Kuan Der Lee

Targeted methylation of two tumor suppressor genes is sufficient to transform mesenchymal stem cells into cancer stem/initiating cells

Although DNA hypermethylation within promoter CpG islands is highly correlated with tumorigenesis, it has not been established whether DNA hypermethylation within a specific tumor suppressor gene (TSG) is sufficient to fully transform a somatic stem cell. In this study, we addressed this question using a novel targeted DNA methylation technique to methylate the promoters of HIC1 and RassF1A, two well-established TSGs, along with a two-component reporter system to visualize successful targeting of human bone marrow-derived mesenchymal stem cells (MSC) as a model cell system. MSCs harboring targeted promoter methylations of HIC1/RassF1A displayed several features of cancer stem/initiating cells including loss of anchorage dependence, increased colony formation capability, drug resistance, and pluripotency. Notably, inoculation of immunodeficient mice with low numbers of targeted MSC resulted in tumor formation, and subsequent serial xenotransplantation and immunohistochemistry confirmed the presence of stem cell markers and MSC lineage in tumor xenografts. Consistent with the expected mechanism of TSG hypermethylation, treatment of the targeted MSC with a DNA methyltransferase inhibitor reversed their tumorigenic phenotype. To our knowledge, this is the first direct demonstration that aberrant TSG hypermethylation is sufficient to transform a somatic stem cell into a fully malignant cell with cancer stem/initiating properties.

A KRAS mutation status-stratified randomized phase II trial of gemcitabine and oxaliplatin alone or in combination with cetuximab in advanced biliary tract cancer

BACKGROUND Previous clinical trials have not proved that adding epidermal growth factor receptor inhibitors to chemotherapy confers a survival benefit for patients with advanced biliary tract cancer (ABTC). Whether the KRAS mutation status of tumor cells confounded the results of past studies is unknown. PATIENTS AND METHODS ABTC patients stratified by KRAS status, Eastern Cooperative Oncology Group performance status, and primary tumor location were randomized 1 : 1 to receive GEMOX (800 mg/m(2) gemcitabine and 85 mg/m(2) oxaliplatin) or C-GEMOX (500 mg/m(2) cetuximab plus GEMOX) every 2 weeks. The primary end point was objective response rate (ORR). RESULTS The study enrolled 122 patients between December 2010 and May 2012 (62 treated with C-GEMOX and 60 with GEMOX). Compared with GEMOX alone, C-GEMOX was associated with trend to better ORR (27% versus 15%; P = 0.12) and progression-free survival (PFS, 6.7 versus 4.1 months; P = 0.05), but not overall survival (OS, 10.6 versus 9.8 months; P = 0.91). KRAS mutations, which were detected in 36% of tumor samples, did not affect the trends of difference in ORR and PFS between C-GEMOX and GEMOX. The two treatment arms had similar adverse events, except that more patients had skin rashes, allergic reactions, and neutropenia in the C-GEMOX arm. Of patients with C-GEMOX, the presence of a grade 2 or 3 skin rash was associated with significantly better ORR, PFS, and OS. CONCLUSIONS Addition of cetuximab did not significantly improve the ORR of GEMOX chemotherapy in ABTC, although a trend of PFS improvement was observed. The trend of improvement did not correlate with KRAS mutation status. CLINICAL TRIALS NUMBER This study is registered at ClinicalTrials.gov (NCT01267344). All patients gave written informed consent.

