′‐UTR transgenic mice and in the liver cancer cell line HepG2, stably transfected with the 3′‐UTR or a control vector. We demonstrated that transgenic mice expressing the versican 3′‐UTR developed HCC and increased expression of versican isoforms V0 and V1. HepG2 cells transfected with versican 3′‐UTR displayed increased proliferation, survival, migration, invasion, colony formation, and enhanced endothelial cell growth, but decreased apoptosis. We found that versican 3′‐UTR could bind to miRNAs miR‐133a, miR‐199a*, miR‐144, and miR‐431 and also interacted with CD34 and fibronectin. As a consequence, expression of versican, CD34, and fibronectin was up‐regulated by ectopic transfection of the versican 3′‐UTR, which was confirmed in HepG2 cells and in transgenic mice as compared with wild‐type controls. Transfection with siRNAs targeting the versican 3′‐UTR abolished the effects of the 3′‐UTR. Taken together, these results demonstrate that versican V0 and V1 isoforms play important roles in HCC development and that versican mRNAs compete with endogenous RNAs in regulating miRNA functions.—Fang, L., Du, W. W., Yang, X., Chen, K., Ghanekar, A., Levy, G., Yang, W., Yee, A. J., Lu, W.‐Y., Xuan, J. W., Gao, Z., Xie, F., He, C., Deng, Z., Yang, B. B. Versican 3′‐untranslated region (3′‐UTR) functions as a ceRNA in inducing the development of hepatocellular carcinoma by regulating miRNA activity. FASEB J. 27, 907–919 (2013). www.fasebj.org

MicroRNA miR-24 enhances tumor invasion and metastasis by targeting PTPN9 and PTPRF to promote EGF signaling

Summary MicroRNAs are known to play regulatory roles in gene expression associated with cancer development. We analyzed levels of the microRNA miR-24 in patients with breast carcinoma and found that miR-24 was higher in breast carcinoma samples than in benign breast tissues. We generated constructs expressing miR-24 and studied its functions using both in vitro and in vivo techniques. We found that the ectopic expression of miR-24 promoted breast cancer cell invasion and migration. In vivo experiments in mice indicated that the expression of miR-24 enhanced tumor growth, invasion into local tissues, metastasis to lung tissues and decreased overall mouse survival. In the miR-24-expressing cells and tumors, EGFR was highly phosphorylated, whereas expression of the phosphatases tyrosine-protein phosphatase non-receptor type 9 (PTPN9) and receptor-type tyrosine-protein phosphatase F (PTPRF) were repressed. We confirmed that miR-24 could directly target both PTPN9 and PTPRF. Consistent with this, we found that the levels of phosphorylated epidermal growth factor receptor (pEGFR) were higher whereas the levels of PTPN9 and PTPRF were lower in the patients with metastatic breast carcinoma. Ectopic expression of PTPN9 and PTPRF decreased pEGFR levels, cell invasion, migration and tumor metastasis. Furthermore, we found that MMP2, MMP11, pErk, and ADAM15 were upregulated, whereas TIMP2 was downregulated; all of which supported the roles of miR-24 in tumor invasion and metastasis. Our results suggest that miR-24 plays a key role in breast cancer invasion and metastasis. miR-24 could potentially be a target for cancer intervention.

The European Organisation for Research and Treatment of Cancer Quality of Life Questionnaire for patients with bone metastases: the EORTC QLQ-BM22

AIM The aim of this study was to develop a bone metastases module to supplement the European Organisation for Research and Treatment of Cancer Core Questionnaire (EORTC QLQ-C30) or the EORTC QLQ-C15-PAL for patients with bone metastases. METHODS Phases 1-2 of module development were conducted in Canada, Australia and Germany according to EORTC QOL group guidelines. Phase 3 was conducted in nine countries in seven languages. RESULTS Sixty-one health-related quality of life (HRQOL) issues were generated from health care professionals (n=152) and patients (n=413). This resulted in a 22-item provisional module. Further testing in 170 patients from nine countries resulted in the EORTC QLQ-BM22 module, containing 22 items, conceptualised into both symptom scales, with five painful sites and three pain characteristics, and also functional scales, with eight functional interference and six psychosocial aspects. CONCLUSION This study provides a provisional comprehensive HRQOL measurement tool for future trials, which will continue to undergo further validation.

