Layered Double Hydroxide-Based PdCux@LDH Alloy Nanozyme for a Singlet Oxygen-Boosted Sonodynamic Therapy.

Redox nanozymes have demonstrated tremendous promise in disrupting cellular homeostasis toward cancer therapy, but a dysfunctional competition of diverse activities makes it normally restricted by the complex tumor microenvironment (TME). As palladium nanocrystals can achieve the precise regulation of the enzyme-like activity by regulating exposed crystal planes, noble metal nanoalloys can enhance the enzyme-like activity by promoting electron transfer and enhanced active sites. Herein, bimetallic nanoalloys with optimized enzymatic activity were intelligently designed via the interaction between the Pd and layered double hydroxide, denoted as PdCux@LDH. This PdCux@LDH is able to produce long-lived singlet oxygen (1O2) with high efficiency and selectivity for ultrasound-improved cancer therapy. In addition, this PdCux@LDH nanozyme demonstrated unique surface-dependent multienzyme-mimicking activities for catalyzing cascade reactions: oxidase (OXD)- and catalase (CAT)-mimicking activities. Interestingly, ultrasound (US) stimulation can further improve the dual-enzyme-mimicking activities and impart superior reactive oxygen species (ROS) generation activity, thereby further consuming nicotinamide adenine dinucleotide (NADH) to cause mitochondrial dysfunction, resulting in a highly efficient alloy nanozyme-mediated cancer therapy. This work opens a new research avenue to apply nanozymes for effective sonodynamic therapies (SDT).

Analytic and Clinical Validation of an Ultrasensitive, Quantitative Polymerase Chain Reaction Assay for EGFR Mutation Analysis With Circulating Tumor DNA.

CONTEXT: - The mutation analysis of epidermal growth factor receptor (EGFR) has become a common test to guide therapeutic decision making for lung cancer. Molecular testing with circulating tumor DNA in plasma allows diagnosis of mutations when tumor tissue is not available as well as monitoring treatment response with repeat biopsies. OBJECTIVES: - To develop a timely and cost-effective assay that can accurately detect EGFR mutations in circulating tumor DNA and to evaluate the analytic and clinical performance of the assay. DESIGN: - Analytic assessment was conducted with a set of reference materials carrying classic EGFR mutations. A recently developed Poisson distribution-based approach was employed to understand the assay sensitivity. Clinical evaluation was performed with 224 pairs of plasma and matched tissues from patients with stage I to IV disease. EGFR mutation rates of 390 consecutive plasma samples processed in the central service laboratory were compared with previously reported prevalence in an Asian population. RESULTS: - Our results suggested that limit of detection for the EGFR quantitative polymerase chain reaction assay was 10 mutation copies, and the lowest detectable copy numbers could be extended to a single-digit level. The clinical sensitivity was 53.3% for all stages combined and 81.4% for late stages, with a high specificity of 100%. Clinical observations showed an overall positive finding rate of 32.5% and 41.4% for stage IV disease, which is consistent with previously reported EGFR mutation prevalence in an Asian population. CONCLUSIONS: - Our results supported the clinical utility of the ultrasensitive, quantitative polymerase chain reaction assay for EGFR mutation analysis with circulating tumor DNA.

Gli promotes epithelial-mesenchymal transition in human lung adenocarcinomas.

Adenocarcinoma is the most common type of lung cancer. Epithelial-mesenchymal transition (EMT) is required for tumor invasion/metastasis and the components that control this process are potential therapeutic targets. This study we examined the role of Gli in lung adenocarcinoma and whether its activation regulates metastasis through EMT in lung adenocarcinoma. We found that tumors with high Gli expression had significantly lower E-Cadherin expression in two independent cohorts of patients with lung adenocarcinoma that we studied. In vitro up-regulation of SHh resulted in increased cell migration while small molecule inhibitors of Smo or Gli significantly reduced cell mobility both in a wound healing assay and in a 3D cell invasion assay. Inhibition of Gli in vivo decreased tumor growth and induced an increase in E-Cadherin expression. Our results indicate that Gli may be critical for lung adenocarcinoma metastasis and that a novel Gli inhibitor shows promise as a therapeutic agent by preventing cell migration and invasion in vitro and significantly reducing tumor growth and increasing E-Cadherin expression in vivo.

