BACKGROUND: Identification of biomarkers associated with the prognosis of different cancer subtypes is critical to achieve better therapeutic assistance. In colorectal cancer (CRC) the discovery of stable and consistent survival markers remains a challenge due to the high heterogeneity of this class of tumors. In this work, we identified a new set of gene markers for CRC associated to prognosis and risk using a large unified cohort of patients with transcriptomic profiles and survival information.
RESULTS: We built an integrated dataset with 1273 human colorectal samples, which provides a homogeneous robust framework to analyse genome-wide expression and survival data. Using this dataset we identified two sets of genes that are candidate prognostic markers for CRC in stages III and IV, showing either up-regulation correlated with poor prognosis or up-regulation correlated with good prognosis. The top 10 up-regulated genes found as survival markers of poor prognosis (i.e. low survival) were: DCBLD2, PTPN14, LAMP5, TM4SF1, NPR3, LEMD1, LCA5, CSGALNACT2, SLC2A3 and GADD45B. The stability and robustness of the gene survival markers was assessed by cross-validation, and the best-ranked genes were also validated with two external independent cohorts: one of microarrays with 482 samples; another of RNA-seq with 269 samples. Up-regulation of the top genes was also proved in a comparison with normal colorectal tissue samples. Finally, the set of top 100 genes that showed overexpression correlated with low survival was used to build a CRC risk predictor applying a multivariate Cox proportional hazards regression analysis. This risk predictor yielded an optimal separation of the individual patients of the cohort according to their survival, with a p-value of 8.25e-14 and Hazard Ratio 2.14 (95% CI: 1.75-2.61).
CONCLUSIONS: The results presented in this work provide a solid rationale for the prognostic utility of a new set of genes in CRC, demonstrating their potential to predict colorectal tumor progression and evolution towards poor survival stages. Our study does not provide a fixed gene signature for prognosis and risk prediction, but instead proposes a robust set of genes ranked according to their predictive power that can be selected for additional tests with other CRC clinical cohorts.

AIM: Growth arrest and DNA-damage-inducible 45 beta (GADD45β) is a member of the gene family associated with cell growth control, apoptosis, and DNA damage repair. The aim of present study was to determine the potential effects of GADD45β deletion on prostate hyperplasia progression.
MAIN METHODS: LNCaP cells were incubated with testosterone propionate (1 μM) for 48 h and specific siRNA used to suppress GADD45β expression in vitro. For in vivo experiments, testosterone (3 mg/kg, IP) was injected into wild-type (WT) and GADD45β knockout (GADD45β
KEY FINDINGS: GADD45β-silenced LNCaP cells showed suppressed testosterone-induced 5α-reductase 2 and androgen receptor expression compared to control LNCaP cells. Moreover, after 21 days of testosterone treatment, prostate weight and stromal tissue increment were relatively lower in GADD45β
SIGNIFICANCE: Although the exact correlation between GADD45β and prostate hyperplasia remains to be established, the present GADD45βdeletion suppressed testosterone-induced prostate hyperplasia which was accompanied by inhibition of 5α-reductase 2-related protein expression.

Purified Brazilin from Sappan wood extract has been reported with significant antitumor effect, especially on human T24 cells and bladder cancer mouse models. Brazilin can significantly induce expression of c-Fos and GADD45β and transfection expression of c-Fos and GADD45β in T24 cells can induce significant cell morphology changes, reduced viability and cell death, while transfection of siRNA-c-Fos and siRNA-GADD45β can reverse the induced cell death. Co-transfection of both c-Fos and GADD45β into T24 cells resulted in a significantly additive effect when compared to single transfection with only c-Fos or GADD45β. Meanwhile, transfection of interfering siRNA-c-Fos or siRNA-GADD45β can partially rescue the cell viability and siRNA co-transfection showed increased rescue rate. The transfection expression and interference with pcDNA3.1-c-Fos/siRNA-c-Fos or pcDNA3.1-GADD45β/siRNA-GADD45β did not affect each other's expression. Moreover, analysis of c-Fos and GADD45β regulated genes and signal pathways showed that no common regulated genes or pathways were present. All the results indicated that c-Fos and GADD45β mediate independent Brazilin-inducible genes and pathways. © 2018 IUBMB Life, 70(11):1101-1110, 2018.

BACKGROUND: Matrine is a naturally occurring alkaloid extracted from the Chinese herb Sophora flavescens. It has been demonstrated to exhibit antiproliferative properties, promote apoptosis, and inhibit cell invasion in a number of cancer cell lines by modulating the NF-κB pathway to downregulate the expression of MMP2 and MM9. It has also been shown to improve the efficacy of chemotherapy when it is combined with other chemotherapy drugs. However, the therapeutic potential of matrine for prostate cancer needs to be further studied.
METHODS: We analyzed KEGG pathways of differential gene expression between matrine-treated and untreated prostate cancer cell lines and identified GADD45B as one of major target genes of matrine based on its role in apoptosis and prognosis value for prostate cancer patients in TCGA database. We further analyzed the expression of GADD45B protein in a tissue microarray and mRNA in TCGA database, and tested the synergistic impacts of matrine and GADD45B overexpression on proliferation, apoptosis, migration and invasion of prostate cancer cell DU145.
RESULTS: Matrine promoted the expression of GADD45B, a tumor suppressive gene that is involved in the regulation of cell cycle, DNA damage repair, cell survival, aging, apoptosis and other cellular processes through p38/JNK, ROS-GADD45B-p38, or other signal pathways. Although GADD45B is elevated in prostate cancer tissues, levels of GADD45B in prostate tumor tissues are reduced at late stage of tumor invasion, and higher levels of GADD45B predict better survivals of prostate cancer patients.
CONCLUSIONS: Matrine may be used to treat prostate cancer patients to increase the levels of GADD45B to inhibit tumor invasion and improve patient survivals.

