Bang H. Hoang

Dickkopf 3 Inhibits Invasion and Motility of Saos-2 Osteosarcoma Cells by Modulating the Wnt-β-Catenin Pathway

Osteosarcoma (OS) is a primary malignancy of bone with a tendency to metastasize early. Despite intensive chemotherapy and surgical resection, approximately 30% of patients still develop distant metastasis. Our previous work using clinical OS samples suggested that expression of the Wnt receptor LRP5 might be associated with tumor metastasis. In the present study, we used a Dickkopf (Dkk) family member and a dominant-negative LRP5 receptor construct to modulate Wnt signaling in OS cells. Saos-2 cells, which ectopically express Dkk-3, do not undergo apoptosis and exhibit enhanced resistance to serum starvation and chemotherapy-induced cytotoxicity. Transfection of Dkk-3 and dominant-negative LRP5 into Saos-2 cells significantly reduces invasion capacity and cell motility. This blockade is associated with changes in cell morphology consistent with a less invasive phenotype. In addition, Dkk-3 and dominant-negative LRP5 also induce changes in beta-catenin localization consistent with an increase in cell-cell adhesion. Taken together, these results support a possible role for Wnt signaling in the pathobiology and progression of human OS.

Expression of Dickkopf genes is strongly reduced in malignant melanoma

The Dickkopf (DKK) genes were originally identified as factors inducing head formation in Xenopus. The genes code for inhibitors that are involved in Wnt signaling. We speculate that loss of DKK expression plays a role in development or progression of malignant melanoma. Thus, we evaluated melanoma cell lines and tissue samples of malignant melanoma for loss of DKK, especially DKK-3 transcription. We found that DKK-1, -2 and -3 were downregulated or lost in all cell lines and in most of the tumor samples analysed. Reduced DKK-3 expression occurred as early as in primary tumors detected by both immunohistochemical and reverse transcription–polymerase chain reaction RT–PCR analysis. Functional assays with stable DKK-3 transfected cell lines revealed that DKK-3 expression increased cell-cell adhesion and decreased cell migration. Further, downregulation of fibronectin, snail-1 and re-expression of E-cadherin was found in the DKK-3 expressing cell clones supporting a role of DKK-3 in tumor progression. Our studies thus indicate that loss of DKK-3 expression may contribute to melanoma progression.

Expression of Frzb/Secreted Frizzled-Related Protein 3, a Secreted Wnt Antagonist, in Human Androgen-Independent Prostate Cancer PC-3 Cells Suppresses Tumor Growth and Cellular Invasiveness

The ability of Frzb/secreted Frizzled-related protein 3 (sFRP3) to inhibit Wnt signaling and the localization of Frzb/sFRP3 on chromosome 2q to a region frequently deleted in cancers have led some investigators to hypothesize that Frzb/sFRP3 is a tumor suppressor gene. Here, we examined the biological effects of Frzb/sFRP3 on an androgen-independent prostate cancer cell model. We showed that expression of Frzb/sFRP3 in PC-3 cells resulted in decreased colony formation in soft agar and a dramatic inhibition of tumor growth in a xenograft mouse model. When cellular morphology was examined, PC-3 cells expressing Frzb/sFRP3 exhibited an increase in cell-cell contact formation accompanied by a pronounced induction of epithelial markers E-cadherin and keratin-8 and down-regulation of mesenchymal markers N-cadherin, fibronectin, and vimentin. This phenomenon suggested a reversal of epithelial-to-mesenchymal transition and a less invasive phenotype. Indeed, further in vitro studies with a Matrigel assay showed that Frzb/sFRP3 decreased the invasive capacity of PC-3 cells. These changes in the biology of PC-3 cells are associated with a decrease in the expression and activities of both matrix metalloproteinase (MMP)-2 and MMP-9 as well as decreases in AKT activation, cytosolic beta-catenin levels, T-cell factor transcription activity, and expression of Slug and Twist. In addition, transfection of PC-3 with a dominant-negative low-density lipoprotein receptor-related protein 5 (DN-LRP5) coreceptor showed similar biological effects as Frzb/sFRP3 transfection. Together, these data suggest that Frzb/sFRP3 and DN-LRP5 exhibit antitumor activity through the reversal of epithelial-to-mesenchymal transition and inhibition of MMP activities in a subset of prostate cancer.

Expression of LDL receptor-related protein 5 (LRP5) as a novel marker for disease progression in high-grade osteosarcoma.

The Wingless‐type (Wnt) family of proteins and its coreceptor LRP5 have recently been implicated in human skeletal development. Wnt pathway modulates cell fate and cell proliferation during embryonic development and carcinogenesis through activation of receptor‐mediated signaling. Osteosarcoma (OS) is a bone‐forming tumor of mesenchymal origin whose growth control has been linked to autocrine or paracrine stimulation by several growth factor families. We examined 4 OS cell lines for WNT1, WNT4, WNT5A, WNT7A, WNT11, FZD1‐10 and LRP5 expression by reverse transcription polymerase chain reaction (RT‐PCR). In addition, RT‐PCR for LRP5 expression was performed in 44 OS patient samples and the findings were correlated with clinical data. Expression profiling of Wnts and their receptors revealed the presence of several isoforms in OS cell lines. Overall, 22/44 (50%) of OS patient samples showed evidence of LRP5 expression. Presence of LRP5 correlated significantly with tumor metastasis (p = 0.005) and the chondroblastic subtype of OS (p = 0.045). In addition, patients whose tumors were positive for LRP5 showed a trend toward decreased event‐free survival (p = 0.066). No significant association was found between LRP5 expression and age, gender, site of disease, site of metastasis or degree of chemotherapy‐induced tumor necrosis. Sequencing of exon 3 of LRP5 in 10 OS patient‐derived cell cultures showed no activating mutation of LRP5. These results showed that expression of LRP5 is a common event in OS and strongly suggest a role for LRP and Wnt signaling in the pathobiology and progression of this disease.

