Xuenong Zou

Identification of miRs-143 and -145 that is associated with bone metastasis of prostate cancer and involved in the regulation of EMT.

The principal problem arising from prostate cancer (PCa) is its propensity to metastasize to bone. MicroRNAs (miRNAs) play a crucial role in many tumor metastases. The importance of miRNAs in bone metastasis of PCa has not been elucidated to date. We investigated whether the expression of certain miRNAs was associated with bone metastasis of PCa. We examined the miRNA expression profiles of 6 primary and 7 bone metastatic PCa samples by miRNA microarray analysis. The expression of 5 miRNAs significantly decreased in bone metastasis compared with primary PCa, including miRs-508-5p, -145, -143, -33a and -100. We further examined other samples of 16 primary PCa and 13 bone metastases using real-time PCR analysis. The expressions of miRs-143 and -145 were verified to down-regulate significantly in metastasis samples. By investigating relationship of the levels of miRs-143 and -145 with clinicopathological features of PCa patients, we found down-regulations of miRs-143 and -145 were negatively correlated to bone metastasis, the Gleason score and level of free PSA in primary PCa. Over-expression miR-143 and -145 by retrovirus transfection reduced the ability of migration and invasion in vitro, and tumor development and bone invasion in vivo of PC-3 cells, a human PCa cell line originated from a bone metastatic PCa specimen. Their upregulation also increased E-cadherin expression and reduced fibronectin expression of PC-3 cells which revealed a less invasive morphologic phenotype. These findings indicate that miRs-143 and -145 are associated with bone metastasis of PCa and suggest that they may play important roles in the bone metastasis and be involved in the regulation of EMT Both of them may also be clinically used as novel biomarkers in discriminating different stages of human PCa and predicting bone metastasis.

HEF1 promotes epithelial mesenchymal transition and bone invasion in prostate cancer under the regulation of microRNA‐145

The principal problem arising from prostate cancer (PCa) is its propensity to metastasize to bones, and its crucial to understand the mechanism of tumor progression to metastasis in order to develop therapies that may reduce the morbidity and mortality of PCa patients. Although we had identified that microRNA(miR)‐145 could repress bone metastasis of PCa via regulating epithelial–mesenchymal transition (EMT) in previous study, it is still unknown how miR‐145 regulated EMT. In the present study, we constructed a luciferase reporter system and identified HEF1 as a direct target of miR‐145. More importantly, HEF1 was shown to promote migration, invasion and EMT of PC‐3 cells, a human PCa cell line originated from a bone metastatic PCa specimen. And HEF1 was also shown to partially mediate miR‐145 suppression of EMT and invasion. Furthermore, inhibition of HEF1 repressed bone invasion of PC‐3 cells in vivo. Expression of HEF1 was negatively correlated with miR‐145 in primary PCa and bone metastatic specimens, but HEF1 was higher in samples which were more likely to commit to bone metastasis or those with higher free prostate‐specific antigen (fPSA) levels and Gleason scores. Taken together, these findings indicate that HEF1 promotes EMT and bone invasion in prostate cancer by directly targeted by miR‐145, and miR‐145 suppresses EMT and invasion, at least in part, through repressing HEF1. J. Cell. Biochem. 114: 1606–1615, 2013.

Wild-type p53 suppresses the epithelial-mesenchymal transition and stemness in PC-3 prostate cancer cells by modulating miR‑145

The principal problem arising from prostate cancer (PCa) is its propensity to metastasize to bone and the mechanism(s) need to be further elucidated. The tumor suppressor p53 plays an important role in regulating the epithelial-mesenchymal transition (EMT) and cancer cell stemness, which have been proposed to play critical roles in cancer metastasis. MiR-145, a direct target of p53, represses bone metastasis of PCa and is involved in regulating EMT and cancer cell stemness. However, it is unknown whether wild‑type p53 (WT-p53) plays a role in regulating invasion, EMT and cancer cell stemness of PCa cells and whether miR-145 mediates the function of WT-p53. In the present study, we found that ectopic expression of WT-p53 inhibited the migration and invasion, and enhanced the adhesion of p53-null PC-3 cells derived from PCa bone metastasis. Furthermore, WT-p53 suppressed the expression of the mesenchymal markers fibronectin, vimentin, N-cadherin, ZEB2 and upregulated the expression of the epithelial marker E-cadherin in PC-3 cells. Moreover, WT-p53 also suppressed colony formation, tumor sphere formation and expression of CSC markers and stemness factors including CD44, Oct4, c-Myc and Klf4 in PC-3 cells. Importantly, WT-p53 upregulated the expression of miR-145, and the inhibitory effects of WT-p53 on migration, invasion, EMT and stemness of PC-3 cells were reversed by anti-miR-145. Together, our findings demonstrate that WT-p53 suppresses migration, invasion, EMT and stemness in PC-3 cells at least partially through modulating miR-145. These results suggest that loss of WT-p53 may promote the bone metastasis of PCa at least partially through repressing miR-145 to elevate EMT and stemness of cancer cells.