The effect of versican G3 domain on local breast cancer invasiveness and bony metastasis

IntroductionIncreased versican expression has been associated with local breast cancer invasiveness and a more aggressive tumor phenotype. The cellular mechanisms are not fully understood and this study evaluated versican G3 domain with its EGF-like motifs in influencing tumor invasion and metastasis.MethodsOne recombinant construct was synthesized (a signal peptide for product secretion and the versican G3 domain). The construct was stably transfected into human breast carcinoma MT-1 cells. Cell viability in vitro was evaluated in low serum and serum starvation conditions. In vivo study of tumor growth was evaluated in a nude mouse model. G3 effects on rodent vascular endothelial cells were evaluated in vitro on cell survival, apoptosis, migration, and vascular formation. The effects of VEGF, fibronectin, and G3 on vascular formation were examined. An intracardiac injection model of metastatic human breast carcinoma tested the effect of G3 on distant bony and soft tissue metastasis. Analysis of metastatic burden included histology, radiographs, and micro-CT quantification of osteolysis.ResultsA greater viability of cancer cells was observed in low serum and serum-free conditions in the presence of versican G3. Larger subcutaneous tumors were obtained in the G3 group following tumor cell injection into CD1 mice. G3 induced a greater degree of rodent vascular endothelial cell proliferation and migration in vitro. Simultaneous presence of fibronectin, VEGF, and G3 promoted endothelial cell migration in wound-healing assays as compared to the treatments containing none, one or two of these molecules. Systemic tumor burden to distant bony and soft tissue metastatic sites was greater in the G3 group using the intracardiac injection metastatic modelConclusionVersican G3 domain appears to be important in local and systemic tumor invasiveness of human breast cancer. Effects include enhancing cell viability, proliferation, migration and enhancing local tumor growth. Potential effects on angiogenesis include enhancing vascular endothelial proliferation, migration, and vessel formation. The interactions between tumor cells, surrounding stromal components and neo-vascularization in breast cancer may include interactions with VEGF and fibronectin. The propensity of versican G3 to influence tumor invasion to bone and the mechanisms of G3 mediated osteolysis warrants ongoing studies.

Versican G3 Promotes Mouse Mammary Tumor Cell Growth, Migration, and Metastasis by Influencing EGF Receptor Signaling

Increased versican expression in breast tumors is predictive of relapse and has negative impact on survival rates. The C-terminal G3 domain of versican influences local and systemic tumor invasiveness in pre-clinical murine models. However, the mechanism(s) by which G3 influences breast tumor growth and metastasis is not well characterized. Here we evaluated the expression of versican in mouse mammary tumor cell lines observing that 4T1 cells expressed highest levels while 66c14 cells expressed low levels. We exogenously expressed a G3 construct in 66c14 cells and analyzed its effects on cell proliferation, migration, cell cycle progression, and EGFR signaling. Experiments in a syngeneic orthotopic animal model demonstrated that G3 promoted tumor growth and systemic metastasis in vivo. Activation of pERK correlated with high levels of G3 expression. In vitro, G3 enhanced breast cancer cell proliferation and migration by up-regulating EGFR signaling, and enhanced cell motility through chemotactic mechanisms to bone stromal cells, which was prevented by inhibitor AG 1478. G3 expressing cells demonstrated increased CDK2 and GSK-3β (S9P) expression, which were related to cell growth. The activity of G3 on mouse mammary tumor cell growth, migration and its effect on spontaneous metastasis to bone in an orthotopic model was modulated by up-regulating the EGFR-mediated signaling pathway. Taken together, EGFR-signaling appears to be an important pathway in versican G3-mediated breast cancer tumor invasiveness and metastasis.

Ergosterol peroxide isolated from Ganoderma lucidum abolishes microRNA miR-378-mediated tumor cells on chemoresistance.