EMX2 Is a Predictive Marker for Adjuvant Chemotherapy in Lung Squamous Cell Carcinomas.

BACKGROUND: Squamous cell carcinomas (SCC) account for approximately 30% of non-small cell lung cancer (NSCLC). Current staging methods do not adequately predict outcome for this disease. EMX2 is a homeo-domain containing transcription factor known to regulate a key developmental pathway. This study assessed the significance of EMX2 as a prognostic and predictive marker for resectable lung SCC. METHODS: Two independent cohorts of patients with lung SCC undergoing surgical resection were studied. EMX2 protein expression was examined by immunohistochemistry, Western blot, or immunofluorescence. EMX2 expression levels in tissue specimens were scored and correlated with patient outcomes. Chemo-sensitivity of lung SCC cell lines stably transfected with EMX2 shRNAs to cisplatin, carboplatin, and docetaxel was examined in vitro. RESULTS: EMX2 expression was down-regulated in lung SCC tissue samples compared to their matched adjacent normal tissues. Positive EMX2 expression was significantly associated with improved overall survival in stage I lung SCC patients, and in stage II/IIIA lung SCC patients receiving adjuvant chemotherapy. EMX2 expression was also associated with expression of EMT markers in both lung SCC cell lines and tissue samples. Knock-down of EMX2 expression in lung SCC cells promoted chemo-resistance and cell migration. CONCLUSIONS: EMX2 expression is down-regulated in lung SCC and its down-regulation is associated with chemo-resistance in lung SCC cells, possibly through regulation of Epithelial-to-Mesenchymal Transition (EMT). EMX2 may serve as a novel prognostic marker for stage I lung SCC patients and a prediction marker for stage II/IIIA lung SCC patients receiving adjuvant chemotherapy.

Measurement of genome-wide DNA methylation predicts survival benefits from chemotherapy in non-small cell lung cancer.

PURPOSE: Novel molecular predictive biomarkers for chemotherapy have been screened and validated in non-small cell lung cancer (NSCLC). However, there was no report on the correlation of genome-wide DNA methylation with survival benefit from chemotherapy in NSCLC. METHODS: A sandwich enzyme-linked immunosorbent assay (ELISA) method was first established, optimized and validated. A total of 191 NSCLC samples were analyzed using the sandwich ELISA for the association between the relative genome-wide DNA methylation level and the survival outcomes from chemotherapy. RESULTS: The analytical performance of the sandwich ELISA method was satisfying and suitable for analysis. Using the sandwich ELISA method, we found that the genome-wide DNA methylation level in NSCLC cancer tissues was significantly lower than that in adjacent normal tissues, which further validated the assay. We found that there was no significant correlation between genome-wide DNA methylation level and patients' histology, stage and progression free survivals. However, in patients with high methylation level, those without chemotherapy had significantly better overall survival than those receiving chemotherapy. In patients receiving chemotherapy, those with low genome-wide DNA methylation level had significantly better overall survival than those with relatively high DNA methylation level. CONCLUSIONS: Genome-wide DNA hypomethylation as a sign of genomic instability may predict overall survival benefit from chemotherapy in NSCLC.

The homeobox gene EMX2 is a prognostic and predictive marker in malignant pleural mesothelioma.