IL-6 expression predicts treatment response and outcome in squamous cell carcinoma of the esophagus

BackgroundThe identification of potential tumor markers can improve therapeutic planning and patient management. The aim of this study was to highlight the significance of IL-6 in esophageal squamous cell carcinoma (SCC).MethodsWe retrospectively analyzed the clinical outcomes of 173 patients with esophageal SCC, and examined the correlation between IL-6 levels and clinical outcomes in esophageal cancer patients. Furthermore, the human esophageal SCC cell line CE81T was selected for cellular and animal experiments to investigate changes in tumor behavior and treatment response after manipulation of IL-6 expression.ResultsIn clinical outcome analysis, positive IL-6 staining and poor treatment response was significantly associated with shorter survival. Furthermore, the frequency of IL-6 immunoreactivity was significantly higher in esophageal cancer specimens than in non-malignant epithelium, and this staining was positively linked to the development of distant metastasis (p = 0.0003) and lower treatment response rates (p = 0.0001).By ELISA analysis, IL-6 serum levels were significantly elevated in patients developing disease failure.When IL-6 expression was inhibited, aggressive tumor behavior and radiation resistance could be overcome in vitro and in vivo. The underlying changes included increased cell death, less epithelial-mesenchymal transition and attenuated STAT3 activation. IL-6 inhibition was also associated with attenuated angiogenesis in tumor-bearing mice.ConclusionsIL-6 was significantly associated with poor prognosis in patients with esophageal cancer. Targeting this cytokine could be a promising strategy for treatment of esophageal cancer, as evidenced by inhibition of aggressive tumor behavior and treatment resistance.

The incidence and risk of developing a second primary esophageal cancer in patients with oral and pharyngeal carcinoma: a population-based study in Taiwan over a 25 year period

BackgroundThe incidence of oral and pharyngeal (including oral cavity, oropharynx and hypopharynx) carcinoma increases rapidly in Asia and South Pacific because of betel quid chewing. Thus far, large-scale epidemiological studies are not available yet to stratify these patients by their risks of developing a second primary cancer in the digestive tract including esophagus, stomach, colon, and rectum.MethodsA population-based study was conducted using the database from the Taiwan National Cancer Registry for the period 1979-2003. We quantified standardized incidence ratios (SIRs) and cumulative incidence of second primary cancers among 33,787 patients with initial diagnoses of oral and pharyngeal carcinoma.ResultsAmong these four digestive tract organs, the esophagus was the only site of second cancer with excess risk in patients with oral and pharyngeal carcinoma. The incidence and risk of developing a second primary esophageal cancer differed by the site of the primary index tumor, most frequently seen in hypopharyngeal cancer (71/4,218 = 1.68%, SIR = 22.76, 95% CI 17.77-28.70), followed by oropharyngeal cancer (30/3,403 = 0.88%, SIR = 14.29, 95% CI 9.64-20.39) and the least in oral cavity cancer (99/26,166 = 0.38%, SIR = 5.57, 95% CI 4.53-6.78). In addition, the risk was extraordinarily high for patients with a follow-up interval ≤ 1 year and those with first primary cancer diagnosed at age ≤50. These patients may justify more close surveillance.ConclusionThe present study represents the first population-based study in Asia attempting to stratify the patients of oral and pharyngeal carcinoma by their risk of developing a second esophageal cancer. It helps identify patients at high risk and tailor the application of intense follow-up surveillance to the estimated risk in each individual case.

Gene expression profiling for analysis acquired oxaliplatin resistant factors in human gastric carcinoma TSGH-S3 cells: the role of IL-6 signaling and Nrf2/AKR1C axis identification.