Multiple types of high throughput genomics data create a potential opportunity to identify driver patterns in ovarian cancer, which will acquire some novel and clinical biomarkers for appropriate diagnosis and treatment to cancer patients. To identify candidate driver genes and the corresponding driving patterns for resistant and sensitive tumors from the heterogeneous data, we combined gene co-expression modules with mutation modulators and proposed the method to identify driver patterns. Firstly, co-expression network analysis is applied to explore gene modules for gene expression profiles through weighted correlation network analysis (WGCNA). Secondly, mutation matrix is generated by integrating the CNV data and somatic mutation data, and a mutation network is constructed from the mutation matrix. Thirdly, candidate modulators are selected from significant genes by clustering vertexs of the mutation network. Finally, a regression tree model is utilized for module network learning, in which the obtained gene modules and candidate modulators are trained for the driving pattern identification and modulators regulatory exploration. Many identified candidate modulators are known to be involved in biological meaningful processes associated with ovarian cancer, such as CCL11, CCL16, CCL18, CCL23, CCL8, CCL5, APOB, BRCA1, SLC18A1, FGF22, GADD45B, GNA15, GNA11, and so on.

Cellular FLICE inhibitory protein (c-FLIP) is a key anti-apoptotic regulator that associates with the signaling complex downstream of NF-κB, negatively interfering with apoptotic signaling. The role of c-FLIP downregulation by negative regulation of NF-κB signaling during apoptosis is poorly understood. Here, we demonstrate that NF-κB-mediated c-FLIPL negatively regulates the JNK signaling pathway, and that cordycepin treatment of human renal cancer cells leads to apoptosis induction through c-FLIPL inhibition. TNF-α-induced inflammatory microenvironments stimulated NF-κB signaling and the c-FLIP long form (c-FLIPL) in TK-10 cells. Specifically, cordycepin inhibited TNF-α-mediated NF-κB activation, which induced renal cancer cell apoptosis. Cordycepin downregulated GADD45B and c-FLIPL, but upregulated MKK7 and phospho-JNK, by preventing nuclear mobilization of NF-κB. Furthermore, siRNA-mediated knockdown of GADD45B in cordycepin-treated TK-10 cells considerably increased MKK7 compared to cordycepin alone. siRNA-mediated knockdown of c-FLIPL prevented TNF-α-induced JNK inactivation, whereas c-FLIPL overexpression inhibited cordycepin-mediated JNK activation. The JNK inhibitor SP600125 strongly inhibited Bax expression. In nude mice, cordycepin significantly decreased tumor volume. Taken together, the results indicate that cordycepin inhibits TNF-α-mediated NF-κB/GADD45B signaling, which activates the MKK7-JNK signaling pathway through inhibition of c-FLIPL expression, thus inducing TK-10 cell apoptosis.

Lung cancer is the most common cause of cancer-related mortality worldwide. The GADD45 gene family plays important roles in a variety of the responses to cell injury including cell cycle checkpoints, apoptosis, DNA repair and anti-tumor immunity. The 19p13.3-GADD45B encoded protein product is involved in apoptosis and inhibiting tumor growth. To evaluate the association of 19p13.3-GADD45B common variants and lung cancer risk, the present study containing 544 Chinese lung cancer cases and 550 cancer-free controls was conducted. Three htSNPs (haplotype-tagging single nucleotide polymorphism) (rs7354, rs14384, and rs3783501) covering 95% of the common haplotype diversity in 19p13.3-GADD45B and interaction of 19p13.3-GADD45B and 19q13.3-PPP1R13L and 19q13.3-CD3EAP variants and smoking-duration were explored. Genotype and allele frequencies and haplotype distributions of the 19p13.3-GADD45B 3 htSNPs were not associated with lung cancer risk after adjustment for smoking status. 19p13.3-GADD45B rs7354 was associated with lung cancer risk among ≤20 (years) smokers [C/A-A/A versus CC, OR (95% CI) = 3.20 (1.11-9.20), P = 0.025] in a dominant model stratified by smoking duration. MDR (multifactor dimensionality reduction) analyses showed that smoking history as main effect and three-way models (smoking duration, 19p13.3-GADD45B rs3783501, 19q13.3-CD3EAP rs967591) (P = 0.001-0.002) indicated statistically significant association with lung cancer risk. The study identified evidence implicating DNA damage response genes on chromosome 19 in etiology of smoke-exposed lung cancer. In conclusion, our findings demonstrate that 19p13.3-GADD45B rs7354 variant and interaction between 19p13.3-GADD45B rs3783501 and 19q13.3-CD3EAP rs967591 may play a role in association with smoke-exposed lung cancer among Chinese. 19p13.3-GADD45B variants should be further evaluated in large prospective studies with molecular pathological annotations of lung cancer.