OBJECTIVES: Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm with a poor prognosis and limited treatment options. EMX2 is a homeobox transcription factor that may regulate key developmental pathways known to promote tumorigenesis. In this study, we evaluated the prognostic and predictive significance of EMX2 expression in MPM. MATERIALS AND METHODS: Fifty surgically resected MPM specimens were studied. Quantitative real-time RT-PCR was used to analyze EMX2 mRNA expression. Association of EMX2 levels with clinical outcomes was evaluated with using the Kaplan-Meier method and a multivariate Cox proportional hazards regression model. RESULTS: EMX2 expression was significantly associated with IMIG stage (p<0.001) and smoking history (p=0.006). Cox hazard regression modeling identified low-EMX2 expression as a negative prognostic factor in progression-free survival by both univariate (p=0.002) and multivariate analysis (p=0.002). Kaplan-Meier analysis revealed significant differences in progression-free survival between low- and high-EMX expressing groups in all patients (p=0.001), and also when grouped by early (I/II) stage disease (p<0.001), patients undergoing pleurectomy (p<0.001) and patients with an epitheliod subtype (p<0.004). Furthermore, EMX2 expression predicted response to neoadjuvant chemotherapy. High-EMX2 expression was associated with decreased progression-free survival after neoadjuvant therapy, suggesting that induction therapy should be avoided in these patients. CONCLUSIONS: EMX2 expression is downregulated in advanced cases of malignant pleural mesothelioma and may serve as an important prognostic and predictive molecular biomarker of progression-free survival.

Hedgehog/Gli promotes epithelial-mesenchymal transition in lung squamous cell carcinomas.

BACKGROUND: Squamous cell carcinomas (SCC) account for approximately 30% of non-small cell lung cancer. Investigation of the mechanism of invasion and metastasis of lung SCC will be of great help for the development of meaningful targeted therapeutics. This study is intended to understand whether the activation of Hedgehog (Hh) pathway is involved in lung SCC, and whether activated Hh signaling regulates metastasis through epithelial-mesenchymal transition (EMT) in lung SCC. METHODS: Two cohorts of patients with lung SCC were studied. Protein expression was examined by immunohistochemistry, Western blot, or immunofluorescence. Protein expression levels in tissue specimens were scored and correlations were analyzed. Vismodegib and a Gli inhibitor were used to inhibit Shh/Gli activity, and recombinant Shh proteins were used to stimulate the Hh pathway in lung SCC cell lines. Cell migration assay was performed in vitro. RESULTS: Shh/Gli pathway components were aberrantly expressed in lung SCC tissue samples. Gli1 expression was reversely associated with the expression of EMT markers E-Cadherin and ß-Catenin in lung SCC specimens. Inhibition of the Shh/Gli pathway suppressed migration and up-regulated E-Cadherin expression in lung SCC cells. Stimulation of the pathway increased migration and down-regulated E-Cadherin expression in lung SCC cells. CONCLUSIONS: Our results suggested that the Shh/Gli pathway may be critical for lung SCC recurrence, metastasis and resistance to chemotherapy. Inhibition of the Shh/Gli pathway activity/function is a potential therapeutic strategy for the treatment of lung SCC patients.

miR-30b and miR-30c expression predicted response to tyrosine kinase inhibitors as first line treatment in non-small cell lung cancer.

BACKGROUND: Aberrantly expressed microRNAs are a hallmark of cancer, and microRNA expression profiling is associated with tumor progression and response to chemotherapy, suggesting their potential application as prognostic and predictive biomarkers. The role of microRNAs in lung cancer remains elusive. It has been recently reported that epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (MET) tyrosine kinase can regulate expression of specific microRNAs including miR-30b, miR-30c, miR-221, miR-222, miR-103 and miR-203, and induce tumorigenesis and gefitinib resistance in lung cancers. We intend to study the role of miR-30b and miR-30c expression in predicting response to tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC). METHODS: We have therefore retrospectively examined expression of miR-30b miR-30c in 41 formalin fixed paraffin embedded tissue samples from NSCLC patients when TKIs were used as first line therapy. RESULTS: We found a significant correlation between expression of miR-30b and miR-30c. Furthermore, miR-30b and miR-30c expression correlated with short-term response. Kaplan-Meier analysis further revealed that the expression of miR-30b and miR-30c predicted progression free survival and the overall survival rate in the examined cohort. CONCLUSION: Our study identified miR-30b and miR-30c as useful prognostic predictors in NSCLC patients who underwent first line treatment with TKIs.

Down-regulation of SIX3 is associated with clinical outcome in lung adenocarcinoma.

