Gene-Function-Based Clusters Explore Intricate Networks of Gene Expression of Circulating Tumor Cells in Patients with Colorectal Cancer.

Colorectal cancer (CRC) is a complex disease characterized by dynamically deregulated gene expression and crosstalk between signaling pathways. In this study, a new approach based on gene-function-based clusters was introduced to explore the CRC-associated networks of gene expression. Each cluster contained genes involved in coordinated regulatory activity, such as RAS signaling, the cell cycle process, transcription, or translation. A retrospective case-control study was conducted with the inclusion of 119 patients with histologically confirmed colorectal cancer and 308 controls. The quantitative expression data of 15 genes were obtained from the peripheral blood samples of all participants to investigate cluster-gene and gene-gene interactions. DUSP6, MDM2, and EIF2S3 were consistently selected as CRC-associated factors with high significance in all logistic models. CPEB4 became an insignificant factor only when combined with the clusters for cell cycle processes and for transcription. The CPEB4/DUSP6 complex was a prerequisite for the significance of MMD, whereas EXT2, RNF4, ZNF264, WEE1, and MCM4 were affected by more than two clusters. Intricate networks among MMD, RAS signaling factors (DUSP6, GRB2, and NF1), and translation factors (EIF2S3, CPEB4, and EXT2) were also revealed. Our results suggest that limited G1/S transition, uncontrolled DNA replication, and the cap-independent initiation of translation may be dominant and concurrent scenarios in circulating tumor cells derived from colorectal cancer. This gene-function-based cluster approach is simple and useful for revealing intricate CRC-associated gene expression networks. These findings may provide clues to the metastatic mechanisms of circulating tumor cells in patients with colorectal cancer.

CircularRNA as novel biomarkers in liver diseases.

The liver is an essential organ that is primarily responsible for digestion and eliminating toxic substances from the body. After the industrial revolution, Western diet and lifestyle changes have increased the incidence of several liver diseases, including non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver cirrhosis, and hepatocellular carcinoma (HCC). NAFLD and NASH are mostly asymptomatic at early stages, and the disease progression from NAFLD to life-threatening HCC remains not fully understood. Circular RNA (circRNA) is consist of a circular structure, and the circRNA-microRNA(miRNA)-mRNA axes have been shown to be involved in several cellular events, including apoptosis, vascularization, metastasis, etc. The highly stable structure of circRNAs has enabled themselves to be used as putative biomarkers of several diseases. Here, we conducted a literature review and discussed the identified roles of circRNAs in NAFLD, NASH, liver cirrhosis, and HCC. For example, deficiency of circRNA_0046366 and circRNA_0046367 has been shown as the characteristics of NAFLD, and restoration of these circRNAs ameliorates the oxidative stress, lipotoxicity, and disease severity in NAFLD. Silencing of circ_0071410 was shown to alleviate hepatic stellate activation, the key step of liver cirrhosis. CDR1 and circ_0067934 can facilitate the invasion and metastasis of HCC, while circMTO1 negatively regulates the progression of HCC. Although several research works have been conducted, the whole picture of circRNA-related underlying mechanisms is unclear. Future works using high-throughput bioinformatic approaches will be needed to delineate the role of circRNAs in liver diseases and to further develop novel diagnostics and therapeutics.

The subpopulation of CD44-positive cells promoted tumorigenicity and metastatic ability in lung adenocarcinoma.

BACKGROUND: Lung cancer is one of the major causes of carcinoma-related deaths in the world. Importantly, lung adenocarcinoma (LAC) is the most common type with poor outcome. However, the progressive clinical phenotype and biomolecular signature of lung cancer presenting the cancer stem-like and metastatic characteristics are still unclear. METHODS: In this study, we identified CD44 marker in lung cancers. The capabilities, including tumorigenic and migration assays, were analyzed in CD44 expression and CD44 expression subgroups. Meanwhile, the potential bio-signature and properties of lung tumor stem-like cells were further studied. RESULTS: The high expression of CD44 subpopulation (CD44-positive) in isolated lung cancer cells showed significantly higher abilities of tumorigenic colonies, tumor-sphere formation, and migratory properties when compared with the CD44 expression group. These subgroups of CD44-positive lung cancer cells further demonstrated the metastatic potential with epithelial-mesenchymal transition (EMT), as well as the high expression of Twist and Snail gene profile. Importantly, the overexpression of Snail with gene vector in CD44 expression cells further significantly promoted the properties of lung tumor stem-like cells. CONCLUSION: The results of this study highlighted the role of CD44-posivite subpopulation in modulating tumor initiation and EMT-based metastatic ability of lung malignancy.

