Correction: Tsoi et al. Overexpression of BQ323636.1 Modulated AR/IL-8/CXCR1 Axis to Confer Tamoxifen Resistance in ER-Positive Breast Cancer. Life 2022, 12, 93.

In the original publication (doi: 10 [...].

SRSF5 Regulates the Expression of BQ323636.1 to Modulate Tamoxifen Resistance in ER-Positive Breast Cancer.

About 70% of breast cancer patients are oestrogen receptor-positive (ER +ve). Adjuvant endocrine therapy using tamoxifen (TAM) is an effective approach for preventing local recurrence and metastasis. However, around half of the patients will eventually develop resistance. Overexpression of BQ323636.1 (BQ) is one of the mechanisms that confer TAM resistance. BQ is an alternative splice variant of NCOR2. The inclusion of exon 11 generates mRNA for NCOR2, while the exclusion of exon 11 produces mRNA for BQ. The expression of SRSF5 is low in TAM-resistant breast cancer cells. Modulation of SRSF5 can affect the alternative splicing of NCOR2 to produce BQ. In vitro and in vivo studies confirmed that the knockdown of SRSF5 enhanced BQ expression, and conferred TAM resistance; in contrast, SRSF5 overexpression reduced BQ expression and, thus, reversed TAM resistance. Clinical investigation using a tissue microarray confirmed the inverse correlation of SRSF5 and BQ. Low SRSF5 expression was associated with TAM resistance, local recurrence and metastasis. Survival analyses showed that low SRSF5 expression was associated with poorer prognosis. We showed that SRPK1 can interact with SRSF5 to phosphorylate it. Inhibition of SRPK1 by a small inhibitor, SRPKIN-1, suppressed the phosphorylation of SRSF5. This enhanced the proportion of SRSF5 interacting with exon 11 of NCOR2, reducing the production of BQ mRNA. As expected, SRPKIN-1 reduced TAM resistance. Our study confirms that SRSF5 is essential for BQ expression. Modulating the activity of SRSF5 in ER +ve breast cancer will be a potential approach to combating TAM resistance.

Modulating the Activity of Androgen Receptor for Treating Breast Cancer.

The androgen receptor (AR) is a steroid hormone receptor widely detected in breast cancer. Evidence suggests that the AR might be a tumor suppressor in estrogen receptor alpha-positive (ERα+ve) breast cancer but a tumor promoter in estrogen receptor alpha-negative (ERα-ve) breast cancer. Modulating AR activity could be a potential strategy for treating breast cancer. For ERα+ve breast cancer, activation of the AR had been demonstrated to suppress the disease. In contrast, for ERα-ve breast cancer, blocking the AR could confer better prognosis to patients. These studies support the feasibility of utilizing AR modulators as anti-cancer drugs for different subtypes of breast cancer patients. Nevertheless, several issues still need to be addressed, such as the lack of standardization in the determination of AR positivity and the presence of AR splice variants. In future, the inclusion of the AR status in the breast cancer report at the time of diagnosis might help improve disease classification and treatment decision, thereby providing additional treatment strategies for breast cancer.

Checkpoint Kinase 2 Inhibition Can Reverse Tamoxifen Resistance in ER-Positive Breast Cancer.

Breast cancer is a heterogeneous disease. Tamoxifen is frequently used to treat ER-positive breast cancer. Our team has identified a novel splice variant of NCOR2, BQ323636.1 (BQ), that mediates tamoxifen resistance. However, the upstream factors that modulate BQ expression are not apparent. This study reveals that tamoxifen treatment causes induction of DNA damage which can enhance BQ expression. We show that DNA damage can activate the ATM/CHK2 and ATR/CHK1 signalling cascades and confirm that ATM/CHK2 signalling is responsible for enhancing the protein stability of BQ. siRNA or a small inhibitor targeting CHK2 resulted in the reduction in BQ expression through reduced phosphorylation and enhanced poly-ubiquitination of BQ. Inhibition of CHK2 by CCT241533 could reverse tamoxifen resistance in vitro and in vivo. Using clinical samples in the tissue microarray, we confirmed that high p-CHK2 expression was significantly associated with high nuclear BQ expression, tamoxifen resistance and poorer overall and disease-specific survival. In conclusion, tamoxifen treatment can enhance BQ expression in ER-positive breast cancer by activating the ATM/CHK2 axis. Targeting CHK2 is a promising approach to overcoming tamoxifen resistance in ER-positive breast cancer.

