Aberrant activation of Wnt signalling through hypermethylation of Wnt inhibitor genes is involved in several human malignancies, including acute myeloid leukaemia (AML). It remains unclear whether hypermethylation of Wnt inhibitors is associated with molecular gene mutations in the development of AML.
We investigated the association of the promoter hypermethylation of six Wnt inhibitors (Wif-1, SFRP1, SFRR2, SFRP4, SFRP5, and DKK1) with gene aberrations in the leukaemogenesis of 269 AML patients.
In total, 166 patients (61.7%) had hypermethylation of at least one Wnt inhibitor. The majority (68.5%) of patients with Wnt inhibitor hypermethylation had concurrent Class II gene mutations that affect transcription factors or cofactors. There was a close association of Wif-1 hypermethylation with t(15;17) (P=0.0005) and CEBPA mutation (P<0.0001), DKK1 hypermethylation with t(8;21) (P<0.0001) and ASXL1 mutation (P=0.0078), SFRP-1 hypermethylation with t(8;21) (P<0.0001), SFRP-2 hypermethylation with AML1/RUNX1 mutation (P=0.0012), and SFRP-5 hypermethylation with MLL/PTD (P=0.0505). On the other side, hypermethylation of Wnt inhibitors was always negatively associated with NPM1 mutation and FLT3/ITD.
There was distinct association between hypermethylation of individual Wnt inhibitors and specific gene aberrations, especially Class II mutations. The Wnt inhibitor hypermethylation might interact with genetic alterations in the leukaemogenesis.
Wnt pathway inhibitors; methylation; genetic alteration; acute myeloid leukaemia
The etiology of esophageal squamous cell cancer (ESCC) in high prevalence regions of China remains unclear.
Endoscopic biopsies were conducted among 7381 inhabitants aged from 25 to 65 of Anyang, China.
In this study, 2.57, 0.20 and 0.16% of the participants had mild, moderate and severe squamous dysplasia, respectively; 0.19 and 0.08% showed squamous carcinoma in situ and invasive ESCC. Using deep well (depth >100 meters) as water source (odds ratio=0.72, 95% confidence interval: 0.54–0.96) was negatively associated with ESCC and its precursors, whereas tobacco and alcohol use were not significantly associated with ESCC.
Water source and other factors in this region need further evaluation by longitudinal studies.
prevalence; risk factor; esophageal cancer; precursor lesion; China
We investigated common genetic variation in the entire ESR1 and EGF genes in relation to endometrial cancer risk, myometrial invasion and endometrial cancer survival. We genotyped a dense set of single-nucleotide polymorphisms (SNPs) in both genes and selected haplotype tagging SNPs (tagSNPs). The tagSNPs were genotyped in 713 Swedish endometrial cancer cases and 1567 population controls and the results incorporated into logistic regression and Cox proportional hazards models. We found five adjacent tagSNPs covering a region of 15 kb at the 5′ end of ESR1 that decreased the endometrial cancer risk. The ESR1 variants did not, however, seem to affect myometrial invasion or endometrial cancer survival. For the EGF gene, no association emerged between common genetic variants and endometrial cancer risk or myometrial invasion, but we found a five-tagSNP region that covered 51 kb at the 5′ end of the gene where all five tagSNPs seemed to decrease the risk of dying from endometrial cancer. One of the five tagSNPs in this region was in strong linkage disequilibrium (LD) with the untranslated A61G (rs4444903) EGF variant, earlier shown to be associated with risk for other forms of cancer.