PURPOSE: Human melanoma is a highly aggressive incurable cancer due to intrinsic cellular resistance to apoptosis, reprogramming, proliferation and survival during tumour progression. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, plays a role in carcinogenesis in many cancer types. However, the cytotoxic molecular mechanisms and gene expression profiles promoted by SFN in human melanoma remain unknown.
METHODS: Three different cell lines were used: two human melanoma A375 and 501MEL and human epidermal melanocytes (HEMa). Cell viability and proliferation, cell cycle analysis, cell migration and invasion and protein expression and phosphorylation status of Akt and p53 upon SFN treatment were determined. RNA-seq of A375 was performed at different time points after SFN treatment.
RESULTS: We demonstrated that SFN strongly decreased cell viability and proliferation, induced G
CONCLUSION: Overall, the data show that SFN cytotoxicity in melanoma derives from complex and concurrent mechanisms during carcinogenesis, which makes it a promising cancer prevention agent.

Glioma Stem-like Cells (GSCs) isolated from patient derived tumors have high metabolic activity and survive in the absence of exogenous growth factors. We recently demonstrated that acute D609 (Tricyclodecan-9-yl-xanthogenate), a PC-PLC inhibitor with anti-oxidative property, can decrease the ATP content & GADD45β protein in GSCs cultured without growth factors, but not in the presence of growth factors. In this study we examined the effect of chronic D609 treatment on GSCs cultured in complete medium containing growth factors. Our results show that chronic exposure of GSCs to D609 decreased the ATP content and reduced the expression of GADD45β protein. Furthermore, cyclin D1 content and the phosphorylation of retinoblastoma protein also diminished, resulting in the arrest of cells in G1 phase of cell cycle after D609 treatment. In addition, the expression of Olig2, a protein responsible for the progression of glioblastoma was reduced by D609. Together these results indicate that chronic D609 treatment can inhibit the growth of glioma cells by arresting cells in G1 phase of cell cycle and/or reducing Olig2 expression.

Most patients with multiple myeloma treated with current therapies, including immunomodulatory drugs, eventually develop relapsed/refractory disease. Clinical activity of lenalidomide relies on degradation of Ikaros and the consequent reduction in IRF4 expression, both required for myeloma cell survival and involved in the regulation of MYC transcription. Thus, we sought to determine the combinational effect of an MYC-interfering therapy with lenalidomide/dexamethasone. We analyzed the potential therapeutic effect of the combination of the BET bromodomain inhibitor CPI203 with the lenalidomide/dexamethasone regimen in myeloma cell lines. CPI203 exerted a dose-dependent cell growth inhibition in cell lines, indeed in lenalidomide/dexamethasone-resistant cells (median response at 0.5 μM: 65.4%), characterized by G1 cell cycle blockade and a concomitant inhibition of MYC and Ikaros signaling. These effects were potentiated by the addition of lenalidomide/dexamethasone. Results were validated in primary plasma cells from patients with multiple myeloma co-cultured with the mesenchymal stromal cell line stromaNKtert. Consistently, the drug combination evoked a 50% reduction in cell proliferation and correlated with basal Ikaros mRNA expression levels (

Prostate cancer (PCa) is the second leading cause of death from cancer in men. The mechanism underlying tumorigenesis and development of PCa is largely unknown. Here, we identified Kinesin family member 14 (KIF14) as a novel candidate oncogene in PCa. We found that KIF14 was overexpressed in multiple PCa cell lines and primary PCa tissues. Knockdown of KIF14 in DU145 and PC3 prostate cancer cells suppressed cell proliferation, induced cell cycle arrest and apoptosis. Transcriptome analysis by RNA-sequencing demonstrated that KIF4 suppression led to transcriptional changes of genes involved in p53 and TGF-beta signaling pathway. In addition, upregulated expression of GADD45A, GADD45B, p21, PIDD and Shisa5, which contribute to growth arrest and apoptosis induction, and downregulated CCNB1 that promotes cell cycle progression were confirmed by quantitative real-time PCR after KIF4 knockdown. We further found that KIF14 protein level was positively correlated with T stage and Gleason Score. Patients with higher KIF14 expression had shorter overall survival time than those with lower KIF14 expression. Thus, our data indicate that KIF14 could act as a potential oncogene that contributes to tumor progression and poor prognosis in PCa, which may represent a novel and useful prognostic biomarker for PCa.

BACKGROUND: Ovarian cancer (OC) is a gynecological oncology that has a poor prognosis and high mortality. This study is conducted to identify the key genes implicated in the prognosis of OC by bioinformatic analysis.
METHODS: Gene expression data (including 568 primary OC tissues, 17 recurrent OC tissues, and 8 adjacent normal tissues) and the relevant clinical information of OC patients were downloaded from The Cancer Genome Atlas database. After data preprocessing, cluster analysis was conducted using the ConsensusClusterPlus package in R. Using the limma package in R, differential analysis was performed to identify feature genes. Based on Kaplan-Meier (KM) survival analysis, prognostic seed genes were selected from the feature genes. After key prognostic genes were further screened by cluster analysis and KM survival analysis, they were performed functional enrichment analysis and multivariate survival analysis. Using the survival package in R, cox regression analysis was conducted for the microarray data of GSE17260 to validate the key prognostic genes.
RESULTS: A total of 3668 feature genes were obtained, among which 75 genes were identified as prognostic seed genes. Then, 25 key prognostic genes were screened, including AXL, FOS, KLF6, WDR77, DUSP1, GADD45B, and SLIT3. Especially, AXL and SLIT3 were enriched in ovulation cycle. Multivariate survival analysis showed that the key prognostic genes could effectively differentiate the samples and were significantly associated with prognosis. Additionally, GSE17260 confirmed that the key prognostic genes were associated with the prognosis of OC.
CONCLUSION: AXL, FOS, KLF6, WDR77, DUSP1, GADD45B, and SLIT3 might affect the prognosis of OC.