Due to an altered expression of oncogenic factors and tumor suppressors, aggressive cancer cells have an intrinsic or acquired resistance to chemotherapeutic agents. This typically contributes to cancer recurrence after chemotherapy. microRNAs are short non-coding RNAs that are involved in both cell self-renewal and cancer development. Here we report that tumor cells transfected with miR-378 acquired properties of aggressive cancer cells. Overexpression of miR-378 enhanced both cell survival and colony formation, and contributed to multiple drug resistance. Higher concentrations of chemotherapeutic drugs were needed to induce death of miR-378-transfected cells than to induce death of control cells. We found that the biologically active component isolated from Ganoderma lucidum could overcome the drug-resistance conferred by miR-378. We purified and identified the biologically active component of Ganoderma lucidum as ergosterol peroxide. We demonstrated that ergosterol peroxide produced greater activity in inducing death of miR-378 cells than the GFP cells. Lower concentrations of ergosterol peroxide were needed to induce death of the miR-378-transfected cells than in the control cells. With further clinical development, ergosterol peroxide represents a promising new reagent that can overcome the drug-resistance of tumor cells.

Traditional assessment of health outcome following total knee arthroplasty was confounded by response shift phenomenon.

OBJECTIVES To examine the existence, direction, and effect of response shift as measured by the total score of Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) and SF-36 physical and mental component score domains at 6 months and 1 year after primary total knee arthroplasty (TKA). STUDY DESIGN AND SETTING Consecutive candidates participated in the study. In line with the Then-test design, two sets of questionnaires were completed at 6 months and 1 year postoperatively. Patients were divided into three groups on the basis of minimal clinically important differences (MCIDs). Parametric and nonparametric statistics and generalized mixed effects models were used. RESULTS Two hundred and thirty-six patients completed the study. The magnitude of response shift increased over time for SF-36 physical and mental component scores. The traditional unadjusted presurgical and 1-year postsurgical assessment failed to reveal any mental health improvement, whereas the adjusted treatment effect demonstrated statistically significant changes. CONCLUSION Response shift phenomenon increases with time in patients undergoing orthopedic interventions. The traditional pre- and post-assessment of joint replacement surgery may be confounded by a change in perspective and in internal standards of measurement in patients undergoing surgery. Response shift has substantial impact on measuring recovery in this population.

Insulin like growth factor binding protein-7 reduces growth of human breast cancer cells and xenografted tumors

Previously, we have shown that insulin-like growth factor binding protein-7 (IGFBP-7) expression is inversely correlated with disease progression in breast cancer and is associated with poor outcome. To further investigate the role of IGFBP-7 in the growth and metastatic behavior of breast cancer, primary breast tumors and metastatic tumors derived from the same patients were analyzed for IGFBP-7 expression. Immunohistochemical analysis revealed that IGFBP-7 is downregulated in half of the human metastatic breast tumors tested. IGFBP-7 has been linked to suppression of oncogenic pathways and can directly restore cellular senescence in melanomas, leading to their regression. It is possible that breast tumors with metastatic potential have escaped from IGFBP-7-induced suppression by its down-regulation. Twenty-two human primary breast tumor specimens were transplanted into human-bone NOD/SCID mice. One of the two triple negative primary breast tumors was serially xenotransplanted more than five times. Each serial transplant resulted in increased tumor take and rate of growth. Expression of IGFBP-7 was downregulated upon each serial implantation. To investigate the role of IGFBP-7 in breast tumor suppression, IGFBP-7 was overexpressed in the triple negative MDA-MB-468 human breast cancer line by stable transfection of a pSec-tag2-IGFBP-7 vector. The parental MDA-MB-468 breast cancer cells expressed extremely low levels of endogenous IGFBP-7. The production of IGFBP-7 protein by the MDA-MB-468 cells stably transfected with IGFBP-7 was confirmed by immunoblotting with anti-IGFBP-7 antibody. Ectopic overexpression of IGFBP-7 significantly reduced the growth of the IGFBP-7 transfected MDA-MB-468 cells compared to the parental MDA-MB-468 cells. We also assessed the role of IGFBP-7 on cell migration, a key determinant of malignant progression and metastasis. When parental MDA-MB-468 cells were treated with various amounts of conditioned medium derived from the IGFBP-7 overexpressing cell line, a significant difference in cell migration rate was observed between untreated and treated cells. IGFBP-7 strongly suppressed the phosphorylation of the mitogen-activated protein kinases (MAPK) ERK-1/2, suggesting that IGFBP-7 mediates its anti-proliferative effects through negative feedback signaling. Levels of phospho-ERK-1/2 were higher in the parental MDA-MB-468 than in IGFBP-7-expressing cells derived from it. When injected subcutaneously into NOD/SCID mice, the increased expression of IGFBP-7 in the MDA-MB-468 transfected cells reduced the rate of tumor growth in comparison to the parental MDA-MB-468 controls. These results suggest that the growth of breast cancer could be prevented by the forced expression of IGFBP-7 protein.