ESR1; EGF; polymorphism; endometrial cancer; survival
Molecular markers involved in DNA repair can help to predict survival in gastric cancer patients treated with 5-FU plus platinum chemotherapy. Excision repair cross-complementing 1 (ERCC1) and thymidylate synthase (TS) mRNA expression levels were assessed in advanced gastric cancer tumour samples using real-time quantitative PCR in 76 patients treated with a modified FOLFOX (biweekly oxaliplatin plus 5-FU and folinic acid) regimen. Median survival time in patients with low ERCC1 levels was significantly longer than in those with high levels (15.8 vs 6.2 months; P<0.0001). Patients with high TS levels had longer survival than those with low levels (12.2 vs 10.1 months; P=0.01). Forty-eight patients with low ERCC1 and high TS levels had a median survival of 16.1 months (P<0.0001). The hazard ratio for patients with high ERCC1 expression was 9.4 (P<0.0001). In patients with high mRNA levels of ERCC1, alternative chemotherapy regimens should be considered.
ERCC1; TS; advanced gastric cancer; FOLFOX
p53 codon 72 polymorphism; cervical cancer; Chinese
There is evidence that tumours produce substances such as cytokines and microvesicular bodies bearing bioactive molecules, which support the carcinogenic process. Furthermore, chemotherapy has also been shown to modify these exudates and in doing so, neutralise their tumourigenic influence.
In the current study, we have investigated the effect of chemotherapy agents on modifying the cytokine profile and microvesicular cargo of supernatants derived from cancer cell lines. In addition, we have explored the effect of these tumour-derived supernatants on angiogenesis, and how chemotherapy can alter the supernatants rendering them less pro-angiogenic.
Herein, we show that supernatants contain a rich cocktail of cytokines, a number of which are potent modulators of angiogenesis. They also contain microvesicular bodies containing RNA transcripts that code for proteins involved in transcription, immune modulation and angiogenesis. These supernatants altered intracellular signalling molecules in endothelial cells and significantly enhanced their tubulogenic character; however, this was severely compromised when supernatants from tumours treated with chemotherapy was used instead.
This study suggests tumour exudates and bioactive material from tumours can influence cellular functions, and that treatment with some chemotherapy can serve to negate these pro-tumourigenic processes.
tumour-supernatants; chemotherapy; immunotherapy; angiogenesis; immune-modulation; microvesicles
To determine whether non-viral nasopharyngeal carcinoma (NPC) risk factors might be associated with (and mediated through) Epstein–Barr virus (EBV) serological responses linked to NPC risk, we evaluated predictors of risk of anti-EBNA1 IgA seropositivity and other markers among unaffected relatives from a large NPC family study in Taiwan.
Multivariate logistic regression conditioned on family was used to examine the associations between sociodemographic, dietary, lifestyle, and occupational variables and risk of anti-EBV EBNA1 IgA positivity, anti-VCA IgA, and anti-DNase positivity.
Among 2393 unaffected relatives from 319 multiplex families, 1180 (49.3%) were anti-EBV EBNA1 IgA seropositive. None of the associations with anti-EBNA1 IgA were statistically significant, except for being 31–50 years of age (vs <30, adjusted ORs 0.51–0.57). For one or more EBV serological markers, there were suggestive associations for older age, GuangDong firm salted fish, betel use, current alcohol use, and male gender.
Overall, we found little evidence to suggest that non-viral NPC risk factors significantly alter EBV serological patterns, suggesting that non-viral NPC risk factors act through pathways independent of EBV serological responses.
Epstein–Barr virus; EBNA1; VCA; IgA; DNase; nasopharyngeal carcinoma
Cancerous inhibitor of protein phosphatase 2A (CIP2A) drives cellular transformation. The objective of this study was to detect the potential effects of CIP2A in renal cell carcinomas (RCCs).
A total of 107 RCC patients were involved in the study. Cancerous inhibitor of protein phosphatase 2A expression was investigated by real-time PCR and immunohistochemistry. In vitro, we examined the expression of CIP2A and c-Myc and tested the migration and invasion capability of A498 and KRC/Y cells with scratch migration assay and Matrigel invasion assay after down-regulating CIP2A expression using siRNA.
