Chung-Man Leung

Silencing of miR-1-1 and miR-133a-2 cluster expression by DNA hypermethylation in colorectal cancer

MicroRNAs are small non-coding RNA molecules that play important roles in the multistep process of colorectal carcinoma (CRC) development. The present study evaluated the relationship between miR-1-1 and miR-133a-2 expression and DNA methylation, and its putative biological role in CRC. The results indicated that DNA methylation regulated the expression of the miR-1-1 and miR-133a-2 cluster in CRC cell lines. Expression of miR-1 and miR-133a was further evaluated in 64 paired tissue samples (CRC tumor and adjacent normal mucosa) using the stem-loop real-time polymerase chain reaction. The miR-1-133a cluster displayed significantly lower expression in CRC tissue compared to adjacent normal mucosa (P<0.001). The results also indicated frequent hypermethylation of the CpG islands upstream of miR-1-133a (54.6%). Liver metastatic tissues exhibited significantly lower miR-1 (P<0.001) and miR-133a (P<0.001) expression compared to adjacent normal mucosa. Expression of the miR-1-133a cluster inversely correlated with TAGLN2 in the tumor specimens. In conclusion, epigenetic repression of the miR-1-133a cluster may play a critical role in colorectal cancer metastasis by silencing TAGLN2.

ATG4B promotes colorectal cancer growth independent of autophagic flux

Autophagy is reported to suppress tumor proliferation, whereas deficiency of autophagy is associated with tumorigenesis. ATG4B is a deubiquitin-like protease that plays dual roles in the core machinery of autophagy; however, little is known about the role of ATG4B on autophagy and proliferation in tumor cells. In this study, we found that ATG4B knockdown induced autophagic flux and reduced CCND1 expression to inhibit G1/S phase transition of cell cycle in colorectal cancer cell lines, indicating functional dominance of ATG4B on autophagy inhibition and tumor proliferation in cancer cells. Interestingly, based on the genetic and pharmacological ablation of autophagy, the growth arrest induced by silencing ATG4B was independent of autophagic flux. Moreover, dephosphorylation of MTOR was involved in reduced CCND1 expression and G1/S phase transition in both cells and xenograft tumors with depletion of ATG4B. Furthermore, ATG4B expression was significantly increased in tumor cells of colorectal cancer patients compared with adjacent normal cells. The elevated expression of ATG4B was highly correlated with CCND1 expression, consistently supporting the notion that ATG4B might contribute to MTOR-CCND1 signaling for G1/S phase transition in colorectal cancer cells. Thus, we report that ATG4B independently plays a role as a positive regulator on tumor proliferation and a negative regulator on autophagy in colorectal cancer cells. These results suggest that ATG4B is a potential biomarker and drug target for cancer therapy.

Co-modulated behavior and effects of differentially expressed miRNA in colorectal cancer

BackgroundMicroRNAs (miRNAs) are short noncoding RNAs (approximately 22 nucleotides in length) that play important roles in colorectal cancer (CRC) progression through silencing gene expression. Numerous dysregulated miRNAs simultaneously participate in the process of colon cancer development. However, the detailed mechanisms and biological functions of co-expressed miRNA in colorectal carcinogenesis have yet to be fully elucidated.ResultsThe objective of this study was to identify the dysfunctional miRNAs and their target mRNAs using a wet-lab experimental and dry-lab bioinformatics approach. The differentially expressed miRNA candidates were identified from 2 miRNA profiles, and were confirmed in CRC clinical samples using reported target genes of dysfunctional miRNAs to perform functional pathway enrichment analysis. Potential target gene candidates were predicted by an in silico search, and their expression levels between normal and colorectal tumor tissues were further analyzed using real-time polymerase chain reaction (RT-PCR).We identified 5 miRNAs (miR-18a, miR-31, miR-96, miR-182, and miR-224) and 10 miRNAs (miR-1, miR-9, miR-10b, miR-133a, miR-143, miR-137, miR-147b, miR-196a/b, and miR-342) that were significantly upregulated and downregulated in colon tumors, respectively. Bioinformatics analysis showed that the known targets of these dysregulated miRNAs simultaneously participated in epithelial-to-mesenchymal transition (EMT), cell growth, cell adhesion, and cell cycles. In addition, we identified that several pivotal target gene candidates may be comodulated by dysfunctional miRNAs during colon cancer progression. Finally, 7 candidates were proven to be differentially expressed, and had an anti-correlationship with dysregulated miRNA in 48 CRC samples.ConclusionFifteen dysfunctional miRNAs were engaged in metastasis-associated pathways through comodulating 7 target genes, which were identified by using a multi-step approach. The roles of these candidate genes are worth further exploration in the progression of colon cancer, and could potentially be targets in future therapy.