BACKGROUND: Lung cancer is a common cancer and the leading cause of cancer-related death worldwide. SIX3 is a human homologue of the highly conserved sine oculis gene family essential during embryonic development in vertebrates, and encodes a homeo-domain containing transcription factor. Little is known about the role of SIX3 in human tumorigenesis. This study is to assess the expression/function of SIX3 and the significance of SIX3 as a prognostic biomarker in lung adenocarcinoma. METHODS: Quantitative real-time RT-PCR was used to analyze SIX3 mRNA expression and quantitative methylation specific PCR (MSP) was used to examine promoter methylation. MTS and colony formation assays were performed to examine cell proliferation. Wound healing assays were used to assess cell migration, and microarrays were utilized to examine genes regulated by SIX3 in lung cancer cells. Association of SIX3 expression levels with clinical outcomes of patients with lung adenocarcinoma was evaluated using the Kaplan-Meier method and a multivariate Cox proportional hazards regression model. RESULTS: SIX3 was down-regulated in lung adenocarcinoma tissues compared to their matched adjacent normal tissues, and this down-regulation was associated with methylation of the SIX3 promoter. SIX3 was also methylation-silenced in lung cancer cell lines. Restoration of SIX3 in lung cancer cells lacking endogenous SIX3 suppressed cell proliferation and migration, and downregulated a number of genes involved in proliferation and metastasis such as S100P, TGFB3, GINS3 and BAG1. Moreover, SIX3 mRNA expression was associated with significantly improved overall survival (OS) and progression-free survival (PFS) in adenocarcinoma patients and patients with bronchioloalveolar carcinoma (BAC) features. CONCLUSIONS: SIX3 may play an important role as a novel suppressor in human lung cancer. SIX3 has potential as a novel prognostic biomarker for patients with lung adenocarcinomas.

Inhibition of the Wnt palmitoyltransferase porcupine suppresses cell growth and downregulates the Wnt/ß-catenin pathway in gastric cancer.

Similarly to the Wnt protein palmitoyltransferase, porcupine (PPN) is essential to the activation of the Wnt/ß-catenin signaling pathway. However, little is known about the role of PPN activity in human gastric cancer, one of the most common causes of cancer-related mortality. Real-time quantitative PCR was used to detect the expression levels of PPN in paired gastric cancer tissues. Cell proliferation, migration and invasion assays were performed following treatment using a newly developed small molecule PPN inhibitor (inhibitors of Wnt production, IWP-2) in the gastric cancer MKN28 cell line. Expression of downstream target genes and transcriptional activity of the Wnt/ß-catenin signaling pathway were examined following IWP-2 treatment in MKN28. We identified that PPN was overexpressed in human gastric cancer tissue samples and cell lines. Following treatment of the gastric cancer cell line MKN28 with IWP-2, we detected that IWP-2 decreased MKN28 cell proliferation, migration and invasion, and elevated caspase 3/7 activity. Further analysis demonstrated that IWP-2 downregulated the transcriptional activity of the Wnt/ß-catenin signaling pathway and downregulated the expression levels of downstream Wnt/ß-catenin target genes in MKN28 cells. As current Wnt pathway-targeting strategies used for anticancer therapy have mainly focused on Wnt-receiving cells, our data shed light on the potential use of Wnt palmitoyltransferase PPN inhibitors to abrogate Wnt production in Wnt-producing cells, thus providing a potential therapeutic option for gastric cancer.

Detection of E2A-PBX1 fusion transcripts in human non-small-cell lung cancer.