Targeting Ribosome Biogenesis to Combat Tamoxifen Resistance in ER+ve Breast Cancer.

Breast cancer is a heterogeneous disease. Around 70% of breast cancers are estrogen receptor-positive (ER+ve), with tamoxifen being most commonly used as an adjuvant treatment to prevent recurrence and metastasis. However, half of the patients will eventually develop tamoxifen resistance. The overexpression of c-MYC can drive the development of ER+ve breast cancer and confer tamoxifen resistance through multiple pathways. One key mechanism is to enhance ribosome biogenesis, synthesising mature ribosomes. The over-production of ribosomes sustains the demand for proteins necessary to maintain a high cell proliferation rate and combat apoptosis induced by therapeutic agents. c-MYC overexpression can induce the expression of eIF4E that favours the translation of structured mRNA to produce oncogenic factors that promote cell proliferation and confer tamoxifen resistance. Either non-phosphorylated or phosphorylated eIF4E can mediate such an effect. Since ribosomes play an essential role in c-MYC-mediated cancer development, suppressing ribosome biogenesis may help reduce aggressiveness and reverse tamoxifen resistance in breast cancer. CX-5461, CX-3543 and haemanthamine have been shown to repress ribosome biogenesis. Using these chemicals might help reverse tamoxifen resistance in ER+ve breast cancer, provided that c-MYC-mediated ribosome biogenesis is the crucial factor for tamoxifen resistance. To employ these ribosome biogenesis inhibitors to combat tamoxifen resistance in the future, identification of predictive markers will be necessary.

Overexpression of BQ323636.1 Modulated AR/IL-8/CXCR1 Axis to Confer Tamoxifen Resistance in ER-Positive Breast Cancer.

NCOR2 is a co-repressor for estrogen receptor (ER) and androgen receptor (AR). Our group previously identified a novel splice variant of NCOR2, BQ323636.1 (BQ), that mediates tamoxifen resistance via interference of NCOR2 repression on ER. Luciferase reporter assay showed BQ overexpression could enhance the transcriptional activity of androgen response element (ARE). We proposed that BQ employs both AR and ER to confer tamoxifen resistance. Through in silico analysis, we identified interleukin-8 (IL-8) as the sole ERE and ARE containing gene responsiveness to ER and AR activation. We confirmed that BQ overexpression enhanced the expression of IL-8 in ER+ve breast cancer cells, and AR inhibition reduced IL-8 expression in the BQ overexpressing cell lines, suggesting that AR was involved in the modulation of IL-8 expression by BQ. Moreover, we demonstrated that IL-8 could activate both AKT and ERK1/2 via CXCR1 to confer tamoxifen resistance. Targeting CXCR1/2 by a small inhibitor repertaxin reversed tamoxifen resistance of BQ overexpressing breast cancer cells in vitro and in vivo. In conclusion, BQ overexpression in ER+ve breast cancer can enhance IL-8 mediated signaling to modulate tamoxifen resistance. Targeting IL-8 signaling is a promising approach to overcome tamoxifen resistance in ER+ve breast cancer.

Targeting the IL-6/STAT3 Signalling Cascade to Reverse Tamoxifen Resistance in Estrogen Receptor Positive Breast Cancer.

