Development and validation of an early diagnosis model for bone metastasis in non-small cell lung cancer based on serological characteristics of the bone metastasis mechanism.

Background: Bone metastasis significantly impact the prognosis of non-small cell lung cancer (NSCLC) patients, reducing their quality of life and shortening their survival. Currently, there are no effective tools for the diagnosis and risk assessment of early bone metastasis in NSCLC patients. This study employed machine learning to analyze serum indicators that are closely associated with bone metastasis, aiming to construct a model for the timely detection and prognostic evaluation of bone metastasis in NSCLC patients. Methods: The derivation cohort consisted of 664 individuals with stage IV NSCLC, diagnosed between 2015 and 2018. The variables considered in this study included age, sex, and 18 specific serum indicators that have been linked to the occurrence of bone metastasis in NSCLC. Variable selection used multivariate logistic regression analysis and Lasso regression analysis. Six machine learning methods were utilized to develop a bone metastasis diagnostic model, assessed with Area Under the Curve (AUC), Decision Curve Analysis (DCA), sensitivity, specificity, and validation cohorts. External validation used 113 NSCLC patients from the Medical Alliance (2019-2020). Furthermore, a prospective validation study was conducted on a cohort of 316 patients (2019-2020) who were devoid of bone metastasis, and followed-up for at least two years to assess the predictive capabilities of this model. The model's prognostic value was evaluated using Kaplan-Meier survival curves. Findings: Through variable selection, 11 serum indictors were identified as independent predictive factors for NSCLC bone metastasis. Six machine learning models were developed using age, sex, and these serum indicators. A random forest (RF) model demonstrated strong performance during the training and internal validation cohorts, achieving an AUC of 0.98 (95% CI 0.95-0.99) for internal validation. External validation further confirmed the RF model's effectiveness, yielding an AUC of 0.97 (95% CI 0.94-0.99). The calibration curves demonstrated a high level of concordance between the anticipated risk and the observed risk of the RF model. Prospective validation revealed that the RF model could predict the occurrence of bone metastasis approximately 10.27 ± 3.58 months in advance, according to the results of the SPECT. An online computing platform (https://bonemetastasis.shinyapps.io/shiny_cls_1model/) for this RF model is publicly available and free-to-use by doctors and patients. Interpretation: This study innovatively employs age, gender, and 11 serological markers closely related to the mechanism of bone metastasis to construct an RF model, providing a reliable tool for the early screening and prognostic assessment of bone metastasis in NSCLC patients. However, as an exploratory study, the findings require further validation through large-scale, multicenter prospective studies. Funding: This work is supported by the National Natural Science Foundation of China (NO.81974315); Shanghai Municipal Science and Technology Commission Medical Innovation Research Project (NO.20Y11903300); Shanghai Municipal Health Commission Health Industry Clinical Research Youth Program (NO.20204Y034).

Exploring the impact of PDGFD in osteosarcoma metastasis through single-cell sequencing analysis.