Survival in Patients Operated on for Pathologic Fracture: Implications for End-of-Life Orthopedic Care

PURPOSE Life expectancy is routinely used as part of the decision-making process in deciding the value of surgery for the treatment of bone metastases. We sought to investigate the validity of frequently used indices in the prognostication of survival in patients with metastatic bone disease. METHODS The study prospectively assessed 191 patients who underwent surgery for metastatic bone disease. Diagnostic, staging, nutritional, and hematologic parameters cited to be related to life expectancy were evaluated. Preoperatively, the surgeon recorded an estimate of projected life expectancy for each patient. The time until death was recorded. RESULTS Kaplan-Meier survival analyses indicated that the survival estimate, primary diagnosis, use of systemic therapy, Eastern Cooperative Oncology Group (ECOG) performance status, number of bone metastases, presence of visceral metastases, and serum hemoglobin, albumin, and lymphocyte counts were significant for predicting survival (P < .004). Cox regression analysis indicated that the independently significant predictors of survival were diagnosis (P < .006), ECOG performance status (P < .04), number of bone metastases (P < .008), presence of visceral metastases (P < .03), hemoglobin count (P < .009), and survival estimate (P < .00005). Diagnosis, ECOG performance status, and visceral metastases covaried with surgeon survival estimate. Linear regression and receiver-operator characteristic assessment confirmed that clinician estimation was the most accurate predictor of survival, followed by hemoglobin count, number of visceral metastases, ECOG performance status, primary diagnosis, and number of bone metastases. Nevertheless, survival estimate was accurate in predicting actual survival in only 33 (18%) of 181 patients. CONCLUSION A better means of prognostication is needed. In this article, we present a sliding scale for this purpose.

Wnt Inhibitory Factor 1 Decreases Tumorigenesis and Metastasis in Osteosarcoma

It has been reported that the progression of osteosarcoma was closely associated with the aberrant activation of canonical Wnt signaling. Wnt inhibitory factor-1 (WIF-1) is a secreted Wnt inhibitor whose role in human osteosarcoma remains unknown. In this study, WIF-1 expression in NHOst and osteosarcoma cell lines was determined by real-time reverse transcription-PCR, methylation-specific PCR, and Western blotting analysis. In addition, tissue array from patient samples was examined for WIF-1 expression by immunohistochemistry. Compared with normal human osteoblasts, WIF-1 mRNA and protein levels were significantly downregulated in several osteosarcoma cell lines. The downregulation of WIF-1 mRNA expression is associated with its promoter hypermethylation in these tested cell lines. Importantly, WIF-1 expression was also downregulated in 76% of examined osteosarcoma cases. These results suggest that the downregulation of WIF-1 expression plays a role in osteosarcoma progression. To further study the potential tumor suppressor function of WIF-1 in osteosarcoma, we established stable 143B cell lines overexpressing WIF-1. WIF-1 overexpression significantly decreased tumor growth rate in nude mice as examined by the s.c. injection of 143B cells stably transfected with WIF-1 and vector control. WIF-1 overexpression also markedly reduced the number of lung metastasis in vivo in an orthotopic mouse model of osteosarcoma. Together, these data suggest that WIF-1 exerts potent antiosteosarcoma effect in vivo in mouse models. Therefore, the reexpression of WIF-1 in WIF-1–deficient osteosarcoma represents a potential novel treatment and preventive strategy. Mol Cancer Ther; 9(3); 731–41

Expression pattern of two Frizzled-related genes, Frzb-1 and Sfrp-1, during mouse embryogenesis suggests a role for modulating action of Wnt family members.

Wnt proteins have been implicated in regulating growth and pattern formation in a variety of tissues during embryonic development. We previously identified Frzb‐1, a gene which encodes a secreted protein with homology in the ligand binding domain to the Wnt receptor Frizzled, but lacking the domain encoding the putative seven transmembrane segments. Frzb‐1 has recently been shown to bind to Wnt proteins in vitro, and to inhibit the activity of Xenopus Wnt‐8 in vivo. We report now that mFrzb‐1 and Wnt transcripts display both complementary and overlapping expression patterns at multiple sites throughout embryonic development. By Northern analysis, the expression of mFrzb‐1 in the developing mouse embryo is greatest from 10.5 to 12.5 days postcoitum (dpc). In the early embryo, mFrzb‐1 is expressed in the primitive streak, presomitic mesoderm, somites, and brain. Later, mFrzb‐1 exhibits sharp boundaries of expression in the limb bud, branchial arches, facial mesenchyme, and in cartilaginous elements of the appendicular skeleton. We conclude from these experiments that Frzb‐1is expressed at a time and location to modulate the action of Wnt family members during development of the limbs and central nervous system. Dev. Dyn. 1998;212:364–372.

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 G1 arrest. The G1 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 G1 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 β-catenin to the SKP2 promoter. Together, our results suggest that mechanisms of WIF1-induced G1 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. [Mol Cancer Ther 2009;8(2):458–68]

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

BackgroundAberrations 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.ResultsThe 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.ConclusionThese 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.

Analysis of prognostic factors in Ewing sarcoma using a population-based cancer registry.

Ewing sarcoma is a high‐grade malignancy that most often occurs in children. Because its occurrence in adults has been historically low, few studies have been published on the epidemiology of Ewing sarcoma in this group of patients. By using data from a large, population‐based cancer registry, the authors designed the present study to examine the outcome of children and adult patients with Ewing sarcoma and relevant prognostic factors.