Generation of high quality of hepatocyte-like cells from induced pluripotent stem cells with Parp1 but lacking c-Myc.

BACKGROUND: Induced pluripotent stem cells (iPSCs) have a great potential for application in patient-specific therapy. The reprogramming method that does not involve c-Myc reduces tumorigenic risk, but also largely reduces the efficiency of generation of iPSCs, especially for those reprogrammed from damaged cells. Poly(ADP-ribose) polymerase 1 (Parp1) catalyzes a reaction of poly(ADP-ribosylation) and has been reported to enhance cell reprogramming. METHODS: Using Oct-4/Sox2/Klf4/Parp1 (OSKP) reprogramming method, reprogramming factors plus Parp1 were capable of generation of iPSCs from adult fibroblasts and further toward to differentiate from iPSCs status into hepatocyte-like cells. RESULTS: Our results showed that Oct-4/Sox2/Klf4/Parp1 (OSKP)-derived iPSC exhibited regular pluripotent properties, long-term passages and more stable cellular-divided period. These OSKP-derived iPSCs can effectively differentiate into hepatocyte-like cells (OSKP-iPSC-Heps), and present high mRNA levels of Sox17, HNF3b, and HNF4a in OSKP-iPSC-Heps. The mature hepatic functions, including CYP3A4, LDL uptake, glycogen synthesis and urea secretion were analyzed and well detected in OSKP-iPSC-Heps on day 14 post-differentiation. CONCLUSION: In conclusion, we demonstrated that Parp1 promoted reprogramming process to generate the high quality of iPSCs, which could be used as a high quality source of hepatocytes.

Improvement of non-alcoholic steatohepatitis by hepatocyte-like cells generated from iPSCs with Oct4/Sox2/Klf4/Parp1.

The prevalence of nonalcoholic fatty liver disease (NAFLD) is usually increased with age. Non-alcoholic steatohepatitis (NASH), a serious form of NAFLD, may lead to cirrhosis and end-stage liver diseases. Induced pluripotent stem cells (iPSCs) hold promising potential in personalized medicine. Although obviation of c-Myc reduces tumorigenic risk, it also largely reduced the generation of iPSCs. Recently, Poly(ADP-ribose) polymerase 1 (Parp1) has been reported to enhance cell reprogramming. In this study, we demonstrated that forced expression of Oct4/Sox2/Klf4/Parp1 (OSKP) effectively promoted iPSC generation from senescent somatic cells from 18-month-old mouse. The iPSCs presented regular pluripotent properties, ability to form smaller teratoma with smaller size, and the potential for tridermal differentiation including hepatocyte-like cells (OSKP-iPSC-Heps). Resembled to fetal hepatocytes but not senescent hepatocytes, these OSKP-iPSC-Heps possessed antioxidant ability and were resistant to oxidative insult induced by H2O2 or exogenous fatty acid. Intrasplenic transplantation of OSKP-iPSC-Heps ameliorated the triglyceride over-accumulation and hepatitis, prevented the production of inflammatory cytokines and oxidative substances, and reduced apoptotic cells in methionine/choline-deficient diet (MCDD)-fed mice. In conclusion, we demonstrated that Parp-1 promoted iPSC generation from senescent cells, which can be used for the treatment of NASH after hepatic-specific differentiation. These findings indicated that patient-derived iPSC-Heps may offer an alternative option for treatment of NASH and NASH-associated end-stage liver diseases.