The Gadd45a stress sensor gene is a member in the Gadd45 family of genes that includes Gadd45b & Gadd45g. To investigate the effect of GADD45A in the development of CML, syngeneic wild type lethally irradiated mice were reconstituted with either wild type or Gadd45a null myeloid progenitors transduced with a retroviral vector expressing the 210-kD BCR-ABL fusion oncoprotein. Loss of Gadd45a was observed to accelerate BCR-ABL driven CML resulting in the development of a more aggressive disease, a significantly shortened median mice survival time, and increased BCR-ABL expressing leukemic stem/progenitor cells (GFP+Lin- cKit+Sca+). GADD45A deficient progenitors expressing BCR-ABL exhibited increased proliferation and decreased apoptosis relative to WT counterparts, which was associated with enhanced PI3K-AKT-mTOR-4E-BP1 signaling, upregulation of p30C/EBPα expression, and hyper-activation of p38 and Stat5. Furthermore, Gadd45a expression in samples obtained from CML patients was upregulated in more indolent chronic phase CML samples and down regulated in aggressive accelerated phase CML and blast crisis CML. These results provide novel evidence that Gadd45a functions as a suppressor of BCR/ABL driven leukemia and may provide a unique prognostic marker of CML progression.

Digestive cancers are major causes of mortality and morbidity worldwide. Fisetin, a naturally occurring flavonoid, has been previously shown anti-proliferative, anti-cancer, neuroprotective, and antioxidant activities. In our study, the anti-tumor activities in addition to regulatory effects of fisetin on some cancer cell lines were investigated. Data presented here showed that fisetin induces growth inhibition, and apoptosis in hepatic (HepG-2), colorectal (Caco-2) and pancreatic (Suit-2) cancer cell lines. Gene expression results showed that 1307 genes were significantly regulated in their expression in hepatic and pancreatic cell lines. 350 genes were commonly up-regulated and 353 genes were commonly down-regulated. Additionally, 604 genes were oppositely expressed in both tumor cells. CDK5 signaling, NRF2-mediated oxidative stress response, glucocorticoid signaling, and ERK/MAPK signaling were among most prominent signaling pathways modulating the growth inhibitory effects of fisetin on hepatic and pancreatic cancer cells. The present analysis showed, for the first time, that the anti-tumor effect of fisetin was mediated mainly through modulation of multiple signaling pathways and via activation of CDKN1A, SEMA3E, GADD45B and GADD45A and down-regulation of TOP2A, KIF20A, CCNB2 and CCNB1 genes.

GADD45β (Growth Arrest and DNA Damage inducible protein) is a stress activated protein which plays an important role in regulating apoptosis, proliferation, DNA repair and potentially may have a role in cancer. In this study we examined the role of anti-oxidative stress on the expression of GADD45β in glioma stem-like cells (GSC). We show that patient derived GSCs have high survival in the absence of exogenous growth factors. Addition of D609 (Tricyclodecan-9-yl-xanthogenate), a known anti-oxidative compound, to GSCs reduced the cellular ATP content with significant effects observed when GSCs were cultured in growth factor free medium. D609 exposure also resulted in a decrease in the protein and an increase in mRNA of GADD45β with a concomitant decline in the survival of cells. However, under similar conditions the phosphorylation of p38 MAP kinase (stress activated MAP kinase), a downstream target of GADD45β, was significantly enhanced in response to D609. Therefore it appears that GADD45β might play a role in glioma stem cell survival and that p38 MAP kinase may not be directly activated by GADD45β. Together these observations suggest that anti-oxidative compounds like D609 can target GADD45β which may be one strategy to curtail the growth of glioma stem like cells.

The aim of this study was to investigate the differences in the gene expression profiles of radiation-sensitive (RS) and radiation-resistant (RR) sinonasal squamous cell carcinoma (SNSCC) and to identify prognostic markers for the radiation reaction of SNSCC. We first examined the differentially expressed genes (DEGs) in RS and RR SNSCC tissues by analyzing clinical samples with GeneChip Human Transcriptome Array 2.0 (HTA 2.0).To understand the functional significance of the molecular changes, we examined the DEGs with Gene Ontology (GO) and pathway analyses to identify the core genes. The expression of several core genes (CCND2, COL5A2, GADD45B, and THBS2) was confirmed with reverse transcription quantitative PCR (RT-qPCR) in a larger series of tissues. We identified 208 DEGs, of which 76 were upregulated and 132 downregulated in the RS tissues relative to the RR tissues. The DEGs were mainly involved in the regulation of cell proliferation, the NF-kappaB signaling pathway, the cell adhesion molecule signaling pathway, and the extracellular matrix-receptor interaction signaling pathway. RT-qPCR confirmed that the CCND2, COL5A2, GADD45B, and THBS2 genes were significantly differentially expressed in the RS and RR tissues, consistent with the GeneChip data. These results extend our understanding of the molecular mechanisms underlying the sensitivity of SNSCC to radiation. The DEGs are involved in the differential response to radiation therapy and the dysregulated core genes identified in this study can be used to predict radiation sensitivity in SNSCC.