Cancerous inhibitor of protein phosphatase 2A was over-expressed in RCC tissues. Clear cell RCC showed an even higher-CIP2A expression level than papillary or chromophobe RCC did. The CIP2A immunostaining level was positively correlated with primary tumour stage, lymph node metastasis, distant metastasis, TNM stage and histological grade (all P<0.05). High-CIP2A expression implied poor survival for patients (P<0.05). Cancerous inhibitor of protein phosphatase 2A depletion by siRNA down-regulated c-Myc expression and attenuated the migration and invasion of RCC cells.
Higher-CIP2A expression positively correlates with the aggressive phenotype of RCCs, and predicts poor prognosis for patients. Cancerous inhibitor of protein phosphatase 2A may be a novel target for prevention and treatment of RCC metastasis and recurrence.
CIP2A; RCC; metastasis; prognosis; survival
It has been suggested that the relative importance of oestrogen-metabolising pathways may affect the risk of oestrogen-dependent tumours including endometrial cancer. One hypothesis is that the 2-hydroxy pathway is protective, whereas the 16α-hydroxy pathway is harmful.
We conducted a case–control study nested within three prospective cohorts to assess whether the circulating 2-hydroxyestrone : 16α-hydroxyestrone (2-OHE1 : 16α-OHE1) ratio is inversely associated with endometrial cancer risk in postmenopausal women. A total of 179 cases and 336 controls, matching cases on cohort, age and date of blood donation, were included. Levels of 2-OHE1 and 16α-OHE1 were measured using a monoclonal antibody-based enzyme assay.
Endometrial cancer risk increased with increasing levels of both metabolites, with odds ratios in the top tertiles of 2.4 (95% CI=1.3, 4.6; Ptrend=0.007) for 2-OHE1 and 1.9 (95% CI=1.1, 3.5; Ptrend=0.03) for 16α-OHE1 in analyses adjusting for endometrial cancer risk factors. These associations were attenuated and no longer statistically significant after further adjustment for oestrone or oestradiol levels. No significant association was observed for the 2-OHE1 : 16α-OHE1 ratio.
Our results do not support the hypothesis that greater metabolism of oestrogen via the 2-OH pathway, relative to the 16α-OH pathway, protects against endometrial cancer.
endometrial cancer; nested case–control study; oestrogen metabolites; 2-hydroxyestrone; 16α-hydroxyestrone, post-menopause
Reduced expression of class 1 human leucocyte antigens (HLA1) is often a mechanism by which tumours evade surveillance by the host immune system. This is often associated with an immune function that is unable to mount appropriate responses against disease, which can result in a state that favours carcinogenesis.
In the current study, we have explored the effects of Bacillus Calmette-Guerin (BCG) on the cytokine output of leucocytes, which is a key determinant in generating antitumour action, and have also assessed the effect of these cytokine cocktails on HLA1 expression in solid tumour cell lines.
BCG potently activated a broad range of leucocytes, and also enhanced the production of cytokines that were Th1-predominant. Supernatants from BCG-treated leucocytes significantly increased the expression of HLA1 on the surface of cancer cell lines, which correlated with increased cytolytic T-cell activity. We also showed that the increased HLA1 expression was associated with activation of intracellular signalling pathways, which was triggered by the increases in the Th1-cytokines interferon-γ and tumour necrosis factor-α, as counteracting their effects negated the enhancement.
These studies reaffirm the role of BCG as a putative immunotherapy through their cytokine-modifying effects on leucocytes and their capacity to enhance tumour visibility.
HLA1; BCG; immunovisibility; immunotherapy; cytokines
Persistent activation of signal transducers and activators of transcription 3 (STAT3) is commonly detected in many types of cancer, including colon cancer. To date, whether STAT3 is activated and the effects of STAT3 inhibition by a newly developed curcumin analogue, GO-Y030, in colon cancer stem cells are still unknown.
Flow cytometry was used to isolate colon cancer stem cells, which are characterised by both aldehyde dehydrogenase (ALDH)-positive and CD133-positive subpopulations (ALDH+/CD133+). The levels of STAT3 phosphorylation and the effects of STAT3 inhibition by a newly developed curcumin analogue, GO-Y030, that targets STAT3 in colon cancer stem cells were examined.