Sox2, a stemness gene, regulates tumor-initiating and drug-resistant properties in CD133-positive glioblastoma stem cells.

BACKGROUND: Glioblastoma multiforme (GBM) is the most lethal type of adult brain cancer and performs outrageous growth and resistance regardless of adjuvant chemotherapies, eventually contributing to tumor recurrence and poor outcomes. Considering the common heterogeneity of cancer cells, the imbalanced regulatory mechanism could be switched on/off and contribute to drug resistance. Moreover, the subpopulation of GBM cells was recently discovered to share similar phenotypes with neural stem cells. These cancer stem cells (CSCs) promote the potency of tumor initiation. As a result, targeting of glioma stem cells has become the dominant way of improving the therapeutic outcome against GBM and extending the life span of patients. Among the biomarkers of CSCs, CD-133 (prominin-1) has been known to effectively isolate CSCs from cancer population, including GBM; however, the underlying mechanism of how stemness genes manipulate CSC-associated phenotypes, such as tumor initiation and relapse, is still unclear. METHODS: Tumorigenicity, drug resistance and embryonic stem cell markers were examined in primary CD133-positive (CD133(+)) GBM cells and CD133(+) subpopulation. Stemness signature of CD133(+) GBM cells was identified using microarray analysis. Stem cell potency, tumorigenicity and drug resistance were also tested in differential expression of SOX2 in GBM cells. RESULTS: In this study, high tumorigenic and drug resistance was noticed in primary CD-133(+) GBM cells; meanwhile, plenty of embryonic stem cell markers were also elevated in the CD-133+ subpopulation. Using microarray analysis, we identified SOX2 as the most enriched gene among the stemness signature in CD133(+) GBM cells. Overexpression of SOX2 consistently enhanced the stem cell potency in the GBM cell lines, whereas knockdown of SOX2 dramatically withdrew CD133 expression in CD133(+) GBM cells. Additionally, we silenced SOX2 expression using RNAi system, which abrogated the ability of tumor initiation as well as drug resistance of CD133(+) GBM cells, suggesting that SOX2 plays a crucial role in regulating tumorigenicity in CD133(+) GBM cells. CONCLUSION: SOX2 plays a crucial role in regulating tumorigenicity in CD133(+) GBM cells. Our results not only revealed the genetic plasticity contributing to drug resistance and stemness but also demonstrated the dominant role of SOX2 in maintenance of GBM CSCs, which may provide a novel therapeutic target to overcome the conundrum of poor survival of brain cancers.

Enhanced antioxidant capacity of dental pulp-derived iPSC-differentiated hepatocytes and liver regeneration by injectable HGF-releasing hydrogel in fulminant hepatic failure.

Acute hepatic failure (AHF) is a severe liver injury leading to sustained damage and complications. Induced pluripotent stem cells (iPSCs) may be an alternative option for the treatment of AHF. In this study, we reprogrammed human dental pulp-derived fibroblasts into iPSCs, which exhibited pluripotency and the capacity to differentiate into tridermal lineages, including hepatocyte-like cells (iPSC-Heps). These iPSC-Heps resembled human embryonic stem cell-derived hepatocyte-like cells in gene signature and hepatic markers/functions. To improve iPSC-Heps engraftment, we next developed an injectable carboxymethyl-hexanoyl chitosan hydrogel (CHC) with sustained hepatocyte growth factor (HGF) release (HGF-CHC) and investigated the hepatoprotective activity of HGF-CHC-delivered iPSC-Heps in vitro and in an immunocompromised AHF mouse model induced by thioacetamide (TAA). Intrahepatic delivery of HGF-CHC-iPSC-Heps reduced the TAA-induced hepatic necrotic area and rescued liver function and recipient viability. Compared with PBS-delivered iPSC-Heps, the HGF-CHC-delivered iPSC-Heps exhibited higher antioxidant and antiapoptotic activities that reduced hepatic necrotic area. Importantly, these HGF-CHC-mediated responses could be abolished by administering anti-HGF neutralizing antibodies. In conclusion, our findings demonstrated that HGF mediated the enhancement of iPSC-Hep antioxidant/antiapoptotic capacities and hepatoprotection and that HGF-CHC is as an excellent vehicle for iPSC-Hep engraftment in iPSC-based therapy against AHF.