PURPOSE: This study was aimed at developing and validating a quantitative multigene assay for predicting tumor recurrence after gastric cancer surgery.
EXPERIMENTAL DESIGN: Gene expression data were generated from tumor tissues of patients who underwent surgery for gastric cancer (n = 267, training cohort). Genes whose expression was significantly associated with activation of YAP1 (a frequently activated oncogene in gastrointestinal cancer), 5-year recurrence-free survival, and 5-year overall survival were first identified as candidates for prognostic genes (156 genes, P < 0.001). We developed the recurrence risk score (RRS) by using quantitative RT-PCR to identify genes whose expression levels were significantly associated with YAP1 activation and patient survival in the training cohort.
RESULTS: We based the RRS assay on 6 genes, IGFBP4, SFRP4, SPOCK1, SULF1, THBS, and GADD45B, whose expression levels were significantly associated with YAP1 activation and prognosis in the training cohort. The RRS assay was further validated in an independent cohort of 317 patients. In multivariate analysis, the RRS was an independent predictor of recurrence [HR, 1.6; 95% confidence interval (CI), 1.02-2.4; P = 0.03]. In patients with stage II disease, the RRS had an HR of 2.9 (95% CI, 1.1-7.9; P = 0.03) and was the only significant independent predictor of recurrence.
CONCLUSIONS: The RRS assay was a valid predictor of recurrence in the two cohorts of patients with gastric cancer. Independent prospective studies to assess the clinical utility of this assay are warranted. Clin Cancer Res; 22(24); 6228-35. ©2016 AACR.

Globally, cervical cancer is the most common malignancy affecting women. The main treatment methods for this type of cancer include conization or hysterectomy procedures. Sulforaphane (SFN) is a natural, compound-based drug derived from dietary isothiocyanates which has previously been shown to possess potent anti-tumor and chemopreventive effects against several types of cancer. The present study investigated the effects of SFN on anti-proliferation and G₂/M phase cell cycle arrest in cervical cancer cell lines (Cx, CxWJ, and HeLa). We found that cytotoxicity is associated with an accumulation of cells in the G₂/M phases of the cell-cycle. Treatment with SFN led to cell cycle arrest as well as the down-regulation of Cyclin B1 expression, but not of CDC2 expression. In addition, the effects of GADD45β gene activation in cell cycle arrest increase proportionally with the dose of SFN; however, mitotic delay and the inhibition of proliferation both depend on the dosage of SFN used to treat cancer cells. These results indicate that SFN may delay the development of cancer by arresting cell growth in the G₂/M phase via down-regulation of Cyclin B1 gene expression, dissociation of the cyclin B1/CDC2 complex, and up-regulation of GADD45β proteins.

In Western countries, the incidence of testicular germ cell cancers (GCC) is steadily rising over the last decades. Mostly, men between 20 and 40 years of age are affected. In general, patients suffering from GCCs are treated by orchiectomy and radio- or chemotherapy. Due to resistance mechanisms, intolerance to the therapy or denial of chemo- / radiotherapy by the patients, GCCs are still a lethal threat, highlighting the need for alternative treatment strategies.In this study, we revealed that germ cell cancer cell lines are highly sensitive to the histone deacetylase inhibitor romidepsin in vitro and in vivo, highlighting romidepsin as a potential therapeutic option for GCC patients.Romidepsin-mediated inhibition of histone deacetylases led to disturbances of the chromatin landscape. This resulted in locus-specific histone-hyper- or hypoacetylation. We found that hypoacetylation at the ARID1A promotor caused repression of the SWI/SNF-complex member ARID1A. In consequence, this resulted in upregulation of the stress-sensors and apoptosis-regulators GADD45B, DUSP1 and CDKN1A. RNAi-driven knock down of ARID1A mimicked in parts the effects of romidepsin, while CRISPR/Cas9-mediated deletion of GADD45B attenuated the romidepsin-provoked induction of apoptosis and cell cycle alterations.We propose a signaling cascade involving ARID1A, GADD45B and DUSP1 as mediators of the romidepsin effects in GCC cells.

BACKGROUND: African American (AA) colon cancer patients have a worse prognosis than Caucasian (CA) colon cancer patients, however, reasons for this disparity are not well understood. To determine if tumor biology might contribute to differential prognosis, we measured recurrence risk and gene expression using the Oncotype DX® Colon Cancer Assay (12-gene assay) and compared the Recurrence Score results and gene expression profiles between AA patients and CA patients with stage II colon cancer.
METHODS: We retrieved demographic, clinical, and archived tumor tissues from stage II colon cancer patients at four institutions. The 12-gene assay and mismatch repair (MMR) status were performed by Genomic Health (Redwood City, California). Student's t-test and the Wilcoxon rank sum test were used to compare Recurrence Score data and gene expression data from AA and CA patients (SAS Enterprise Guide 5.1).
RESULTS: Samples from 122 AA and 122 CA patients were analyzed. There were 118 women (63 AA, 55 CA) and 126 men (59 AA, 67 CA). Median age was 66 years for AA patients and 68 for CA patients. Age, gender, year of surgery, pathologic T-stage, tumor location, the number of lymph nodes examined, lymphovascular invasion, and MMR status were not significantly different between groups (p = 0.93). The mean Recurrence Score result for AA patients (27.9 ± 12.8) and CA patients (28.1 ± 11.8) was not significantly different and the proportions of patients with high Recurrence Score values (≥41) were similar between the groups (17/122 AA; 15/122 CA). None of the gene expression variables, either single genes or gene groups (cell cycle group, stromal group, BGN1, FAP, INHBA1, Ki67, MYBL2, cMYC and GADD45B), was significantly different between the racial groups. After controlling for clinical and pathologic covariates, the means and distributions of Recurrence Score results and gene expression profiles showed no statistically significant difference between patient groups.
CONCLUSION: The distribution of Recurrence Score results and gene expression data was similar in a cohort of AA and CA patients with stage II colon cancer and similar clinical characteristics, suggesting that tumor biology, as represented by the 12-gene assay, did not differ between patient groups.