Oxaliplatin treatment is a mainstay of treatment for advanced gastrointestinal tract cancer, but the underlying mechanisms of acquired oxaliplatin resistance remain largely obscured. We previously demonstrated that increased DNA repair capacity and copper-transporting ATPase 1 (ATP7A) level contributed to oxaliplatin resistance in the human gastric carcinoma cell line TSGH-S3 (S3). In the present study, we applied gene array technology to identify additional resistance factors in S3 cells. We found that interleukin-6 (IL-6), aldo-keto reductase 1C1 (AKR1C1), and AKR1C3 are the top 3 upregulated genes in S3 cells when compared with parent TSGH cells. Despite a higher level of endogenous IL-6 in S3, IL-6 receptor (IR-6R, gp-80, and gp-130) levels were similar between TSGH and S3 cells. The addition of exogenous IL-6, IL-6 targeted siRNA, or neutralizing antibodies neither affected Stat3 activation, a downstream target of IL-6, nor changed oxaliplatin sensitivity in S3 cells. However, manipulation of AKR1C activity with siRNA or AKR1C inhibitors significantly reversed oxaliplatin resistance. AKR1Cs are classical antioxidant response element (ARE) genes that can be transcriptionally upregulated by nuclear factor erythroid 2-related factor 2 (Nrf2). Knockdown of Nrf2 not only decreased the levels of AKR1C1, AKR1C2, and AKR1C3 mRNA and protein but also reversed oxaliplatin resistance in S3 cells. Taken together, these results suggest that activation of the Nrf2/AKR1C axis may contribute to oxaliplatin resistance in S3 cells but that the IL-6 signaling pathway did not contribute to resistance. Manipulation of Nrf2/AKR1Cs activity may be useful for management of oxaliplatin-refractory gastric cancers.

The predictive role of E2-EPF ubiquitin carrier protein in esophageal squamous cell carcinoma

The ubiquitin proteasome pathway has been implicated in carcinogenesis. However, the role of E2-EPF ubiquitin carrier protein (UCP) in esophageal cancer remains relatively unstudied. In the study, we examined the mRNA level of circulating tumor cells from 60 esophageal cancer patients by membrane arrays consisting of a panel of potential markers including UCP, compared to 40 normal populations. The predictive capacity of UCP was also assessed by immunohistochemical staining of a retrospective series of 84 biopsied esophageal squamous cell carcinomas in relation to clinical outcome. In addition, we studied in vitro biological changes including tumor growth, metastatic capacity, and the sensitivity to irradiation and cisplatin, after experimental manipulation of UCP expression in esophageal cancer cells. By the data of 25-gene membrane array analysis, UCP was the only factor significantly associated with the extent of tumor burden in esophageal cancer patients. Our immunochemistry findings further indicate that UCP positivity was linked to poor response to neoadjuvant therapy and worse survival. In cell culture, inhibited UCP significantly decrease tumor growth and the capacity for metastasis. The epithelial–mesenchymal transition (EMT) induced by VHL/HIF-1α-TGF-β1 pathway might be the underlying mechanism responsible to the more aggressive tumor growth in UCP-positive esophageal cancer. Our results suggest that UCP was significantly associated with poor prognosis of esophageal cancer and may be a new molecular target for therapeutic intervention for esophageal squamous cell carcinoma.

Changes in DNA methylation are associated with the development of drug resistance in cervical cancer cells

Background and proposeChanges in DNA methylation are associated with changes in somatic cell fate without the alteration of coding sequences. In addition to its use as a traceable biomarker, reversible DNA methylation could also serve as a therapeutic target. In particular, if the development of drug resistance is associated with changes in DNA methylation, then demethylation might reverse the resistance phenotype. The reversion of the drug-resistance might then be feasible if the association between abnormal DNA methylation and the development of drug-resistance could be identified.MethodsMethylation differences between the drug-resistance cervical cancer cell, SiHa, and its derived oxaliplatin-resistant S3 cells were detected by methylation specific microarray. The drug-resistance cells were treated with demethylation agent to see if the resistance phenotype were reversed. Targeted methylation of one of the identified locus in normal cell is expected to recapitulate the development of resistance and a two-component reporter system is adopted to monitor the increase of DNA methylation in live cells.ResultsIn this report, we identified methylation changes, both genome-wide and within individual loci, in the oxaliplatin-resistant cervical cancer cell S3 compared with its parental cell line SiHa. Treatment of S3 with a demethylation agent reversed increases in methylation and allowed the expression of methylation-silenced genes. Treatment with the demethylation agent also restored the sensitivity of S3 to cisplatin, taxol, and oxaliplatin to the same level as that of SiHa. Finally, we found that methylation of the target gene Casp8AP2 is sufficient to increase drug resistance in different cells.ConclusionsThese results suggest that global methylation is associated with the development of drug resistance and could serve as a biomarker and therapeutic target for drug resistance in cervical cancer.