Breast cancer is the most common female cancer. About 70% of breast cancer patients are estrogen receptor α (ERα) positive (ER+) with tamoxifen being the most commonly used anti-endocrine therapy. However, up to 50% of patients who receive tamoxifen suffer recurrence. We previously identified BQ323636.1 (BQ), a novel splice variant of NCOR2, can robustly predict tamoxifen resistance in ER+ primary breast cancer. Here we show that BQ can enhance IL-6/STAT3 signalling. We demonstrated that through interfering with NCOR2 suppressive activity, BQ favours the binding of ER to IL-6 promoter and the binding of NF-ĸB to IL-6 receptor (IL-6R) promoter, leading to the up-regulation of both IL-6 and IL-6R and thus the activation of STAT3. Knockdown of IL-6R could compromise tamoxifen resistance mediated by BQ. Furthermore, Tocilizumab (TCZ), an antibody that binds to IL-6R, could effectively reverse tamoxifen resistance both in vitro and in vivo. Analysis of clinical breast cancer samples confirmed that IL-6R expression was significantly associated with BQ expression and tamoxifen resistance in primary breast cancer, with high IL-6R expression correlating with poorer survival. Multivariate Cox-regression analysis confirmed that high IL-6R expression remained significantly associated with poor overall as well as disease-specific survival in ER+ breast cancer.

Targeting microtubules sensitizes drug resistant lung cancer cells to lysosomal pathway inhibitors.

Oncogene-addicted cancers are predominantly driven by specific oncogenic pathways and display initial exquisite sensitivity to designer therapies, but eventually become refractory to treatments. Clear understanding of lung tumorigenic mechanisms is essential for improved therapies. Methods: Lysosomes were analyzed in EGFR-WT and mutant cells and corresponding patient samples using immunofluorescence or immunohistochemistry and immunoblotting. Microtubule organization and dynamics were studied using immunofluorescence analyses. Also, we have validated our findings in a transgenic mouse model that contain EGFR-TKI resistant mutations. Results: We herein describe a novel mechanism that a mutated kinase disrupts the microtubule organization and results in a defective endosomal/lysosomal pathway. This prevents the efficient degradation of phosphorylated proteins that become trapped within the endosomes and continue to signal, therefore amplifying downstream proliferative and survival pathways. Phenotypically, a distinctive subcellular appearance of LAMP1 secondary to microtubule dysfunction in cells expressing EGFR kinase mutants is seen, and this may have potential diagnostic applications for the detection of such mutants. We demonstrate that lysosomal-inhibitors re-sensitize resistant cells to EGFR tyrosine-kinase inhibitors (TKIs). Identifying the endosome-lysosome pathway and microtubule dysfunction as a mechanism of resistance allows to pharmacologically intervene on this pathway. Conclusions: We find that the combination of microtubule stabilizing agent and lysosome inhibitor could reduce the tumor progression in EGFR TKI resistant mouse models of lung cancer.

Phosphorylation independent eIF4E translational reprogramming of selective mRNAs determines tamoxifen resistance in breast cancer.

Eukaryotic translation initiation factor 4E (eIF4E) selectively promotes translation of mRNAs with atypically long and structured 5'-UTRs and has been implicated in drug resistance. Through genome-wide transcriptome and translatome analysis we revealed eIF4E overexpression could promote cellular activities mediated by ERα and FOXM1 signalling pathways. Whilst eIF4E overexpression could enhance the translation of both ERα and FOXM1, it also led to enhanced transcription of FOXM1. Polysome fractionation experiments confirmed eIF4E could modulate the translation of ERα and FOXM1 mRNA. The enhancement of FOXM1 transcription was contingent upon the presence of ERα, and it was the high levels of FOXM1 that conferred Tamoxifen resistance. Furthermore, tamoxifen resistance was conferred by phosphorylation independent eIF4E overexpression. Immunohistochemistry on 134 estrogen receptor (ER+) primary breast cancer samples confirmed that high eIF4E expression was significantly associated with increased ERα and FOXM1, and significantly associated with tamoxifen resistance. Our study uncovers a novel mechanism whereby phosphorylation independent eIF4E translational reprogramming in governing the protein synthesis of ERα and FOXM1 contributes to anti-estrogen insensitivity in ER+ breast cancer. In eIF4E overexpressing breast cancer, the increased ERα protein expression in turn enhances FOXM1 transcription, which together with its increased translation regulated by eIF4E, contributes to tamoxifen resistance. Coupled with eIF4E translational regulation, our study highlights an important mechanism conferring tamoxifen resistance via both ERα dependent and independent pathways.