Verification of gene expression profiles for colorectal cancer using 12 internet public microarray datasets.

AIM: To verify gene expression profiles for colorectal cancer using 12 internet public microarray datasets. METHODS: Logistic regression analysis was performed, and odds ratios for each gene were determined between colorectal cancer (CRC) and controls. Twelve public microarray datasets of GSE 4107, 4183, 8671, 9348, 10961, 13067, 13294, 13471, 14333, 15960, 17538, and 18105, which included 519 cases of adenocarcinoma and 88 normal mucosa controls, were pooled and used to verify 17 selective genes from 3 published studies and estimate the external generality. RESULTS: We validated the 17 CRC-associated genes from studies by Chang et al (Model 1: 5 genes), Marshall et al (Model 2: 7 genes) and Han et al (Model 3: 5 genes) and performed the multivariate logistic regression analysis using the pooled 12 public microarray datasets as well as the external validation. The goodness-of-fit test of Hosmer-Lemeshow (H-L) showed statistical significance (P = 0.044) for Model 2 of Marshall et al in which observed event rates did not match expected event rates in subgroups of the model population. Expected and observed event rates in subgroups were similar, which are called well calibrated, in Models 1, 3 and 4 with non-significant P values of 0.460, 0.194 and 1.000 for H-L tests, respectively. A 7-gene model of CPEB4, EIF2S3, MGC20553, MS4A1, ANXA3, TNFAIP6 and IL2RB was pairwise selected, which showed the best results in logistic regression analysis (H-L P = 1.000, R (2) = 0.951, areas under the curve = 0.999, accuracy = 0.968, specificity = 0.966 and sensitivity = 0.994). CONCLUSION: A novel gene expression profile was associated with CRC and can potentially be applied to blood-based detection assays.

Gene expression profile of peripheral blood in colorectal cancer.

AIM: Optimal molecular markers for detecting colorectal cancer (CRC) in a blood-based assay were evaluated. METHODS: A matched (by variables of age and sex) case-control design (111 CRC and 227 non-cancer samples) was applied. Total RNAs isolated from the 338 blood samples were reverse-transcribed, and the relative transcript levels of candidate genes were analyzed. The training set was made of 162 random samples of the total 338 samples. A logistic regression analysis was performed, and odds ratios for each gene were determined between CRC and non-cancer. The samples (n = 176) in the testing set were used to validate the logistic model, and an inferred performance (generality) was verified. By pooling 12 public microarray datasets(GSE 4107, 4183, 8671, 9348, 10961, 13067, 13294, 13471, 14333, 15960, 17538, and 18105), which included 519 cases of adenocarcinoma and 88 controls of normal mucosa, we were able to verify the selected genes from logistic models and estimate their external generality. RESULTS: The logistic regression analysis resulted in the selection of five significant genes (P < 0.05; MDM2, DUSP6, CPEB4, MMD, and EIF2S3), with odds ratios of 2.978, 6.029, 3.776, 0.538 and 0.138, respectively. The five-gene model performed stably for the discrimination of CRC cases from controls in the training set, with accuracies ranging from 73.9% to 87.0%, a sensitivity of 95% and a specificity of 95%. In addition, a good performance in the test set was obtained using the discrimination model, providing 83.5% accuracy, 66.0% sensitivity, 92.0% specificity, a positive predictive value of 89.2% and a negative predictive value of 73.0%. Multivariate logistic regressions analyzed 12 pooled public microarray data sets as an external validation. Models that provided similar expected and observed event rates in subgroups were termed well calibrated. A model in which MDM2, DUSP6, CPEB4, MMD, and EIF2S3 were selected showed the result in logistic regression analysis (H-L P = 0.460, R2= 0.853, AUC = 0.978, accuracy = 0.949, specificity = 0.818 and sensitivity = 0.971). CONCLUSION: A novel gene expression profile was associated with CRC and can potentially be applied to blood-based detection assays.