Significance of the TGF-β1/IL-6 axis in oral cancer

The aim of the present study was to explore specific molecular markers that could lead to new insights into the identification of innovative treatments in oral cancer. The role of TGF-β1 (transforming growth factor-β1) and its predictive power in the prognosis of oral cancer has been identified. Human oral cancer cell lines, including SCC4 and SCC25, were selected for cellular experiments. Changes in tumour aggressiveness, responses to treatment and the signalling pathway responsible were investigated in vitro. Furthermore, 125 oral cancer tissue specimens were constructed into tissue microarray blocks for immunohistochemical analysis to correlate the expression of TGF-β1 with clinical outcome. Using in vitro experiments, our results revealed that activated TGF-β1 signalling resulted in more aggressive tumour growth, augmented the epithelial–mesenchymal transition and more resistance to treatment. Activated IL-6 (interleukin-6) signalling could be the mechanism underlying the effects of TGF-β1 on oral cancer. Regarding clinical data, the incidence of TGF-β1 immunoreactivity in oral cancer specimens was significantly higher than in non-malignant epithelium and positively linked to IL-6 staining. Furthermore, expression of TGF-β1 was significantly correlated with the risk of lymph node involvement, disease recurrence and shorter survival in patients with pathological stage III–IV oral cancer. In conclusion, the TGF-β1/IL-6 axis had predictive power in the prognosis of oral cancer, and targeting TGF-β1 could represent a promising treatment strategy.

The role of DNA methyltransferase 3b in esophageal squamous cell carcinoma

The identification of potential tumor markers can improve therapeutic planning and patient management. The objective of this study was to highlight the role of DNA methyltransferase 3b (DNMT3b) in esophageal squamous cell carcinoma (SCC).

A phase II study of weekly docetaxel and cisplatin plus oral tegafur/uracil and leucovorin as first-line chemotherapy in patients with locally advanced or metastatic gastric cancer

Background:Docetaxel plus cisplatin and 5-fluorouracil has become a new standard for treating advanced gastric cancer. However, high rates of severe neutropenia limit its application. Modification of the regimen could be the solution to get similar activity but less myelosuppression.Methods:Patients with histologically confirmed, locally advanced, or recurrent/metastatic gastric adenocarcinoma without previous chemotherapy were enrolled. This regimen consisted of docetaxel (Tyxan, TTY, Taipei, Taiwan) 30-min infusion at a dose of 36 mg m−2, followed by cisplatin 30 mg m−2 infusion over 1 h on days 1 and 8, and oral tegafur/uracil 300 mg m−2 per day plus leucovorin 90 mg per day on days 1–14, every 3 weeks. Tumour response was evaluated after every 2 cycles of treatment.Results:From August 2007 to March 2009, 45 patients were enrolled. The median age was 56 years (range: 22–75). Among the 40 patients evaluable for tumour response, one achieved a complete response, 22 had partial responses and 11 had stable disease. The overall response rates of the evaluable and intent-to-treat (ITT) populations were 58% (95% CI: 41–74%) and 53% (95% CI: 38–68%), respectively. The disease control rates in these populations were 85% (95% CI: 70–94%) and 82% (95% CI: 68–92%), respectively. In the ITT analysis, the median time to progression and overall survival were 6.8 and 13.9 months, respectively. Major grade 3–4 toxicities were neutropenia (51%), anaemia (22%), diarrhoea (16%), and infections (20%). No patient died of treatment-related toxicities.Conclusion:Concurrent weekly docetaxel and cisplatin plus oral tegafur/uracil and leucovorin are effective and well tolerated in the treatment of advanced gastric cancer.