BQ323636.1, a Novel Splice Variant to NCOR2, as a Predictor for Tamoxifen-Resistant Breast Cancer.

Purpose: Adjuvant tamoxifen treatment revolutionized the management of estrogen receptor (ER)-positive breast cancers to prevent cancer recurrence; however, drug resistance compromises its clinical efficacy. The mechanisms underlying tamoxifen resistance are not fully understood, and no robust biomarker is available to reliably predict those who will be resistant. Here, we study BQ323636.1, a novel splice variant of the NCOR2 gene, and evaluate its efficacy in predicting tamoxifen resistance in patients with breast cancer.Experimental Design: A monoclonal anti-BQ323636.1 antibody that specifically recognizes the unique epitope of this splice variant was generated for in vitro mechanistic studies and for in vivo analysis by immunohistochemistry on tissue microarrays of two independent cohorts of 358 patients with more than 10 years clinical follow-up data, who had ER-positive primary breast cancer and received adjuvant tamoxifen treatment. An orthotopic mouse model was also used.Results: Overexpression of BQ323636.1 conferred resistance to tamoxifen in both in vitro and in an orthotopic mouse model. Mechanistically, coimmunoprecipitation showed BQ323636.1 could bind to NCOR2 and inhibit the formation of corepressor complex for the suppression of ER signaling. Nuclear BQ3232636.1 overexpression in patients samples was significantly associated with tamoxifen resistance (P = 1.79 × 10-6, sensitivity 52.9%, specificity 72.0%). In tamoxifen-treated patients, nuclear BQ323636.1 overexpression was significantly correlated with cancer metastasis and disease relapse. Nuclear BQ323636.1 was also significantly associated with poorer overall survival (P = 1.13 × 10-4) and disease-specific survival (P = 4.02 × 10-5).Conclusions: These findings demonstrate that BQ323636.1 can be a reliable biomarker to predict tamoxifen resistance in patients with ER-positive breast cancer. Clin Cancer Res; 24(15); 3681-91. ©2018 AACRSee related commentary by Jordan, p. 3480.

Dual-utility NLS drives RNF169-dependent DNA damage responses.

Loading of p53-binding protein 1 (53BP1) and receptor-associated protein 80 (RAP80) at DNA double-strand breaks (DSBs) drives cell cycle checkpoint activation but is counterproductive to high-fidelity DNA repair. ring finger protein 169 (RNF169) maintains the balance by limiting the deposition of DNA damage mediator proteins at the damaged chromatin. We report here that this attribute is accomplished, in part, by a predicted nuclear localization signal (NLS) that not only shuttles RNF169 into the nucleus but also promotes its stability by mediating a direct interaction with the ubiquitin-specific protease USP7. Guided by the crystal structure of USP7 in complex with the RNF169 NLS, we uncoupled USP7 binding from its nuclear import function and showed that perturbing the USP7-RNF169 complex destabilized RNF169, compromised high-fidelity DSB repair, and hypersensitized cells to poly (ADP-ribose) polymerase inhibition. Finally, expression of USP7 and RNF169 positively correlated in breast cancer specimens. Collectively, our findings uncover an NLS-mediated bipartite mechanism that supports the nuclear function of a DSB response protein.

Comparison of fluorescence in-situ hybridisation with dual-colour in-situ hybridisation for assessment of HER2 gene amplification of breast cancer in Hong Kong.