PURPOSE: The overall survival rate for metastatic osteosarcoma hovers around 20%. Responses to second-line chemotherapy, targeted therapies, and immunotherapies have demonstrated limited efficacy in metastatic osteosarcoma. Our objective is to validate differentially expressed genes and signaling pathways between non-metastatic and metastatic osteosarcoma, employing single-cell RNA sequencing (scRNA-seq) and additional functional investigations. We aim to enhance comprehension of metastatic mechanisms and potentially unveil a therapeutic target. METHODS: scRNA-seq was performed on two primary osteosarcoma lesions (1 non-metastatic and 1 metastatic). Seurat package facilitated dimensionality reduction and cluster identification. Copy number variation (CNV) was predicted using InferCNV. CellChat characterized ligand-receptor-based intercellular communication networks. Differentially expressed genes underwent GO function enrichment analysis and GSEA. Validation was achieved through the GSE152048 dataset, which identified PDGFD-PDGFRB as a common ligand-receptor pair with significant contribution. Immunohistochemistry assessed PDGFD and PDGFRB expression, while multicolor immunofluorescence and flow cytometry provided insight into spatial relationships and the tumor immune microenvironment. Kaplan-Meier survival analysis compared metastasis-free survival and overall survival between high and low levels of PDGFD and PDGFRB. Manipulation of PDGFD expression in primary osteosarcoma cells examined invasion abilities and related markers. RESULTS: Ten clusters encompassing osteoblasts, osteoclasts, osteocytes, fibroblasts, pericytes, endothelial cells, myeloid cells, T cells, B cells, and proliferating cells were identified. Osteoblasts, osteoclasts, and osteocytes exhibited heightened CNV levels. Ligand-receptor-based communication networks exposed significant fibroblast crosstalk with other cell types, and the PDGF signaling pathway was activated in non-metastatic osteosarcoma primary lesion. These results were corroborated by the GSE152048 dataset, confirming the prominence of PDGFD-PDGFRB as a common ligand-receptor pair. Immunohistochemistry demonstrated considerably greater PDGFD expression in non-metastatic osteosarcoma tissues and organoids, correlating with extended metastasis-free and overall survival. PDGFRB expression showed no significant variation between non-metastatic and metastatic osteosarcoma, nor strong correlations with survival times. Multicolor immunofluorescence suggested co-localization of PDGFD with PDGFRB. Flow cytometry unveiled a highly immunosuppressive microenvironment in metastatic osteosarcoma. Manipulating PDGFD expression demonstrated altered invasive abilities and marker expressions in primary osteosarcoma cells from both non-metastatic and metastatic lesions. CONCLUSIONS: scRNA-seq illuminated the activation of the PDGF signaling pathway in primary lesion of non-metastatic osteosarcoma. PDGFD displayed an inhibitory effect on osteosarcoma metastasis, likely through the suppression of the EMT signaling pathway.

Identification of the methotrexate resistance-related diagnostic markers in osteosarcoma via adaptive total variation netNMF and multi-omics datasets.

Osteosarcoma is one of the most common malignant bone tumors with high chemoresistance and poor prognosis, exhibiting abnormal gene regulation and epigenetic events. Methotrexate (MTX) is often used as a primary agent in neoadjuvant chemotherapy for osteosarcoma; However, the high dosage of methotrexate and strong drug resistance limit its therapeutic efficacy and application prospects. Studies have shown that abnormal expression and dysfunction of some coding or non-coding RNAs (e.g., DNA methylation and microRNA) affect key features of osteosarcoma progression, such as proliferation, migration, invasion, and drug resistance. Comprehensive multi-omics analysis is critical to understand its chemoresistant and pathogenic mechanisms. Currently, the network analysis-based non-negative matrix factorization (netNMF) method is widely used for multi-omics data fusion analysis. However, the effects of data noise and inflexible settings of regularization parameters affect its performance, while integrating and processing different types of genetic data is also a challenge. In this study, we introduced a novel adaptive total variation netNMF (ATV-netNMF) method to identify feature modules and characteristic genes by integrating methylation and gene expression data, which can adaptively choose an anisotropic smoothing scheme to denoise or preserve feature details based on the gradient information of the data by introducing an adaptive total variation constraint in netNMF. By comparing with other similar methods, the results showed that the proposed method could extract multi-omics fusion features more effectively. Furthermore, by combining the mRNA and miRNA data of methotrexate (MTX) resistance with the extracted feature genes, four genes, Carboxypeptidase E (CPE), LIM, SH3 protein 1 (LASP1), Pyruvate Dehydrogenase Kinase 1 (PDK1) and Serine beta-lactamase-like protein (LACTB) were finally identified. The results showed that the gene signature could reliably predict the prognostic status and immune status of osteosarcoma patients.

[Retracted] MicroRNA­152 inhibits cell proliferation, migration and invasion by directly targeting MAFB in nasopharyngeal carcinoma.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that certain of the cell migration and invasion assay data shown in Fig. 3A and B were strikingly similar to data appearing in different form in other articles by different authors at different research institutes, some of which have been retracted; moreover, there appeared to be some overlapping data examining the western blots featured in Figs. 5B and 6A. Owing to the fact that the contentious data in the above article had already been published, or were already under consideration for publication, prior to its submission to Molecular Medicine Reports, the Editor has decided that this paper should be retracted from the Journal. After having been in contact with the authors, they agreed with the decision to retract the paper. The Editor apologizes to the readership for any inconvenience caused. [Molecular Medicine Reports 15: 948­956, 2017; DOI: 10.3892/mmr.2016.6059].