Gene expression profiling of colorectal tumors and normal mucosa by microarrays meta-analysis using prediction analysis of microarray, artificial neural network, classification, and regression trees.

BACKGROUND: Microarray technology shows great potential but previous studies were limited by small number of samples in the colorectal cancer (CRC) research. The aims of this study are to investigate gene expression profile of CRCs by pooling cDNA microarrays using PAM, ANN, and decision trees (CART and C5.0). METHODS: Pooled 16 datasets contained 88 normal mucosal tissues and 1186 CRCs. PAM was performed to identify significant expressed genes in CRCs and models of PAM, ANN, CART, and C5.0 were constructed for screening candidate genes via ranking gene order of significances. RESULTS: The first screening identified 55 genes. The test accuracy of each model was over 0.97 averagely. Less than eight genes achieve excellent classification accuracy. Combining the results of four models, we found the top eight differential genes in CRCs; suppressor genes, CA7, SPIB, GUCA2B, AQP8, IL6R and CWH43; oncogenes, SPP1 and TCN1. Genes of higher significances showed lower variation in rank ordering by different methods. CONCLUSION: We adopted a two-tier genetic screen, which not only reduced the number of candidate genes but also yielded good accuracy (nearly 100%). This method can be applied to future studies. Among the top eight genes, CA7, TCN1, and CWH43 have not been reported to be related to CRC.

Inhibition of phosphorylated STAT3 by cucurbitacin I enhances chemoradiosensitivity in medulloblastoma-derived cancer stem cells.

INTRODUCTION: CD133 (PROM1) is a potential marker for cancer stem cells (CSCs), including those found in brain tumors. Recently, medulloblastoma (MB)-derived CD133-positive cells were found to have CSC-like properties and were proposed to be important contributors to tumorigenicity, cancer progression, and chemoradioresistance. However, the biomolecular pathways and therapeutic targets specific to MB-derived CSCs remain unresolved. MATERIALS AND METHODS: In the present study, we isolated CD133(+) cells from MB cell lines and determined that they showed increased tumorigenicity, radioresistance, and higher expression of both embryonic stem cell-related and drug resistance-related genes compared to CD133(-) cells. Bioinformatics analysis suggested that the STAT3 pathway might be important in MB and CD133(+) cells. To evaluate the effects of inhibiting the STAT3 pathway, MB-derived CD133(+/-) cells were treated with the potent STAT3 inhibitor, cucurbitacin I. Treatment with cucurbitacin I significantly suppressed the CSC-like properties and stemness gene signature of MB-derived CD133(+) cells. Furthermore, cucurbitacin I treatment increased the apoptotic sensitivity of MB-derived CD133(+) cells to radiation and chemotherapeutic drugs. Notably, cucurbitacin I demonstrated synergistic effects with ionizing radiation to inhibit tumorigenicity in MB-CD133(+)-inoculated mice. RESULTS: These results indicate that the STAT3 pathway plays a key role in mediating CSC properties in MB-derived CD133(+) cells. Targeting STAT3 with cucurbitacin I may therefore represent a novel therapeutic approach for treating malignant brain tumors.

Oct4-related cytokine effects regulate tumorigenic properties of colorectal cancer cells.

Oct4, a member of the POU-domain transcription factor family, has been implicated in the cancer stem cell (CSC)-like properties of various cancers. However, the precise role of Oct4 in colorectal CSC initiation remains uncertain. Numerous studies have demonstrated a strong link between inflammation and tumorigenesis in colorectal cancers. In this study, we demonstrated that Oct4 overexpression enhances CSC-like properties of colorectal cancer cells (CRCs), including sphere formation, cell colony formation, cell migration, invasiveness, and drug resistance. In addition, putative CSC markers, stemness genes, drug-resistant genes, as well as interleukin (IL)-8 and IL-32 were upregulated. Microarray-based bioinformatics of CRCs showed higher expression levels of embryonic stem cell-specific genes in cells that overexpressed Oct4. Neutralization of either IL-8 or IL-32 with specific antibodies partially blocked the tumorigenic effects induced by either Oct4 overexpression or by the addition of conditioned media from Oct4-overexpressing CRCs. In addition, the presence of Oct4-overexpressing CRCs enhanced the tumorigenic potential of parental CRCs in vivo. In summary, these data suggest that IL-8 and IL-32 play a role in regulating the CSC-like properties that promote tumorigenesis of CRCs in both autocrine and paracrine manners.