Our results observed that ALDH+/CD133+ colon cancer cells expressed higher levels of phosphorylated STAT3 than ALDH-negative/CD133-negative colon cancer cells, suggesting that STAT3 is activated in colon cancer stem cells. GO-Y030 and curcumin inhibited STAT3 phosphorylation, cell viability, tumoursphere formation in colon cancer stem cells. GO-Y030 also reduced STAT3 downstream target gene expression and induced apoptosis in colon cancer stem cells. Furthermore, GO-Y030 suppressed tumour growth of cancer stem cells from both SW480 and HCT-116 colon cancer cell lines in the mouse model.
Our results indicate that STAT3 is a novel therapeutic target in colon cancer stem cells, and inhibition of activated STAT3 in cancer stem cells by GO-Y030 may offer an effective treatment for colorectal cancer.
STAT3; curcumin analogue; colon cancer; cancer stem cells; ALDH; CD133
Previous studies indicate that disulfiram (DS), an anti-alcoholism drug, is cytotoxic to cancer cell lines and reverses anticancer drug resistance. Cancer stem cells (CSCs) are the major cause of chemoresistance leading to the failure of cancer chemotherapy. This study intended to examine the effect of DS on breast cancer stem cells (BCSCs).
The effect of DS on BC cell lines and BCSCs was determined by MTT, western blot, CSCs culture and CSCs marker analysis.
Disulfiram was highly toxic to BC cell lines in vitro in a copper (Cu)-dependent manner. In Cu-containing medium (1 μ), the IC50 concentrations of DS in BC cell lines were 200–500 n. Disulfiram/copper significantly enhanced (3.7–15.5-fold) cytotoxicity of paclitaxel (PAC). Combination index isobologram analysis demonstrated a synergistic effect between DS/Cu and PAC. The increased Bax and Bcl2 protein expression ratio indicated that intrinsic apoptotic pathway may be involved in DS/Cu-induced apoptosis. Clonogenic assay showed DS/Cu-inhibited clonogenicity of BC cells. Mammosphere formation and the ALDH1+VE and CD24Low/CD44High CSCs population in mammospheres were significantly inhibited by exposure to DS/Cu for 24 h. Disulfiram/copper induced reactive oxygen species (ROS) generation and activated its downstream apoptosis-related cJun N-terminal kinase and p38 MAPK pathways. Meanwhile, the constitutive NFκB activity in BC cell lines was inhibited by DS/Cu.
Disulfiram/copper inhibited BCSCs and enhanced cytotoxicity of PAC in BC cell lines. This may be caused by simultaneous induction of ROS and inhibition of NFκB.
disulfiram; reactive oxygen species; NFκB; breast cancer stem cells; paclitaxel; MAPK pathway
The epithelial growth factor receptor (EGFR) family of receptor tyrosine kinases has been reported to have an active role in a number of malignancies. Amplifications and overexpression of various EGFR family members, including EGFR, Her2, and ErbB3, have been reported in epithelial ovarian cancer. Although anti-EGFR-targeted therapy has shown limited clinical activity in ovarian cancer to date, a recent report suggests that activation of ErbB3, one of the members of the EGFR family, may support the growth and proliferation of ovarian cancer cells and that ErbB3 may therefore serve as a potential therapeutic target in this disease. Here, we review the EGFR family and the clinical experience with anti-EGFR family member-directed therapies in ovarian cancer to date.
EGFR family; ErbB3; ovarian cancer
Recent genome-wide association studies of colorectal cancer (CRC) have identified common single-nucleotide polymorphisms (SNPs) mapping to 10 independent loci that confer modest increased risk. These studies have been conducted in European populations and it is unclear whether these observations generalise to populations with different ethnicities and rates of CRC.