Th1 immune maturation effects of Nocardia rubra cell-wall skeleton via PI3K/Akt/PAX8 regulatory axis.

Nocardia rubra cell-wall skeleton (Nr-CWS) is reported as an external immunotherapeutic enhancer with the advantage of antitumor effect on human cancers. However, the immune regulatory role of Nr-CWS is not fully illustrated. We studied mouse CD4+ T lymphocytes isolated from mice spleen were induced by Nr-CWS and observed that the differentiation of Th1 CD4+ T cells and the cytokines of IL-2, TNF-α, IFN-γ were all enhanced by Nr-CWS. Furthermore, RNA sequencing was conducted to investigate the different mRNA profiling induced by Nr-CWS. We observed that paired box 8 (PAX8) was significantly up-regulated in Nr-CWS-treated Th1 cells compared to control. As a transcription factor, chromatin immunoprecipitation sequencing was carried out to study the genome-wide distribution of PAX8. Interestingly, we found that the binding domain of PAX8 was elevated by Nr-CWS, and the target genes associated with these binding sites showed a positive correlation between their transcription and PAX8 binding strength. Finally, we determined that Nr-CWS could enhance the activity of the PI3 K/Akt signaling pathway. Akt agonist could mimic the effect of Nr-CWS for PAX8 up-regulation, while Akt inhibitor compromised the expression of PAX8. Taken together, we determined a novel role of Nr-CWS in boosting the activity of Th1 maturation via the PI3 K/Akt/PAX8 axis.

The timing of targeted therapy initiation in metastatic sarcoma as an adjuvant to first-line chemotherapy or a second-line agent.

58 cases of metastatic sarcoma were reviewed retrospectively in order to compare the efficacy and safety of concurrent (n=24, group A) versus sequential (n=34, group B) use of chemotherapy and targeted therapy in metastatic sarcoma. Progression-free survival (PFS) 1 was defined as the duration between initiation of first-line treatment to disease progression or recurrence. PFS' was defined as the duration between initiation of first-line treatment to the failure of chemotherapy and targeted therapy, and overall survival (OS) was defined as the duration between initiation of first-line treatment to the date of last follow-up or death. The results revealed that patients in group A possessed a higher tumor burden compared to those in group B (P=0.049). Survival curves revealed that the median PFS1 (15.2 vs. 5.4 months, P=0.000), median PFS' (15.2 vs. 10.8 months, P=0.049), and median OS (42.3 vs. 25.3 months, P=0.041) of subjects in group A were remarkably longer than those of group B. Subgroup analysis showed that patients in group A experienced more favorable PFS1 (15.2 vs. 3 months, P=0.000), PFS' (15.2 vs. 5.8 months, P=0.003), and OS (35.2 vs. 15.7 months, P=0.011) than those in group B, with findings especially prominent in patients with tumor burden ≥ 10 cm in comparison to patients with tumor burden < 10 cm (P ≥ 0.05). All grades of leukopenia, thrombocytopenia, fatigue, and oral mucositis were more frequently diagnosed in patients of group A compared to those of group B. However, there were no significant differences between the rates of Grade 3-4 adverse events between the two groups. This investigation suggests that the concurrent use of targeted therapy and chemotherapy may be useful and safe as a first-line treatment in patients with metastatic sarcoma who possess a high tumor burden.

Case Report: Sequential Chemotherapy and Immunotherapy Produce Sustained Response in Osteosarcoma With High Tumor Mutational Burden.