Synergistic inhibition of Wnt pathway by HIF-1α and osteoblast-specific transcription factor osterix (Osx) in osteoblasts.

Osterix (Osx) is an osteoblast-specific transcription factor required for osteoblast differentiation. Inhibition of Wnt pathway by Osx highlights the potential for feedback control mechanisms involved in bone formation. Hypoxia-inducible factor-1α (HIF-1α) is a master regulator of hypoxia. HIF-1α has been reported to couple angiogenesis to osteogenesis. Our recent study has demonstrated that Osx and HIF-1α cooperatively regulate VEGF expression in osteoblasts. Effects of hypoxia/HIF-1α on osteoblast proliferation and related mechanisms are not well understood. In this study, osteoblast growth under hypoxia was examined. We observed that osteoblast growth was inhibited under hypoxia. To explore possible mechanisms for hypoxia/HIF-1α to inhibit osteoblast proliferation, we tested the effect of hypoxia/HIF-1α on Wnt pathway. Quantitative RT-PCR results revealed that Wnt target genes such as cyclin D1 and c-Myc were downregulated under hypoxia while HIF-1α was upregulated. Treatment of desferrioxamine, a HIF-1α activator, led to further downregulation of expressions of cyclin D1 and c-Myc in osteoblasts. On the contrary, the inhibition of HIF-1α by siRNA in osteoblasts led to the expression increase of cyclin D1 and c-Myc. These data suggest that HIF-1α inhibits Wnt pathway in osteoblasts. To examine the effect of HIF-1α on Wnt pathway, HIF-1α was cotransfected with β-catenin along with Topflash reporter in transient transfection assay. Our results showed that HIF-1α inhibited β-catenin-induced Topflash reporter activity. Interestingly, a synergistic interplay was observed between Osx and HIF-1α in the inhibition of β-catenin-induced Topflash expression. Our findings indicate that Osx and HIF-1α cooperatively inhibit Wnt pathway. This study revealed additional new information of the cooperation between HIF-1α and Osx in osteoblasts.

HIF-1α inhibits Wnt signaling pathway by activating Sost expression in osteoblasts.

The nature of the cellular and molecular mechanisms for the transition of avascular cartilage replacement with bone during endochondral ossification remains poorly understood. One of the driving forces is hypoxia. As a master regulator of hypoxia, hypoxia-inducible factor-1α (HIF-1α) has been reported to couple angiogenesis to osteogenesis. Our recent study has demonstrated that osteoblast growth is inhibited under hypoxia and that HIF-1α cooperates with Osterix (Osx) to inhibit Wnt pathway. However, molecular mechanisms for inhibitory effects of HIF-1α on Wnt pathway are not well understood. In this study, our quantitative RT-PCR results revealed that the expression of a Wnt antagonist Sclerostin (Sost) was upregulated in osteoblasts during hypoxia while HIF-1α was upregulated. Treatment of desferrioxamine (DFO), a HIF-1α activator, led to further increase of Sost expression, suggesting that HIF-1α may activate Sost expression. The regulation of Sost gene expression by HIF-1α was then investigated. We performed loss-of-function experiments to examine Sost expression by using siRNA approach against HIF-1α, and found that the inhibition of HIF-1α by siRNA in osteoblasts led to the decrease of Sost expression. To address transcriptional regulation of Sost gene by HIF-1α, transient transfection assay was performed and showed that HIF-1α activated Sost-1 kb promoter reporter activity in a dose-dependent manner. To narrow down the minimal region of Sost promoter activated by HIF-1α, we generated a series of deletion mutants of Sost constructs. It was demonstrated that Sost-260 was the minimal region of Sost promoter for HIF-1α activation and that Sost-106 construct, which lack hypoxia response element, abolished HIF-1α-mediated Sost reporter activation. Gel shift assay showed that HIF-1 bound to the promoter sequence of Sost directly. These findings support our hypothesis that HIF-1α activates Sost expression. This study provides a novel molecular mechanism through which HIF-1α inhibits Wnt signaling in osteoblasts.