Postoperative improvement in health-related quality of life: a national comparison of surgical treatment for focal (one- to two-level) lumbar spinal stenosis compared with total joint arthroplasty for osteoarthritis

BACKGROUND CONTEXT The results of single-center studies have shown that surgical intervention for lumbar spinal stenosis yielded comparable health-related quality of life (HRQoL) improvement to total joint arthroplasty (TJA). Whether these results are generalizable to routine clinical practice in Canada is unknown. PURPOSE The primary purpose of this equivalence study was to compare the relative improvement in physical HRQoL after surgery for focal lumbar spinal stenosis (FLSS) compared with TJA for hip and knee osteoarthritis (OA) across six Canadian centers. STUDY DESIGN/SETTING A Canadian multicenter ambispective cohort study. PATIENT SAMPLE A cohort of 371 primary one- to two-level spinal decompression (n=214 with instrumented fusion) for FLSS (n=179 with degenerative lumbar spondylolisthesis [DLS]) was compared with a cohort of primary total hip (n=156) and knee (n=208) arthroplasty for OA. OUTCOME MEASURES The primary outcome was the change in preoperative to 2-year postoperative 36-Item Short Form Health Survey Physical Component Summary (PCS) score as reflected by the number of patients reaching minimal clinically important difference (MCID) and substantial clinical benefit (SCB). METHODS Univariate analyses were conducted to identify baseline differences and factors that were significantly related to outcomes at 2 years. Multivariable regression modeling was used as our primary analysis to compare outcomes between groups. RESULTS The mean age (years) and percent females for the spine, hip, and knee groups were 63.3/58.5, 66.0/46.9, and 65.8/64.3, respectively. All three groups experienced significant improvement of baseline PCS (p<.001). Multivariate analyses, adjusting for baseline differences (age, gender, baseline Mental Component Summary score, baseline PCS), demonstrated no significant differences in PCS outcome between spinal surgery and arthroplasty (combined hip and knee cohorts) patients with an odds ratio of 0.80 (95% confidence interval [CI], 0.57-1.11; p=.17) and 0.79 (95% CI, 0.58-1.09; p=.15) for achieving MCID or SCB, respectively. In subgroup analysis, spine and knee outcomes were not significantly different, with hip arthroplasty superior to both (p<.0001). CONCLUSIONS Significant improvement in physical HRQoL after surgical treatment of FLSS (including DLS) is consistently achieved nationally. Our overall results demonstrate that a comparable number of patients can expect to achieve MCID and SCB 2 years after surgical intervention for FLSS and total knee arthroplasty.

Versican V2 isoform enhances angiogenesis by regulating endothelial cell activities and fibronectin expression

Versican is a proteoglycan expressed in the extracellular matrix, where it regulates a variety of cell activities and affects tumor development. With alternative splicing, there are four versican isoforms, denoted V0, V1, V2 and V3. The V2 isoform is highly expressed in the mature brain but its function in the mature brain has not yet been elucidated. Since brain tumors are among the most angiogenic of human tumors, we investigated whether or not the V2 isoform plays a role in angiogenesis and found that the glioblastoma cell line U87 stably transfected with V2 formed tumors containing extensive vasculature. Although the V2‐expressing cells grew slowly, they survived well in serum‐free medium. They also displayed high adhesive ability to endothelial cells and facilitated tube‐like structure formation. Importantly, fibronectin was up‐regulated by V2 and mediated V2 function. Thus, versican V2 could be a potential target for intervention of brain tumor angiogenesis.