OBJECTIVES: To compare the PathVysion fluorescence in-situ hybridisation assay with the INFORM HER2 Dual in-situ hybridisation assay on 104 invasive breast cancers with a broad spectrum of immunohistochemistry scores. METHODS: This case series involved consecutive patients diagnosed with invasive breast carcinoma with equivocal immunohistochemistry score and referred for further HER2 assessment from the departments of Surgery and/or Clinical Oncology of the two hospitals between January 2013 and February 2014. An additional 10 cases with negative HER2 immunohistochemistry and 11 cases with positive HER2 immunohistochemistry were further included. RESULTS: The results of both fluorescence in-situ hybridisation and dual in-situ hybridisation were available in 99 of 104 cases, respectively. Student'st test showed no statistically significant difference in the mean number of HER2 count, CEP17 copies, or HER2/CEP17 ratio between that obtained by fluorescence in-situ hybridisation and that obtained by dual in-situ hybridisation. Pearson's correlation of results for the two assays was strong for HER2/CEP17 signal ratio (R=0.963, P<0.001) and mean HER2 copies per nucleus (R=0.897, P<0.001). Overall agreement was 96.0% (95 out of 99 cases, ĸ0.882). Three of the four discordant cases were equivocal for either fluorescence in-situ hybridisation or dual in-situ hybridisation. The results of immunohistochemistry 0/1+ and 3+ cases showed 100% concordance between the two assays. The failure rate was 0.96% for fluorescence in-situ hybridisation and 3.85% for dual in-situ hybridisation. Cases that failed for fluorescence in-situ hybridisation were successful for dual in-situ hybridisation and vice versa. CONCLUSIONS: Our study showed that dual in-situ hybridisation is a reliable and useful option for HER2 testing in breast cancer.

Genome-wide association analysis in East Asians identifies breast cancer susceptibility loci at 1q32.1, 5q14.3 and 15q26.1.

In a three-stage genome-wide association study among East Asian women including 22,780 cases and 24,181 controls, we identified 3 genetic loci newly associated with breast cancer risk, including rs4951011 at 1q32.1 (in intron 2 of the ZC3H11A gene; P=8.82×10(-9)), rs10474352 at 5q14.3 (near the ARRDC3 gene; P=1.67×10(-9)) and rs2290203 at 15q26.1 (in intron 14 of the PRC1 gene; P=4.25×10(-8)). We replicated these associations in 16,003 cases and 41,335 controls of European ancestry (P=0.030, 0.004 and 0.010, respectively). Data from the ENCODE Project suggest that variants rs4951011 and rs10474352 might be located in an enhancer region and transcription factor binding sites, respectively. This study provides additional insights into the genetics and biology of breast cancer.

SIRT6 modulates paclitaxel and epirubicin resistance and survival in breast cancer.

In this study, we report the identification of a novel role of SIRT6 in both epirubicin and paclitaxel resistance in breast cancer. We found that SIRT6 protein levels are elevated in paclitaxel- and epirubicin-resistant MCF-7 cells compared with the parental sensitive cells. SIRT6 knockout and depletion sensitized cells to both paclitaxel and epirubicin treatment, whereas SIRT6 ectopic overexpression led to increased resistance to paclitaxel and epirubicin. Moreover, our data suggest that SIRT6 could be mediating epirubicin resistance through enhancing the DNA repair response to epirubicin-induced DNA damage. Clonogenic assays also revealed that mouse embryonic fibroblasts (MEFs) lacking SIRT6 have decreased long-term viability in response to epirubicin. The tumour suppressor FOXO3a increases its levels of acetylation in MEFs depleted of SIRT6, whereas its induction by epirubicin is attenuated in breast cancer cells overexpressing SIRT6. Further cell viability studies demonstrate that deletion of FOXO1/3/4 in MEFs can confer sensitivity to both paclitaxel and epirubicin, suggesting that SIRT6 reduces paclitaxel and epirubicin sensitivity, at least in part, through modulating FOXO acetylation and expression. Consistently, immunohistochemical analysis of 118 breast cancer patient samples revealed that high SIRT6 nuclear staining is significantly associated with poorer overall survival (P = 0.018; Kaplan-Meier analysis). Multivariate Cox analysis demonstrated that nuclear SIRT6 staining remained associated with death after correcting for tumour stage and lymph-node involvement (P = 0.033). Collectively, our data suggest that SIRT6 has a role in paclitaxel and epirubicin sensitivity via targeting FOXO proteins and that SIRT6 could be a useful biomarker and therapeutic target for paclitaxel- and epirubicin-resistant cancer.