Growth arrest DNA damage-inducible gene 45β (GADD45β), which influences cell growth, apoptosis and cellular response to DNA damage, is downregulated in hepatocellular carcinoma (HCC). S-adenosylmethionine (SAMe) serves as an essential methyl donor in multiple metabolic pathways and is a polyamine and glutathione (GSH) precursor. In this study, we assessed the roles of GADD45β and SAMe in cell survival during acute ischemia-hypoxia (I/H). SAMe treatment induced growth of HL-7702 normal hepatic cells, but decreased the viability of HepG2 (p53 wild-type) and Hep3B (p53 null) HCC cells. Cells were exposed to I/H with or without SAMe pre-treatment. I/H exposure alone triggered HCC cell proliferation promoted by autophagy. SAMe pre-treatment restored GADD45β expression and activated HCC cell apoptosis and eliminated I/H-induced HCC cell proliferation. p53 loss blunted the response to SAMe and I/H exposure in Hep3B cells; thus, the inhibitory effect of SAMe on cell proliferation may be reduced in p53-null cells as compared to wild-type cells. These results indicate that GADD45β induction by SAMe inhibits HCC cell proliferation during I/H as a result of increased apoptosis, and that SAMe also protects normal hepatocytes from apoptotic cell death and promotes normal cell regeneration. SAMe should be considered a potential therapeutic agent for the management of HCC.

BACKGROUND: 5-Azacytidine (5-AZA), a DNA methyl transferase inhibitor, is a clinically used epigenetic drug for cancer therapy. Recently, we have shown that 5-AZA upregulates ten-eleven translocation (TET) protein expression in hepatocellular carcinoma (HCC) cells, which induce active demethylation. Vitamin C facilitates TET activity and enhances active demethylation. The aim of this study is to investigate whether vitamin C is able to enhance the effect of 5-AZA on active demethylation and to evaluate its consequence in HCC cell lines.
METHODS: HCC cell lines (Huh7 and HLE) were treated with 5-AZA and vitamin C. After 48 h of treatment, viability (resazurin conversion), toxicity (lactose dehydrogenase (LDH) release), and proliferation ((proliferating cell nuclear antigen (PCNA)) of single- and combined-treated cells were assessed. The effect of the treatment on 5-hydroxymethylcytosine (5hmC) intensity (immunofluorescence (IF) staining), TET, Snail, GADD45B, and P21 mRNA (real-time PCR) and protein expression (Western blot) were investigated.
RESULTS: Our results indicated that vitamin C enhances the anti-proliferative and apoptotic effect of 5-AZA in HCC cell lines. By further analyzing the events leading to cell cycle arrest, we have shown for the first time in HCC that the combination of 5-AZA and vitamin C leads to an enhanced downregulation of Snail expression, a key transcription factor governing epithelial-mesenchymal transition (EMT) process, and cell cycle arrest.
CONCLUSIONS: We conclude that when combined with 5-AZA, vitamin C enhances TET activity in HCC cells, leading to induction of active demethylation. An increase in P21 expression as a consequence of downregulation of Snail accompanied by the induction of GADD45B expression is the main mechanism leading to cell cycle arrest in HCCs.

The JNK and P38α pathways play a crucial role in tissue homeostasis, apoptosis and autophagy under genotoxic stresses, but it is unclear whether single nucleotide polymorphisms (SNPs) of genes in these pathways play a role in platinum-based chemotherapy-induced toxicities in patients with advanced non-small cell lung cancer (NSCLC). We genotyped 11 selected, independent, potentially functional SNPs of nine genes in the JNK and P38α pathways in 689 patients with advanced NSCLC treated with platinum-combination chemotherapy regimens. Associations between these SNPs and chemotherapy toxicities were tested in a discovery group of 345 patients and then validated in a replication group of 344 patients. In both discovery and validation groups as well as their pooled analysis, carriers of GADD45B rs2024144T variant allele had a significantly higher risk for severe hematologic toxicity and carriers of MAPK14 rs3804451A variant allele had a significantly higher risk for both overall toxicity and gastrointestinal toxicity. In addition, carriers of GADD45A rs581000C had a lower risk of anemia, while carriers of GADD45B rs2024144T had a significantly higher risk for leukocytopenia or agranulocytosis. The present study provides evidence that genetic variants in genes involved in the JNK and P38α pathways may predict platinum-based chemotherapy toxicity outcomes in patients with advanced NSCLC. Larger studies of other patient populations are needed to validate our findings.