An association study was performed on 892 CRC cases and 890 controls recruited from the Hong Kong Chinese population, genotyping 32 SNPs, which were either associated with CRC in previous studies or are in close proximity to previously reported risk SNPs.
Twelve of the SNPs showed evidence of an association. The strongest associations were provided by rs10795668 on 10p14, rs4779584 on 15q14 and rs12953717 on 18q21.2. There was significant linear association between CRC risk and the number of independent risk variants possessed by an individual (P=2.29 × 10−5).
These results indicate that some previously reported SNP associations also impact on CRC risk in the Chinese population. Possible reasons for failure of replication for some loci include inadequate study power, differences in allele frequency, linkage disequilibrium structure or effect size between populations. Our results suggest that many associations for CRC are likely to generalise across populations.
colorectal cancer; genetic; association; replication; Chinese
The ratio of digit lengths is fixed in utero, and may be a proxy indicator for prenatal testosterone levels.
We analysed the right-hand pattern and prostate cancer risk in 1524 prostate cancer cases and 3044 population-based controls.
Compared with index finger shorter than ring finger (low 2D : 4D), men with index finger longer than ring finger (high 2D : 4D) showed a negative association, suggesting a protective effect with a 33% risk reduction (odds ratio (OR) 0.67, 95% confidence interval (CI) 0.57–0.80). Risk reduction was even greater (87%) in age group <60 (OR 0.13, 95% CI 0.09–0.21).
Pattern of finger lengths may be a simple marker of prostate cancer risk, with length of 2D greater than 4D suggestive of lower risk.
prostate cancer; hand pattern; case–control study
Recently, microRNAs in cancer development have attracted much attention, but their roles in tumorigenesis are still largely unknown. In this study, a functional role of miR-22 in hepatocellular carcinoma (HCC) development has been identified.
Quantitative real-time PCR was used to determine the level of miR-22 transcript in HCC clinical samples, and its correlation with disease-free survival was determined using Kaplan–Meier method. Restoration of miR-22 expression was carried out in HCC cell lines to assess its influence on HCC cell proliferation and tumourigenicity.
In the 160 paired HCC tissue samples, miR-22 expression was downregulated in HCC, and low miR-22 expression in HCC was predictive of poor survival in HCC patients. Functional studies indicated that ectopic expression of miR-22 significantly inhibits HCC cell proliferation and tumourigenicity. Furthermore, histone deacetylase 4 (HDAC4), known to have critical roles in cancer development, was proved to be directly targeted and regulated by miR-22. Furthermore, HDAC4 was upregulated in miR-22-downregulated HCC tissues, suggesting that downregulation of miR-22 might participate in HCC carcinogenesis and progression through potentiation of HDAC4 expression. In addition, cell proliferation was also suppressed by knockdown of HDAC4 or treatment with HDAC inhibitor trichostatin A in HCC cell lines.
miR-22, downregulated in HCC, has an anti-proliferative effect on HCC cells both in vitro and in vivo. Furthermore, miR-22 may have considerable potential in identification of the prognosis and application of cancer therapy for HCC patients.
miR-22; hepatocellular carcinoma; prognosis; proliferation; HDAC4
O6-methylguanine-DNA methyltransferase (MGMT) expression in glioblastoma correlates with temozolomide resistance. Dose-intense temozolomide schedules deplete MGMT activity in peripheral blood mononuclear cells; however, no published data exist evaluating the effect of temozolomide schedules on intracranial tumour MGMT activity.
Human glioblastoma cells (GBM43) with an unmethylated MGMT promoter were implanted intracranially in immunodeficient rodents. Three weeks later, animals received temozolomide 200 mg m−2 for 5 days (schedule A, standard dose) or 100 mg m−2 for 21 days (schedule B, dose intense).