Background: Immunotherapy has provided an effective method for the treatment of many cancers. However, its efficacy in osteosarcoma is not satisfactory so far. Case Presentation: Here, we presented a case of osteosarcoma treated with sequential chemotherapy and immunotherapy and showed promising therapeutic potential. The 29-year-old female patient presented 9th rib osteosarcoma with suspected right lung lower lobe metastasis. Surgery was performed to remove the primary lesion, and a series of chemotherapies were given afterward in consideration of the response and tolerance. The right lung lower lobe metastasis was under control first but progressed (PD) 9 months after the initiation of therapy. The lesion was surgically removed and subsequent chemotherapy was implemented. The patient had good tolerance with chemotherapy and maintained well for approximately 11 months before the discovery of 11th rib and right lung upper lobe metastases. Surgery was then performed on both lesions and achieved complete response. Post-surgical brief chemotherapy and subsequent long-term immunotherapy (pembrolizumab) maintained continuous remission for 33 months. The patient survived for 60 months with well-controlled disease from the time of confirmed diagnosis. Genetic alterations of all primary and metastatic lesions were investigated by whole-exome sequencing (WES). Substantial similarity in mutational landscape between the primary lesion and 11th rib metastasis and between the two lung metastases were revealed, while substantial heterogeneity was found between the rib lesions and lung metastases. The tumor mutational burden (TMB) for the 9th rib primary lesion, the metastatic 11th rib lesion, and the metastatic right upper and lower lobe nodule tissues was 8.02, 2.38, 4.61, and 0.14 mutations/Mb, respectively. The primary lesion exhibited the most diverse copy number variation (CNV) changes among all lesions. Furthermore, pathway enrichment analysis also suggested significant heterogeneity among the lesions. Conclusions: Surgery with sequential chemotherapy and maintenance immunotherapy was shown to have good response for the first time on osteosarcoma patient who had high TMB tumor lesions and good tolerance for chemotherapy and immunotherapy.

The Plasma Concentration of D-Dimer is Associated with Neoadjuvant-Chemotherapy Efficacy and the Prognosis in Osteosarcoma.

PURPOSE: This retrospective study explored the clinical value of the plasma D-dimer level in osteosarcoma. MATERIALS AND METHODS: We measured the plasma D-dimer level before neoadjuvant chemotherapy (D0) and the plasma D-dimer level after four courses of neoadjuvant chemotherapy (D1) in 103 patients with stage-IIB high-grade osteosarcoma of the limb. The change in the D-dimer level (ΔD) was defined as D1 minus D0. The chi-square test was used to compare categorical variables. Analyses of receiver operating characteristic (ROC) curves were undertaken to determine the optimal cutoff points for D0, D1, and ΔD. The area under the ROC (AUC) of D0, D1, and ΔD was calculated to evaluate their discriminatory abilities in monitoring the response to neoadjuvant chemotherapy (tumor necrosis). Survival curves were generated according to Kaplan-Meier analyses and compared using the Log rank test. Univariate analyses and multivariate analyses were carried out to determine independent prognostic factors. RESULTS: Kaplan-Meier curves showed that a high D-dimer level at D0 and tumor diameter ≥8 cm were associated significantly with worse overall survival (OS) (P<0.05). Multivariate Cox regression analyses revealed a high D-dimer level at D0 (hazard ratio, 3.92; 95% confidence interval, 1.756-5.804; P=0.000) was an independent unfavorable prognostic factor. The chi-square test showed ΔD to be associated significantly with tumor necrosis. Analyses of ROC curves showed the D-dimer level at D0 and ΔD had better ability compared to that at D1 to discriminate the response to neoadjuvant chemotherapy. CONCLUSION: The D-dimer level was correlated with the prognosis and response to chemotherapy in patients with stage-IIB high-grade osteosarcoma of the limb. The D-dimer level may serve as a risk factor of the response to chemotherapy and prognosis of localized osteosarcoma.

SKP1 promotes YAP-mediated colorectal cancer stemness via suppressing RASSF1.

BACKGROUND: Cancer stem cells (CSCs) have been recognized as an important drug target, however, the underlying mechanisms have not been fully understood. SKP1 is a traditional drug target for cancer therapy, while, whether SKP1 promotes colorectal cancer (CRC) stem cells (CRC-SCs) and the underlying mechanisms have remained elusive. METHODS: Human CRC cell lines and primary human CRC cells were used in this study. Gene manipulation was performed by lentivirus system. The mRNA and protein levels of target genes were examined by qRT-PCR and western blot. The sphere-forming and in vitro migration capacities were determined by sphere formation and transwell assay. The self-renewal was determined by limiting dilution assay. The tumorigenicity and metastasis of cancer cells were examined by xenograft model. The promoter activity was examined by luciferase reporter assay. Nuclear run-on and Chromatin immunoprecipitation-PCR (ChIP-PCR) assay were employed to examine the transcription and protein-DNA interaction. Co-immunoprecipitation assay was used to test protein-protein interaction. The relationship between gene expression and survival was analyzed by Kaplan-meier analysis. The correlation between two genes was analyzed by Spearman analysis. Data are represented as mean ± SD and the significance was determined by Student's t test. RESULTS: SKP1 was upregulated in CRC-SCs and predicted poor prognosis of colon cancer patients. Overexpression of SKP1 promoted the stemness of CRC cells reflected by increased sphere-forming, migration and self-renewal capacities as well as the expression of CSCs markers. In contrast, SKP1 depletion produced the opposite effects. SKP1 strengthened YAP activity and knockdown of YAP abolished the effect of SKP1 on the stemness of CRC cells. SKP1 suppressed RASSF1 at both mRNA and protein level. Overexpression of RASSF1 abolished the effect of SKP1 on YAP activity and CRC stemness. CONCLUSION: Our results demonstrated that SKP1 suppresses RASSF1 at both mRNA and protein level, attenuates Hippo signaling, activates YAP, and thereby promoting the stemness of CRC cells.