BACKGROUND: Statins purportedly exert antitumoral effects, but the underlying mechanisms are currently not fully elucidated. The aim of this study was to explore potential statin-induced effects on global gene expression profiles in primary breast cancer.
EXPERIMENTAL DESIGN: This window-of-opportunity phase II trial enrolled 50 newly diagnosed breast cancer patients prescribed atorvastatin (80 mg/day) for 2 weeks presurgically. Pre- and posttreatment tumor samples were analyzed using Significance Analysis of Microarrays (SAM) to identify differentially expressed genes. Similarly, SAM and gene ontology analyses were applied to gene expression data derived from atorvastatin-treated breast cancer cell lines (MCF7, BT474, SKBR3, and MDAMB231) comparing treated and untreated cells. The Systematic Motif Analysis Retrieval Tool (SMART) was used to identify enriched transcription factor-binding sites. Literature Vector Analysis (LitVAn) identified gene module functionality, and pathway analysis was performed using GeneGo Pathways Software (MetaCore; https://portal.genego.com/).
RESULTS: Comparative analysis of gene expression profiles in paired clinical samples revealed 407 significantly differentially expressed genes (FDR = 0); 32 upregulated and 375 downregulated genes. Restricted filtration (fold change ≥1.49) resulted in 21 upregulated and 46 downregulated genes. Significantly upregulated genes included DUSP1, RHOB1, GADD45B, and RGS1. Pooled results from gene ontology, LitVAn and SMART analyses identified statin-induced effects on the apoptotic and MAPK pathways among others. Comparative analyses of gene expression profiles in breast cancer cell lines showed significant upregulation of the mevalonate and proapoptotic pathways following atorvastatin treatment.
CONCLUSIONS: We report potential statin-induced changes in global tumor gene expression profiles, indicating MAPK pathway inhibition and proapoptotic events.

High doses of sodium phenobarbital (NaPB), a constitutive androstane receptor (CAR) activator, have been shown to produce hepatocellular tumors in rodents by a mitogenic mode of action (MOA) involving CAR activation. The effect of 1-week dietary treatment with NaPB on liver weight and histopathology, hepatic CYP2B enzyme activity and CYP2B/3A mRNA expression, replicative DNA synthesis and selected genes related to cell proliferation, and functional transcriptomic and metabolomic analyses was studied in male CD-1 mice, Wistar Hannover (WH) rats, and chimeric mice with human hepatocytes. The treatment of chimeric mice with 1000-1500-ppm NaPB resulted in plasma levels around 3-5-fold higher than those observed in human subjects given therapeutic doses of NaPB. NaPB produced dose-dependent increases in hepatic CYP2B activity and CYP2B/3A mRNA levels in all animal models. Integrated functional metabolomic and transcriptomic analyses demonstrated that the responses to NaPB in the human liver were clearly different from those in rodents. Although NaPB produced a dose-dependent increase in hepatocyte replicative DNA synthesis in CD-1 mice and WH rats, no increase in replicative DNA synthesis was observed in human hepatocyte-originated areas of chimeric mice. In addition, treatment with NaPB had no effect on Ki-67, PCNA, GADD45β, and MDM2 mRNA expression in chimeric mice, whereas significant increases were observed in CD-1 mice and/or WH rats. However, increases in hepatocyte replicative DNA synthesis were observed in chimeric mice both in vivo and in vitro after treatment epidermal growth factor. Thus, although NaPB could activate CAR in both rodent and human hepatocytes, NaPB did not increase replicative DNA synthesis in human hepatocytes of chimeric mice, whereas it was mitogenic to rat and mouse hepatocytes. As human hepatocytes are refractory to the mitogenic effects of NaPB, the MOA for NaPB-induced rodent liver tumor formation is thus not relevant for humans.

Lung cancer is the leading cause of cancer related death and there is no effective treatment to date. Bufalin has been shown effective in inducing apoptosis and DNA damage in lung cancer cells. However, the genetic mechanisms underlying these actions have not been elucidated yet. Cultured NCI-H460 cells were treated with or without 2 μM of bufalin for 24 h. The total RNA was extracted from each treatment for cDNA synthesis and labeling, microarray hybridization, and then followed by flour-labeled cDNA hybridized on chip. The localized concentrations of fluorescent molecules were detected and quantitated and analyzed by Expression Console software (Affymetrix) with default RMA parameters. The key genes involved and their possible interaction pathways were mapped by GeneGo software. About 165 apoptosis-related genes were affected. CASP9 was up-regulated by 5.51 fold and THAP1 by 2.75-fold while CCAR1 was down-regulated by 2.24 fold. 107 genes related to DNA damage/repair were affected. MDC1 was down-regulated by 2.22-fold, DDIT4 by 2.52 fold while GADD45B up-regulated by 3.72 fold. 201 genes related to cell cycles were affected. CCPG1 was down-regulated by 2.11 fold and CDCA7L by 2.71 fold. Many genes about apoptosis, cell cycle regulation and DNA repair are changed significantly following bufalin treatment in NCI-H460 cells. These changes provide an in depth understanding of cytotoxic mechanism of bufalin in genetic level and also offer many potentially useful biomarkers for diagnosis and treatment of lung cancer in future.