BACKGROUND: E2A-PBX1 fusion gene caused by t(1;19)(q23;p13), has been well characterized in acute lymphoid leukemia (ALL). There is no report on E2A-PBX1 fusion transcripts in non-small-cell lung cancer (NSCLC). METHODS: We used polymerase chain reaction (PCR) to detect E2A-PBX1 fusion transcripts in human NSCLC tissue specimens and cell lines. We analyzed correlation of E2A-PBX1 fusion transcripts with clinical outcomes in 76 patients with adenocarcinoma in situ (AIS) and other subgroups. We compared mutation status of k-ras, p53 and EGFR in 22 patients with E2A-PBX1 fusion transcripts. RESULTS: We detected E2A-PBX1 transcripts in 23 of 184 (12.5%) NSCLC tissue specimens and 3 of 13 (23.1%) NSCLC cell lines. Presence of E2A-PBX1 fusion transcripts correlated with smoking status in female patients (P=0.048), AIS histology (P=0.006) and tumor size (P=0.026). The overall survival was associated with gender among AIS patients (P=0.0378) and AIS patients without E2A-PBX1 fusion transcripts (P=0.0345), but not among AIS patients with E2A-PBX1 fusion transcripts (P=0.6401). The overall survival was also associated with status of E2A-PBX1 fusion transcripts among AIS stage IA patients (P=0.0363) and AIS stage IA female patients (P=0.0174). In addition, among the 22 patients with E2A-PBX1 fusion transcripts, 12 (54.5%) patients including all four non-smokers, showed no common mutations in k-ras, p53 and EGFR. CONCLUSIONS: E2A-PBX1 fusion gene caused by t(1;19)(q23;p13) may be a common genetic change in AIS and a survival determinant for female AIS patients at early stage.

Adenoviral delivery of the EMX2 gene suppresses growth in human gastric cancer.

BACKGROUND: EMX2 is a human orthologue of the Drosophila empty spiracles homeobox gene that has been implicated in embryogenesis. Recent studies suggest possible involvement of EMX2 in human cancers; however, the role of EMX2 in carcinogenesis needs further exploration. RESULTS: In this study, we reported that down-regulation of EMX2 expression was significantly correlated with EMX2 promoter hypermethylation in gastric cancer. Restoring EMX2 expression using an adenovirus delivery system in gastric cancer cell lines lacking endogenous EMX2 expression led to inhibition of cell proliferation and Wnt signaling pathway both in vitro and in a gastric cancer xenograft model in vivo. In addition, we observed that animals treated with the adenoviral EMX2 expression vector had significantly better survival than those treated with empty adenoviral vector. CONCLUSION: Our study suggests that EMX2 is a putative tumor suppressor in human gastric cancer. The adenoviral-EMX2 may have potential as a novel gene therapy for the treatment of patients with gastric cancer.

HSulf-1 inhibits cell proliferation and invasion in human gastric cancer.

The HSulf-1 gene encodes an extracellular 6-O-endosulfatase and regulates the sulfation status of heparan sulfate proteoglycans (HSPG). We have demonstrated that promoter hypermethylation is correlated with the HSulf-1 silencing in gastric cancer. To investigate the functional importance of HSulf-1 silencing in gastric cancer, we restored HSulf-1 expression in the gastric cancer cell line MKN28, which lacks endogenous HSulf-1. Following restoration of expression, HSulf-1 inhibited cell proliferation, motility, and invasion in vitro, as well as significantly suppressing the MKN28 xenograft model (P < 0.05). No noticeable changes in proliferation and motility were observed following restoration of HSulf-1 in another gastric cancer cell line, namely AGS cells. Interestingly, in MKN28 cells, which have been reported to be dependent on extracellular Wnt signaling, we found that HSulf-1 inhibited the transcriptional activity of the Wnt / ß-catenin pathway and downregulated its targeted genes. Conversely, in AGS cells, in the constitutive Wnt / ß-catenin pathway is active, HSulf-1 had no effect on the activity of the Wnt / ß-catenin pathway. Furthermore, transfection of Wnt3a cDNA or ß-catenin shRNA resulted in rescue or enhancement, respectively, of the effects of HSulf-1 in MKN28 cells. Furthermore, HSPG epitope analysis confirmed that HSulf-1 affected the structure of heparan sulfate on the cell surface. Together, the results of the present study suggest that extracellular HSulf-1 may function as a negative regulator of proliferation and invasion in gastric cancer by suppressing Wnt / ß-catenin signaling at the cell surface.

HSulf-1 suppresses cell growth and down-regulates Hedgehog signaling in human gastric cancer cells.