Celecoxib enhances radiosensitivity in medulloblastoma-derived CD133-positive cells.

OBJECTS: Cyclooxygenase-2 (COX-2), the enzyme that converts arachidonic acid to prostaglandins, is overexpressed in a variety of tumors, including medulloblastoma (MB). CD133, a transmembrane glycoprotein, has been suggested as a marker for cancer stem cells in brain tumors. The aim of the present study was to investigate the role of celecoxib, a selective COX-2 inhibitor, in enhancing the effects of ionizing radiotherapy (IR) on medulloblastoma-derived CD133-positive cells (MB-CD133(+)). MATERIALS AND METHODS: MB-CD133(+) were isolated from two medulloblastoma cell lines (Daoy and UW228). Then, they were treated with celecoxib in different concentrations, and cell viability was assessed. The assays of cell survival, soft agar, radiosensitivity, colony formation, and apoptotic activity in MB-CD133(+) treated with celecoxib alone, radiation alone, or celecoxib combined with radiation were further evaluated. RESULTS: MB-CD133(+) showed the self-renew ability to form sphere bodies in vitro and regenerate tumors in vivo. The levels of COX-2 mRNA and protein in MB-CD133(+) were significantly higher than those in MB-CD133(-). The treatment of 30 µM celecoxib could effectively inhibit the abilities of cell proliferation and colony formation and increase IR-induced apoptosis in treated MB-CD133(+). Furthermore, in vivo study demonstrated that celecoxib significantly enhanced radiosensitivity in MB-CD133(+)-transplanted grafts. Notably, xenotransplantation analysis demonstrated that the treatment of celecoxib could further suppress the expressions of angiogenic and stemness-related genes in treated MB-CD133(+) grafts of SCID mice. CONCLUSIONS: Celecoxib presents the potential of radiosensitizing effect in MB-derived cancer stem cells. Therefore, it should be warranted in future trials to enhance the radiotherapeutic effects in MB patients.

Differential expression profiling between atypical teratoid/rhabdoid and medulloblastoma tumor in vitro and in vivo using microarray analysis.

OBJECTIVES: Atypical teratoid/rhabdoid tumor (AT/RT) and medulloblastoma (MB) are the most malignant primary brain tumors in early childhood. AT/RT is frequently misdiagnosed as primitive neuroectodermal tumor/medulloblastoma. The biological features and clinical outcomes of AT/RT and MB are extremely different. In this study, we used microarray as a platform to distinguish these two tumors with the definitive diagnostic marker as well as the profiling of expression genes. METHODS: In order to clarify the pathogenesis and find the biological markers for AT/RT, we established a derivative AT/RT primary cell culture. The differential profiling between AT/RT and MB were analyzed by using microarray method. RESULTS: With the use of the microarray method, we demonstrated that 15 genes were significantly changed (at least 5-fold in upregulation and 1/5-fold in downregulation) between AT/RT and MB in tissues and cell lines. The quantitative reverse transcription-polymerase chain reaction analyses further confirmed that mRNA expression levels of SERPINI1 and osteopontin were highly expressed in AT/RT cells and tissues than those in MB. Importantly, our microarray result suggested that AT/RT presents the stemness-like pattern and expression profiling of embryonic stem cells as well as high mRNA expressions of Oct-4, Nanog, Sox-2, and c-Myc. CONCLUSIONS: Our study demonstrated the differential gene expression profiling between AT/RT and MB. Based on the microarray findings, AT/RTs present embryonic stem-like gene recapitulation and further provide novel insights into their underlying biology.