Long Non-coding RNA MRUL Contributes to Osteosarcoma Progression Through the miR-125a-5p/FUT4 Axis.

Osteosarcoma (OS) originates in the skeletal system and has a rising global incidence. Long Non-coding RNAs (lncRNAs) are key regulators of human cancers development and progression. However, their roles in the development of OS are not well understood. This research aimed to investigate the effect of a long non-coding RNA (lncRNA), MRUL, on OS and revealed its potential molecular mechanisms. The bioinformatics analysis demonstrated that lncRNA MRUL was involved in regulating nucleic acid-templated transcription, cellular macromolecule biosynthetic process, immune response, and inflammatory response. In this work, the expression of lncRNA MRUL was detected by quantitative real-time polymerase chain reaction (qRT-RCR) in both cancer tissues and cell lines. We found that lncRNA MRUL was up-regulated in cancer tissues and cell lines. Functional experiments showed that knockdown of lncRNA MRUL inhibited OS cell proliferation, and metastasis. At the same time, we found that lncRNA MRUL interacted with miR-125a-5p to suppress FUT4 expression. Moreover, inhibition of miR-125a-5p abrogated the biological roles of lncRNA MRUL knockdown on OS cell proliferation, migration, and invasion. In conclusion, these results demonstrated that OS-upregulated lncRNA MRUL promoted cell proliferation, and metastasis via negatively regulating miR-125a-5p, and imply that lncRNA MRUL may be a potential biomarker for OS.

Establishment of a serological molecular model for the early diagnosis and progression monitoring of bone metastasis in lung cancer.

BACKGROUND: The prognosis is very poor for lung cancer patients with bone metastasis. Unfortunately, a suitable method has yet to become available for the early diagnosis of bone metastasis in lung cancer patients. The present work describes an attempt to develop a novel model for the early identification of lung cancer patients with bone metastasis risk. METHODS: As the test group, 205 primary lung cancer patients were recruited, of which 127 patients had bone metastasis; the other 78 patients without bone metastasis were set as the negative control. Additionally, 106 healthy volunteers were enrolled as the normal control. Serum levels of several cytokines in the bone microenvironment (CaN, OPG, PTHrP, and IL-6) and bone turnover markers (tP1NP, ß-CTx) were detected in all samples by ECLIA or ELISA assay. Receiver operating characteristic (ROC) curves and multivariate analyses were performed to evaluate diagnostic abilities and to assess the attributable risk of bone metastasis for each of these indicators; the diagnostic model was established via logistic regression analysis. The prospective validation group consisted of 44 patients with stage IV primary lung cancer on whom a follow-up of at least 2 years was conducted, during which serum bone biochemical marker concentrations were monitored. RESULTS: The serological molecular model for the diagnosis of bone metastasis was logit (p). ROC analysis showed that when logit (p) > 0.452, the area under curve of the model was 0.939 (sensitivity: 85.8%, specificity: 89.7%). Model validation demonstrated accuracy with a high degree of consistency (specificity: 85.7%, specificity: 87.5%, Kappa: 0.770). The average predictive time for bone metastasis occurrence of the model was 9.46 months earlier than that of the bone scan diagnosis. Serum OPG, PTHrP, tP1NP, ß-CTx, and the diagnostic model logit (p) were all positively correlated with bone metastasis progression (P < 0.05). CONCLUSIONS: This diagnostic model has the potential to be a simple, non-invasive, and sensitive tool for diagnosing the occurrence and monitoring the progression of bone metastasis in patients with lung cancer.