Treatment of primary basilar invagination by cervical traction and posterior instrumented reduction together with occipitocervical fusion.

Study Design. This retrospective study was conducted to analyze the radiographic and clinical results in seven patients with primary basilar invagination who accepted a combination of continuous cervical traction before operation and posterior screw/rod system reduction together with occipitocervical fusion. Objective. To evaluate the radiographic and clinical outcomes of this treatment regimen in combination of continuous cervical traction and posterior instrumented reduction with pedicle screw/rod system. Summary of Background Data. Primary basilar invagination poses considerable difficulties in the surgical management regarding surgical approach, reduction, and decompression. A variety of methods have been described to treat primary basilar invagination and all methods existed limits. Methods. There were four male and three female patients, and the ages ranged from 12 to 40 years (average age, 22.3 yr). Six patients presented neurologic deficits. The Nurick scale was from 1 grade to 4 grades (average, 2.7 grades). The distance of the odontoid tip in relation to Wackenheim line, atlantodental interval, Klaus height index, craniospinal angle, modified Omega angle, and cervicomedullary angle were measured pretreated and after surgery. When the tip of odontoid process was inferior or approximate to Wackenheim line and McRae line after cervical traction, the operation of reduction and fixation should be accepted. Results. After surgery, the mean Wackenheim value and atlantodental distance were reduced 9.3 mm and 2.0 mm, respectively. The mean Klaus height index, craniospinal angle, Omega angle, and cervicomedullary angle improved 6.5 mm, 17.0°, 11.6°, and 27.4°, respectively. All postoperative data had a significance compared with pretreatment data (P < 0.05). There was a tendency that younger patients were able to obtain more ideal reduction than adults. Of six patients with neurologic symptoms, five patients were normal or nearly normal. All patients achieved solid fusion. Conclusion. This case series demonstrates a safe, easy, and effective treatment regimen for the patients with primary basilar invagination.

Hemivertebra resection and scoliosis correction by a unilateral posterior approach using single rod and pedicle screw instrumentation in children under 5 years of age.

Ten consecutive patients under 5 years of age with congenital scoliosis caused by single-level hemivertebra underwent hemivertebra resection and scoliosis correction by a unilateral posterior approach using single rod and pedicle screw instrumentation. The mean age at the time of surgery was 3.3 years. The mean correction of main curve, segmental curve, and segmental kyphotic angle was 65.9, 62.8, and 78.1%, respectively. The average follow-up duration was 3.5 years. All patients achieved solid fusion on the convex side. No patient required revision surgery. The results indicate that this therapeutic method is less traumatic, simple, and safe.

Malignant Transformation of Benign Intraosseous Schwannoma in the Cervical Spine: A Case Report with an Immunohistochemical Study

Although 3% to 30% of lesions in von Recklinghausen disease undergo malignant transformation, malignant transformation of benign solitary schwannoma is extremely rare. We reported a case of recurrence and malignant transformation in a benign intraosseous schwannoma arising in the cervical spine of a 44-year-old man. The patient presented giant tumor in the C3 vertebral body with aggressive, expansile, and osteolytic destruction and relapsed 2 years after surgical resection and spinal reconstruction. Clinical data, radiologic characteristics, surgical management, histopathologic and immunohistochemical features were noted in the duration of follow-up. The local recurrence, nuclear pleomorphism, epithelioid differentiation, a small number of positive S-100 protein-staining cells, and especially the high percentage of positive cells with p53 (80%) and Ki-67 (75%) proteins support the aggressive nature of the lesion in malignant transformation of benign intraosseous schwannoma in the cervical spine. Immunohistochemistry would be useful as an ancillary technique in diagnosis. It is our practice to suggest that such case has to be carefully resected and the patient followed up.