Detection of hypermethylated DNA or cyclooxygenase-2 messenger RNA in fecal samples of patients with colorectal cancer or polyps.

BACKGROUND: Detection of fecal DNA is a promising approach to colorectal cancer screening. However, the sensitivity of current fecal DNA tests for colorectal polyps is low. We evaluated the feasibility of detecting aberrantly methylated DNA or cyclooxygenase-2 (COX-2) mRNA in feces of patients with colorectal cancer or polyps. METHODS: Fecal samples were collected prior to colonoscopy from 20 patients with colorectal cancer, 30 patients with colorectal polyps, and 30 subjects with normal colonic examination. Presence of hypermethylated DNA in 7 tumor-related genes (APC, ATM, hMLH1, sFRP2, HLTF, MGMT, and GSTP1) in stool was analyzed by methylation-specific PCR. COX-2 mRNA in fecal samples was detected by RT-PCR. RESULTS: With the use of this panel of methylation markers, the sensitivity of detecting colorectal cancer and adenoma was 75% (95% CI 50.9-91.3%) and 68% (95% CI 46.5-85.1%), respectively. Three normal subjects also had methylated DNA detected in stool, which gives a specificity of 90% (95% CI 73.5-97.9%). The mean number of genes methylated in DNA from the stool of patients with colorectal cancer and adenoma was 1.4 and 0.9, respectively. In contrast, COX-2 mRNA was detected in the stool samples of 10 (50%) cancer patients and one (4%) patient with advanced adenoma only. Two (6.7%) stool samples from normal subjects also had COX-2 mRNA detected. CONCLUSION: Detection of aberrantly methylated DNA in fecal samples is more sensitive than COX-2 mRNA for detection of colorectal cancer and adenoma.

Effects of Helicobacter pylori eradication on methylation status of E-cadherin gene in noncancerous stomach.

PURPOSE: Promoter hypermethylation of E-cadherin plays an important role on gastric cancer development. Whereas E-cadherin methylation was frequently detected in the stomach of Helicobacter pylori-infected individuals, we tested whether eradication of H. pylori alters the methylation status of the noncancerous gastric epithelium. EXPERIMENTAL DESIGN: Endoscopic biopsies were taken from the antrum and corpus of H. pylori-infected subjects without gastric cancer. Presence of methylated E-cadherin sequences in the gastric specimens was detected by methylation-specific PCR. Bisulfite DNA sequencing was done to determine the topographical distribution and changes in methylation profiles with H. pylori eradication. RESULTS: Among the 28 H. pylori-infected subjects (median age, 44.5 years), 15 (53.6%) had E-cadherin methylation detected in stomach at baseline. Discordant methylation patterns between the antrum and corpus were noted in six patients. One year after successful H. pylori eradication, there was a significant reduction in the methylation density of the promoter region and exon 1 of the E-cadherin gene as detected by bisulfite DNA sequencing (P < 0.001). CONCLUSION: Promoter methylation in E-cadherin was frequently detected in the stomach of H. pylori-infected individuals. Eradication of H. pylori might possibly reduce the methylation density in E-cadherin gene and the chance of subsequent neoplastic transformation.

H. pylori genotypes and cytokine gene polymorphisms influence the development of gastric intestinal metaplasia in a Chinese population.