Long noncoding RNA OIP5-AS1 mediates resistance to doxorubicin by regulating miR-137-3p/PTN axis in osteosarcoma.

Opa-interacting protein 5 antisense RNA1 (OIP5-AS1) has been demonstrated to facilitate proliferation, metastasis and resistance to treatments in various types of cancers. Nevertheless, the exact mechanisms underlying the roles of OIP5-AS1 in osteosarcoma(OS) drug resistance have not yet been clearly elucidated. Therefore, we sought to investigate the functional involvement of OIP5-AS1 in osteosarcoma. Our results indicated that OIP5-AS1 was dramatically up-regulated in osteosarcoma drug-resistant tissues and cells in comparison with drug-sensitive tissues and cells. Also, the knockdown of OIP5-AS1 was found to have decreased doxorubicin resistance of OS cells. Further analyses revealed that OIP5-AS1 operated as a competitor for endogenous RNA of miR-137-3p as well as regulated pleiotrophin(PTN) expression, which has been reported to be an oncogene in OS in previous research. Furthermore, the loss of miR-137-3p or alternatively, the gain of PTN, both resulted in the abolishment of the inhibitory role of OIP5-AS1 silencing the proliferative activity. Our analyses indicated and helped to determine the role of OIP5-AS1 in contributing to tumorigenesis of osteosarcoma via the miR-137-3p/PTN axis and, therefore outlining its potential for use as a therapeutic target against this cancer.

SKA1 induces de novo MTX-resistance in osteosarcoma through inhibiting FPGS transcription.

De novo methotrexate (MTX)-resistance, whose underlying mechanism remains largely unknown, usually leads to very poor prognosis in patients with osteosarcoma (OS). In this study, we established the de novo MTX-resistant OS cell line SF-86 and identified the candidate gene spindle and kinetochore associated complex subunit 1 (SKA1) as potentially related to de novo MTX-resistance. Analysis of a cohort of 95 OS patients demonstrated that SKA1 overexpression significantly correlated with de novo MTX-resistance and poor 5-year survival. Mechanistically, SKA1 overexpression lead to a downregulation of folylpoly-γ-glutamate synthetase (FPGS), a key enzyme that converts MTX into its active form, MTX-PG. We further demonstrated that SKA1 interacts with DNA-directed RNA polymerase II subunit RPB3. ChIP analysis revealed that RPB3 binds the promoter region of the FPGS gene and triggers FPGS transcription upon MTX treatment in SW1353, a MTX-sensitive OS cell line lacking endogenous SKA1 expression. On the contrary, this process is blocked in SF-86 cells due to the formation of an inhibitory SKA1-RPB3 complex. Furthermore, downregulation of SKA1 levels restores MTX sensitivity in SF-86. Collectively, our study has established the de novo MTX-resistant cell line SF-86 and identified SKA1 as a novel regulator of FPGS, playing a key role in the development of de novo MTX-resistance in OS.

Luteolin attenuates Wnt signaling via upregulation of FZD6 to suppress prostate cancer stemness revealed by comparative proteomics.

The mechanisms underlying luteolin-induced inhibition of prostate cancer (PCa) stemness have remained elusive. Here, we report that luteolin suppresses PCa stemness through Wnt signaling by upregulation of FZD6 (frizzled class receptor 6). Luteolin inhibits PCa cell proliferation, migration, self-renewal as well as the expression of prostate cancer stem cell markers in vitro. Through iTRAQ-based quantitative proteomics study, we identified 208 differentially expressed proteins in luteolin-treated PC-3 cells. Subsequent mechanistic analysis revealed that luteolin inhibits Wnt signaling by transcriptional upregulation of FZD6, and thereby suppressing the stemness of PCa cells. Furthermore, we identified FZD6 as a tumor suppressor that can abolish PCa stemness. In summary, our findings demonstrate that suppression of Wnt signaling by upregulation of FZD6 is a mechanism underlying luteolin-induced inhibition of PCa stemness. Our work suggests a new therapeutic strategy against human prostate cancer caused by aberrant activation of Wnt signaling.