Tumour MGMT activity was depleted by day 6 in both treatment groups compared with baseline. O6-methylguanine-DNA methyltransferase activity returned to baseline by day 22 in the schedule A group, but remained suppressed in the schedule B group. By day 29, MGMT activity had returned to baseline in both groups. Mean tumour volume was significantly decreased compared with untreated controls with either schedule (P<0.01), although neither schedule was superior (P=0.60). Median survival was 64, 42, and 28 days for schedule A, schedule B, and no drug, respectively (P<0.001 A or B vs control, P=NS A vs B).
Dose-intense temozolomide prolongs tumour MGMT activity depletion compared with standard dosing, however, survival was not improved in this model.
temozolomide; glioblastoma; O6-methylguanine-DNA methyltransferase; xenograft
Colorectal cancer is (CRC) one of the commonest cancers and its therapy is still based on few drugs. Currently, no biological criteria are used to choose the most effective of the established drugs for treatment.
A panel of 77 CRC cell lines was tested for sensitivity to 5-fluorouracil (5FU) using the SRB assay. The responses were grouped into three categories and correlated with genetic changes in the cell lines.
The strongest and most clearcut correlation was between 5-fluorouracil response and replication error status (mismatch repair deficiency). All the other significant correlations (loss of heterozygosity for DCC and mutations in TGFbIIR) are secondary to the association with replication error status.
Interpretation and conclusion:
Our findings validate previous analyses based mainly on clinical data, and indicate that replication error status could be a useful guide to 5-fluorouracil-based CRC therapy. Essentially, all previously described correlations with 5FU response are secondary to the association with replication error status.
5-fluorouracil; mismatch repair deficiency; replication error; colorectal cancer; cell lines
MicroRNAs (miRNAs) are small noncoding RNAs that have important roles in numerous cellular processes. Recent studies have shown aberrant expression of miRNAs in prostate cancer tissues and cell lines. On the basis of miRNA microarray data, we found that miR-145 is significantly downregulated in prostate cancer.
Methods and results:
We investigated the expression and functional significance of miR-145 in prostate cancer. The expression of miR-145 was low in all the prostate cell lines tested (PC3, LNCaP and DU145) compared with the normal cell line, PWR-1E, and in cancerous regions of human prostate tissue when compared with the matched adjacent normal. Overexpression of miR-145 in PC3-transfected cells resulted in increased apoptosis and an increase in cells in the G2/M phase, as detected by flow cytometry. Investigation of the mechanisms of inactivation of miR-145 through epigenetic pathways revealed significant DNA methylation of the miR-145 promoter region in prostate cancer cell lines. Microarray analyses of miR-145-overexpressing PC3 cells showed upregulation of the pro-apoptotic gene TNFSF10, which was confirmed by real-time PCR and western analysis.
One of the genes significantly upregulated by miR-145 overexpression is the proapoptotic gene TNFSF10. Therefore, modulation of miR-145 may be an important therapeutic approach for the management of prostate cancer.
miR145; prostate; cell growth; apoptosis; TNFSF10
Portal vein tumour thrombus (PVTT) is highly associated with the progression and metastasis of hepatocellular carcinoma (HCC). However, there are no appropriate cell models of PVTT with which to study the biological and physiological characteristics of PVTT.
Primary cell culture was performed by the use of a successive xenograft line called PVTT-#1, which was obtained from a 60-year-old male HCC patient accompanied by PVTT.
A successive cell line named CSQT-2 was established. The cell line showed aggressive phenotypes in terms of cell growth, survival, migration, xenograft and metastasis. Moreover, an orthotopic transplantation assay showed that PVTT can be generated in nude mice when CSQT-2 cells were inoculated in the liver and that it shows a typical migratory tendency in the vascular branches of portal vein. Moreover, the established CSQT-2 cells also showed varied expression of tumour-initiating cell (TIC) markers such as CD133, CD90 and EpCAM.
Establishment of CSQT-2 may provide a suitable model with which to investigate the molecular mechanisms of PVTT-related HCC.
PVTT; HCC; IVIS; TIC; cell line; metastasis