Cucurbitacin E (CuE) is a natural compound previously shown to have anti-feedant, antioxidant and antitumor activities as well as a potent chemo-preventive action against cancer. The present study investigates its anti-proliferative property using MTT assay; CuE demonstrated cytotoxic activity against malignant glioma GBM 8401 cells and induced cell cycle G2/M arrest in these cells. CuE-treated cells accumulated in metaphase (CuE 2.5-10 μM) as determined using MPM-2 by flow cytometry. We attempted to characterize the molecular pathways responsible for cytotoxic effects of CuE in GBM 8401 cells. We studied the genome-wide gene expression profile on microarrays and molecular networks by using pathway analysis tools of bioinformatics. The CuE reduced the expression of 558 genes and elevated the levels of 1354 genes, suggesting an existence of the common pathways involved in induction of G2/M arrest. We identified the RB (GADD45β and GADD45γ) and the p53 (GADD45α) signaling pathways as the common pathways, serving as key molecules that regulate cell cycle. Results indicate that CuE produced G2/M arrest as well as the upregulation of GADD45 γ and binding with CDC2. Both effects increased proportionally with the dose of CuE, suggesting that the CuE-induced mitosis delay is regulated by GADD45γ overexpression. Our findings suggest that, in addition to the known effects on cancer prevention, CuE may have antitumor activity in glioma therapy.

The underlying molecular pathogenesis in hepatocellular carcinoma remains poorly understood. The transcription factor MEF2D promotes survival in various cell types and it seems to function as an oncogene in leukemia. However, its potential contributions to solid cancers have not been explored. In this study, we investigated MEF2D expression and function in hepatocellular carcinoma, finding that MEF2D elevation in hepatocellular carcinoma clinical specimens was associated with poor prognosis. MEF2D-positive primary hepatocellular carcinoma cells displayed a faster proliferation rate compared with MEF2D-negative cells, and silencing or promoting MEF2D expression in these settings limited or accelerated cell proliferation, respectively. Notably, MEF2D-silencing abolished hepatocellular carcinoma tumorigenicity in mouse xenograft models. Mechanistic investigations revealed that MEF2D-silencing triggered G2-M arrest in a manner associated with direct downregulation of the cell-cycle regulatory genes RPRM, GADD45A, GADD45B, and CDKN1A. Furthermore, we identified MEF2D as an authentic target of miR-122, the reduced expression of which in hepatocellular carcinoma may be responsible for MEF2D upregulation. Together, our results identify MEF2D as a candidate oncogene in hepatocellular carcinoma and a potential target for hepatocellular carcinoma therapy.

In the present study, we investigated the anticancer effects of the mitochondrial inhibitors, metaiodobenzylguanidine (MIBG), metformin and phenformin. 131I-MIBG has been used for scintigraphic detection and the targeted radiotherapy of neuroblastoma (NB), a pediatric malignancy. Non-radiolabeled MIBG has been reported to be cytotoxic to NB cells in vitro and in vivo. However, the mechanisms behind its growth suppressive effects have not yet been fully elucidated. Metformin and phenformin are diabetes medications that are being considered in anticancer therapeutics. We investigated the anticancer mechanisms of action of MIBG and metformin in NB. Our data revealed that both drugs suppressed NB cell growth and that the combination drug treatment was more potent. MIBG reduced MYCN and MYC expression in MYCN-amplified and non-MYCN-amplified NB cells in a dose- and time-dependent manner. Metformin was less effective than MIBG in destabilizing MYC/MYCN. The treatment of NB cells with metformin or MIBG resulted in an increased expression of genes encoding biomarkers for favorable outcome in NB [(ephrin (EFN)B2, EFNB3, EPH receptor B6 (EPHB6), neurotrophic tyrosine kinase, receptor, type 1 (NTRK1), CD44 and Myc-interacting zinc finger protein (MIZ-1)] and tumor suppressor genes [(early growth response 1 (EGR1), EPH receptor A2 (EPHA2), growth arrest and DNA-damage-inducible, beta (GADD45B), neuregulin 1 (NRG1), TP53 apoptosis effector (PERP) and sel-1 suppressor of lin-12-like (C. elegans) (SEL1L)]. Accordingly, metformin and MIBG augmented histone H3 acetylation in these cells. Phenformin also exhibited histone modification and was more effective than metformin in destabilizing MYC/MYCN in NB cells. Our data suggest that the destabilization of MYC/MYCN by MIBG, metformin and phenformin and their effects on histone modification are important mechanisms underlying their anticancer effects.

Injury and growth stimulation both remarkably increase the hepatic expression of Gadd45β. In liver cancer, promoter methylation frequently silences Gadd45β, demonstrating due to a suppressive function that is often proapoptotic. This contrasts with normal hepatocytes, where Gadd45β facilitates cell survival, growth, and proliferation. Gadd45β binds MKK7-downstream of TNFα and its receptors-to prevent this kinase from activating JNK2. Hence, the Gadd45b-/- genotype increases cell injury and decreases cell proliferation during liver regeneration (i.e., compensatory growth and proliferation). Liver hyperplasia (i.e., de novo growth and proliferation) is an alternate form of growth, caused by drugs that activate the nuclear receptor, CAR. As in regeneration, the Gadd45b-/- genotype considerably slows growth during hyperplasia. However, there is no injury and the slowing occurs because Gadd45β normally binds to CAR and activates its transcriptional stimulation. Thus, Gadd45β protects the liver through two entirely different processes: binding MKK7 to block damaging signal transduction or binding CAR to coactivate anabolic transcription.