PAK5 overexpression is associated with lung metastasis in osteosarcoma.

p21-activated kinases (PAKs) are multifunctional effectors of Rho GTPases, which are associated with cytoskeletal organization, cellular morphogenesis, migration and survival. PAKs are overactive in a number of tumor tissues and have attracted attention as a potential target for cancer therapy. In the present study, PAK5 levels were analyzed in primary osteosarcoma (OS) samples (n=65) using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC) methods. In the primary OS tissue, increased PAK5 expression (IHC score >2, n=37) was associated with significantly decreased overall survival (P=0.036) compared with decreased PAK5 expression (IHC score ≤2, n=28). PAK5 expression was identified to be significantly associated with metastasis (P=0.010). The lung is the most common metastasis site for OS. In addition, the level of PAK5 in lung metastasis tissue (n=13) was detected using RT-qPCR and IHC methods. PAK5 expression was increased in lung metastasis tissue compared with in primary OS samples. PAK5 was silenced using short hairpin RNA in OS cell lines. Wound healing, migration and nude mice model assay results consistently demonstrated that PAK5 knockdown was able to significantly inhibit OS migration. In PAK5-knockdown cells, the alteration in the expression of a number of metastasis-associated factors, including epithelial cadherin, vimentin, fibronectin and matrix metalloproteinase 2 (MMP2), was analyzed. Only MMP2 expression was decreased significantly (P<0.05). The expression level of MMP2 was analyzed in primary OS tissue and lung metastasis tissue using RT-qPCR and IHC methods. Expression of MMP2 was identified to be associated with expression of PAK5. The results of the present study suggest that PAK5 promotes OS cell migration and that PAK5 expression may be used to predict lung metastasis.

Pleiotrophin promotes chemoresistance to doxorubicin in osteosarcoma by upregulating P-glycoprotein.

Chemoresistance is a major hindrance to successful treatment of osteosarcoma (OS). Pleiotrophin (PTN), a neurotrophic growth factor, has been linked to the malignant characteristics of various cancer types. We retrospectively examined the correlation between PTN expression and chemoresistance in OS in a cohort of 133 OS patients. Immunohistochemistry revealed that PTN expression correlated with the necrosis rate and local OS recurrence. In a prognostic analysis, high PTN expression was associated with poor overall and disease-free survival, and was an independent adverse prognostic factor for disease-free survival. In doxorubicin-treated OS cells, PTN knockdown enhanced cellular chemosensitivity, increased the apoptosis rate and inhibited clone formation, while PTN overexpression had the opposite effects. In a xenograft model, PTN knockdown and overexpression respectively enhanced and reduced cellular sensitivity to doxorubicin. PTN upregulated anaplastic lymphoma kinase (ALK), p-Glycogen Synthase Kinase (GSK)3ß, ß-catenin and multidrug resistance protein 1/P-glycoprotein (MDR1/P-gp). In rescue assays with the ß-catenin inhibitor XAV939 and the MDR1/P-gp inhibitor verapamil, PTN promoted chemoresistance to doxorubicin in OS cells by activating ALK/GSK3ß/ß-catenin signaling, thereby upregulating MDR1/P-gp. Therefore, PTN could be used as a biomarker predicting chemotherapeutic responses, and downregulating PTN could be a promising therapeutic strategy to prevent chemoresistance in OS patients.

Pterostilbene suppresses human endometrial cancer cells in vitro by down-regulating miR-663b.

Resveratrol has long been known as an antioxidant and a chemopreventive agent. Similar to resveratrol, pterostilbene (PT) is also a phenolic compound extracted from the Vitis species. However, there are few studies on the antitumor effect of PT. Thus, we investigated the effects of PT on the endometrial cancer (EC) cells in vitro and the related molecular mechanisms. Treatment of EC cell lines HTB-111 and Ishikawa with PT (25-100 µmol/L) dose-dependently suppressed the cell viability and induced apoptosis. Using miR microarrays, we examined the miR expression profile in Ishikawa cells with or without PT, and revealed that miR-663b was the most decreased in PT-treated Ishikawa cells. Furthermore, we predicted and verified that the pro-apoptosis factor BCL2L14 is the direct target of miR-663b. Over-expression of miR-663b and knock-down of BCL2L14 counteracted the suppressing effects of PT on HTB-111 and Ishikawa cells. In addition, we evaluated the miR-663b levels in EC tissues of 51 patients using an in situ hybridization technique. With the median of the score of miR-663b as a cut-off value, these EC patients were divided into two groups, and the patients with high miR-663b expression had significantly poor prognosis.