BACKGROUND: Cytokine gene polymorphisms and Helicobacter pylori (HP) genotypes have been linked to gastric cancer development in Western countries. We determined the role of host cytokine polymorphisms and bacterial virulent factors in the development of gastric intestinal metaplasia (IM) in a Chinese population with a high background gastric cancer incidence. METHODS: Three hundred two HP-infected noncancer individuals living in Shandong province of China with available DNA were studied. Polymorphisms in different loci of inflammatory cytokines Interleukin IL-1B, IL-1RN, Interleukin IL-8, IL-10, IL-18, tumor necrosis factor-A (TNF-A), and Transforming growth factor (TGF-B), were determined by allelic discriminating TaqMan polymerase chain reaction (PCR) or a variable number of tandem repeats. Presence of HP virulence factors in cagA, vacA, and babA2 were determined by PCR. Baseline gastric biopsies were assessed for the presence of IM. RESULTS: Among HP-infected subjects, carriers of the IL-1B-511 T allele were associated with a modestly greater prevalence of IM (adjusted OR 2.0, 95% CI 1.0-3.7). There was no association between the presence of IM and polymorphisms in other inflammatory cytokines. Although most subjects from this region harbored the virulent HP strains, carriage of the vacA m1 strain was associated with a significantly higher prevalence of IM (adjusted OR 1.8, 1.1-3.0). The presence of both host (IL-1B-511 T) and HP (vacA m1) genotypes further increased the risk of IM (OR 5.7, 2.0-16) when compared with individuals with the low-risk genotype. CONCLUSION: The carriage of proinflammatory IL-1B-511 and HP vacA m1 genotypes was associated with the development of gastric IM in the Chinese.

Quantitative detection of promoter hypermethylation in multiple genes in the serum of patients with colorectal cancer.

OBJECTIVES: While promoter hypermethylation is a common molecular alteration of human colorectal cancer that could be detected in the bloodstream, we tested the feasibility of quantitative detection of aberrant DNA methylation in multiple genes in the serum samples of colorectal cancer patients. METHODS: The pre-therapeutic serum samples of 49 colorectal cancer patients and 41 age-matched controls with normal colonoscopy were examined. The presence of methylated DNA in APC (adenomatous polyposis coli), hMLH1 (human MutL homolog 1), and HLTF (helicase-like transcription factor) was detected by quantitative methylation-specific PCR (MethyLight). RESULTS: There was a significant difference in the concentration of methylated serum DNA between cancer patients and controls for HLTF (p= 0.015) and hMLH1 (p= 0.0001) genes, but not for APC gene (p= 0.21). In total, 28 patients with colorectal cancer and 4 controls had methylated DNA detected in at least one marker, which gave a sensitivity of 57% and specificity of 90%. All patients with methylation in two methylation markers had advanced (stage III/IV) cancer (p= 0.006) and patients with methylation in at least one marker tended to have a lower probability of survival (p= 0.08). CONCLUSION: The quantitative detection of aberrant DNA methylation in serum may be a promising high-throughput approach for the noninvasive screening and monitoring of colorectal cancer.

Promoter hypermethylation of tumor-related genes in the progression of colorectal neoplasia.

Gene promoter hypermethylation is increasingly recognized to play an important role in cancer development through silencing of gene transcription. This study determined the methylation profiles of primary colorectal cancers and adenomas to elucidate the role of epigenetic changes in different stages of colorectal carcinogenesis. We examined the methylation profiles of 47 sporadic colorectal cancers, 36 colonic adenomas from patients without cancer and 34 colonic biopsies from patients without colonic lesions. Paired adjacent dysplasia tissues obtained from 17 cancer patients were also examined. Promoter hypermethylation in 10 tumor-related genes (APC, ATM, GSTP1, HLTF, MGMT, hMLH1, p14, p15, SOCS-1 and TIMP-3) were studied by methylation-specific PCR. Promoter hypermethylation was frequently detected in more than 40% of colonic cancers and adenomas in APC, ATM, HLTF, MGMT and hMLH1 genes (p < 0.0001 vs. normal). While low level of methylation was detected in p14, p15 and TIMP-3, there was no methylation detected in GSTP1 and SOCS-1. The frequencies of methylation were comparable between tumors and adenomas, and advanced and nonadvanced adenoma. In contrast, K-ras mutation was only detected in advanced adenomas and cancers. Concurrent methylation in >/= 3 genes was found in 66.7% adenomas and 68.1% cancers but not in normal colonic tissues. Methylation was associated with reduced protein expressions in colorectal adenomas and cancers. Moreover, methylation in ATM was more common in older cancer patients (p = 0.002), but there was no significant association between promoter hypermethylation and other clinicopathologic characteristics of cancer. Our study demonstrated the early and specific involvement of promoter hypermethylation in the colorectal adenoma-carcinoma sequence.