Gastric cancer is the second most lethal cancer worldwide. Despite the current surgical and adjuvant therapies, 5-year survival remains less than 20-25% in the US, Europe and China. Therefore, there is an urgent need to identify new therapeutic targets for treating this malignant disease. Accumulating evidence has supported that aberrant activation of the Hedgehog signaling pathway plays a crucial role in tumorigenesis and progression of gastric cancer. Human sulfatase-1 (HSulf-1) is a recently identified enzyme that desulfates cell surface heparan sulfate proteoglycans (HSPGs), which is critical for Hedgehog signal transduction under a highly sulfated state. HSulf-1 has recently emerged as a tumor suppressor gene in certain types of cancer, including ovarian, breast, myeloma and hepatocellular carcinoma; however, its role in gastric cancer remains to be elucidated. Therefore, we established HSulf-1-expressing monoclonal MKN28 gastric cancer cells to investigate its function in gastric cancer. Expression of HSulf-1 significantly suppressed cellular proliferation and growth in MKN28 gastric cancer cells. Notably, HSulf-1 inhibits Gli-mediated transcription and down-regulates the expression of Hedgehog target genes, including GLI1, PTCH1/2, HHIP, CCND1, C-MYC and BCL-2. Collectively, the study provides evidence that HSulf-1 may function as a tumor suppressor in gastric cancer. It suppresses gastric cancer cell proliferation, possibly through abrogating the Hedgehog signaling pathway. The study provides new mechanistic insight into HSulf-1- mediated tumor suppression, and supports the use of HSulf-1 as a potential new therapeutic target in treating gastric cancer.

Use of serum circulating CCNB2 in cancer surveillance.

Cyclin B2 (CCNB2), a member of the cyclin protein family, has been found to be up-regulated in human cancers. To evaluate the potential use of circulating CCNB2 in serum in cancer surveillance, we examined relative expression levels of serum circulating CCNB2 mRNA in 103 cancer patients, 19 normal controls, and 40 benign disease patients using real-time quantitative reverse transcriptase polymerase chain reaction. We found that the relative expression level of circulating CCNB2 mRNA in cancer patients was significantly higher (p<0.0001) than that in normal controls and benign diseases group. Circulating CCNB2 mRNA level was significantly (p<0.001) correlated with cancer stage and metastasis status. Receiver operating characteristic (ROC) analysis showed an area under the curve (AUC) of 0.87 and 0.83 (p<0.05) in identifying cancer patients' metastasis status in lung and digestive tract cancer, respectively. Moreover, we observed that expression levels of circulating CCNB2 mRNA in cancer patients significantly decreased (p=0.0084) after their therapeutic treatments. These data suggest that detection of serum circulating CCNB2 mRNA may have potential clinical applications in screening and monitoring of metastasis and therapeutic treatments.

Use of serum circulating CCNB2 in cancer surveillance.

Cyclin B2 (CCNB2), a member of the cyclin protein family, has been found to be up-regulated in human cancers. To evaluate the potential use of circulating CCNB2 in serum in cancer surveillance, we examined relative expression levels of serum circulating CCNB2 mRNA in 103 cancer patients, 19 normal controls, and 40 benign disease patients using real-time quantitative reverse transcriptase polymerase chain reaction. We found that the relative expression level of circulating CCNB2 mRNA in cancer patients was significantly higher (p<0.0001) than that in normal controls and benign diseases group. Circulating CCNB2 mRNA level was significantly (p<0.001) correlated with cancer stage and metastasis status. Receiver operating characteristic (ROC) analysis showed an area under the curve (AUC) of 0.87 and 0.83 (p<0.05) in identifying cancer patients' metastasis status in lung and digestive tract cancer, respectively. Moreover, we observed that expression levels of circulating CCNB2 mRNA in cancer patients significantly decreased (p=0.0084) after their therapeutic treatments. These data suggest that detection of serum circulating CCNB2 mRNA may have potential clinical applications in screening and monitoring of metastasis and therapeutic treatments.