Knockdown of ubiquitin-specific peptidase 39 inhibited the growth of osteosarcoma cells and induced apoptosis in vitro.

BACKGROUND: Ubiquitin specific peptidase 39 (USP39), an essential factor in the assembly of the mature spliceosome complex, has an aberrant expression in several cancer. However, its function and the corresponding mechanism on human osteosarcoma has not been fully explored yet. METHODS: The mRNA and DNA copies of USP39 were increased in osteosarcoma cancer tissues compared with the one in human normal tissues according to datasets from the publicly available Oncomine database. A further western blot analysis also demonstrated an aberrant endogenous expression of USP39 in three different osteosarcoma cells. Then lentivirus-mediated short hairpin RNA (shRNA) was designed to silence USP39 in human osteosarcoma cell line U2OS, which is used to test the impact of USP39-silencing on cellular proliferation, colony formation, cell cycle distribution and apoptosis. RESULTS: Knockdown of USP39 expression in U2OS cell significantly decreased cell proliferation, impaired colony formation ability. A further analysis indicated suppression of USP39 arrested cell cycle progression at G2/M phase via p21 dependent way. In addition, the results of Annexin V/7-AAD staining suggested the knockdown of USP39 could promote U2OS cell apoptosis through PARP cleavage. CONCLUSIONS: These results uncover the critical role of USP39 in regulating cancer cell mitosis and indicate USP39 is critical for osteosarcoma tumorigenesis.

Elevated expression of serine/threonine phosphatase type 5 correlates with malignant proliferation in human osteosarcoma.

Osteosarcoma is the most common primary malignant bone tumor in adolescents and young adults. However, the involvement of serine/threonine phosphatase type 5 (PP5) in osteosarcoma remains unclear. The aim of this study was to evaluate the functional role of PP5 in osteosarcoma cells. Firstly, we found that PP5 is widely expressed in several human osteosarcoma cell lines. Then we used lentivirus-delivered siRNA to silence PP5 expression in Saos-2 and U2OS cell lines. Knockdown of endogenous PP5 expression by shRNA-expressing lentivirus significantly decreased the viability and proliferation of the osteosarcoma cells. Moreover, FACS analysis showed that knockdown of PP5 expression induced a significant arrest in the G0/G1 phase of the cell cycle, which was associated with the inhibition of cell proliferation. Therefore, knockdown of PP5 is likely to provide a novel alternative to targeted therapy of osteosarcoma and deserves further investigation.

MicroRNA­152 inhibits cell proliferation, migration and invasion by directly targeting MAFB in nasopharyngeal carcinoma.

Aberrant expression of microRNAs (miRs) has been reported to be involved in nasopharyngeal carcinoma (NPC) carcinogenesis and development. The expression and functions of miR­152 have previously been studied in several types of cancer. However, to the best of our knowledge, no previous studies have investigated the effects of miR­152 on NPC. The present study aimed to explore the expression, functions and molecular mechanisms of miR­152 in NPC. The expression levels of miR­152 were detected in NPC tissues and cell lines using quantitative polymerase chain reaction (qPCR). Cell proliferation, migration and invasion were measured by MTT, cell migration and invasion assays, respectively. Dual­luciferase reporter assay was used to determine whether V­maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) was a direct target gene of miR­152. qPCR and western blotting were used to detect the mRNA and protein expression levels of MAFB. In addition, functional assays were performed to explore the effects of endogenous MAFB on NPC. The results of the present study demonstrated that miR­152 was significantly downregulated in NPC tissues and cell lines. Furthermore, ectopic expression of miR­152 suppressed cell proliferation, migration and invasion of NPC cells. Dual­luciferase reporter assay demonstrated that MAFB was a direct target gene of miR­152, and qPCR and western blotting indicated that miR­152 negatively regulated MAFB expression at the mRNA and protein level. Knockdown of MAFB expression markedly suppressed NPC cell proliferation, migration and invasion. These findings suggested that miR­152 may target MAFB to regulate NPC initiation and progression; therefore, it may be investigated as a target for the treatment of NPC.