MicroRNA expression profiles in human breast cancer cells after multifraction and single-dose radiation treatment

MicroRNAs (miRNAs) are small non-coding RNAs that contribute to modulating signaling pathways after radiation exposure and have emerged as a potential therapeutic target or biomarker in the radiation response of cancer. Exposing breast cancer cells to single-dose (SD) or multifractionated (MF) radiation may affect the cells differently. However, the roles of miRNAs in breast cancer cells after the response to SD or MF is not thoroughly understood. Therefore, the purpose of the present study was to comprehensively investigate the response of miRNAs in MDA-MB-361 by using various radiation exposing protocols. Our results revealed that only a small fraction of miRNAs exhibiting differential expressions (>1.5‑fold) was identified after MDA-MB-361 cells were exposed to SD (10 Gy) or MF radiation (2 Gy x 5 MF). In addition, we observed that several miRNAs in the MDA-MB-361 cells frequently exhibited differential responses to various types of radiation treatment. Among these miRNAs, the expression levels of an oncogenic miR-17-92 cluster increased following SD radiation treatment. Conversely, miR-19a-3p, miR-20a-5p, and miR-19b-3p expressions were inhibited by >1.5-fold in the following MF treatment. Further analysis of the miR-17-92 cluster expression levels revealed that miR-17, miR-18a, miR-19a/b and miR-20a were significantly overexpressed and miR-92a was downregulated in breast cancer. Functional annotation demonstrated that target genes of the miR-17-92 cluster were predominantly involved in the regulation of radiation-associated signal pathways such as mitogen-activated protein kinase (MAPK), ErbB, p53, Wnt, transforming growth factor-β (TGF-β), mTOR signaling pathways and cell cycles with an FDR <0.05. Overall, the results of the present study revealed distinct differences in the response of miRNAs to SD and MF radiation exposure, and these radiation-associated miRNAs may contribute to radiosensitivity and can be used as biomarkers for radiotherapy.

Arm Selection Preference of MicroRNA-193a Varies in Breast Cancer

MicroRNAs (miRNAs) are short noncoding RNAs derived from the 3′ and 5′ ends of the same precursor. However, the biological function and mechanism of miRNA arm expression preference remain unclear in breast cancer. We found significant decreases in the expression levels of miR-193a-5p but no significant differences in those of miR-193a-3p in breast cancer. MiR-193a-3p suppressed breast cancer cell growth and migration and invasion abilities, whereas miR-193a-5p suppressed cell growth but did not influence cell motility. Furthermore, NLN and CCND1, PLAU, and SEPN1 were directly targeted by miR-193a-5p and miR-193a-3p, respectively, in breast cancer cells. The endogenous levels of miR-193a-5p and miR-193a-3p were significantly increased by transfecting breast cancer cells with the 3′UTR of their direct targets. Comprehensive analysis of The Cancer Genome Atlas database revealed significant differences in the arm expression preferences of several miRNAs between breast cancer and adjacent normal tissues. Our results collectively indicate that the arm expression preference phenomenon may be attributable to the target gene amount during breast cancer progression. The miRNA arm expression preference may be a means of modulating miRNA function, further complicating the mRNA regulatory network. Our findings provide a new insight into miRNA regulation and an application for breast cancer therapy.

Comprehensive microRNA profiling of prostate cancer cells after ionizing radiation treatment

MicroRNAs (miRNAs) are small, non-coding RNAs that negatively regulate gene expression and have emerged as potential biomarkers in radiation response to human cancer. Only a few miRNAs have been identified in radiation response to prostate cancer and the involvement of the radiation-associated miRNA machinery in the response of prostate cancer cells to radiation is not thoroughly understood. Therefore, the purpose of the present study was to comprehensively investigate the expression levels, arm selection preference and isomiRs of radiation-response miRNAs in radiation-treated PC3 cells using a next-generation sequencing (NGS) approach. Our data revealed that the arm selection preference and 3′ modification of miRNAs may be altered in prostate cancer after radiation exposure. In addition, the proportion of AA dinucleotide modifications at the end of the read gradually increased in a time-dependent manner after PC3 radiation treatment. We also identified 6 miRNAs whose expression increased and 16 miRNAs whose expression decreased after exposure to 10 Gy of radiation. A pathway enrichment analysis revealed that the target genes of these radiation-induced miRNAs significantly co-modulated the radiation response pathway, including the mitogen-activated protein kinase (MAPK), Wnt, transforming growth factor-β (TGF-β) and ErbB signaling pathways. Furthermore, analysis of The Cancer Genome Atlas (TCGA) database revealed that the expression of these radiation-induced miRNAs was frequently dysregulated in prostate cancer. Our study identified radiation-induced miRNA candidates which may contribute to radiosensitivity and can be used as biomarkers for radiotherapy.

Impact of Intracranial Extension on Survival in Stage IV Nasopharyngeal Carcinoma: Identification of a Subset of Patients with Better Prognosis

OBJECTIVE T-stage is an imperfect prognostic indicator for patients with nasopharyngeal carcinoma. We evaluated the effect of extent of intracranial involvement on survival after conventional radiotherapy in patients with Stage T4 nasopharyngeal carcinoma. METHODS We conducted a retrospective analysis of the results of computed tomography, magnetic resonance imaging and treatment outcomes in 84 consecutive patients with Stage T4 nasopharyngeal carcinoma during the period September 1993 to December 2002 in Taiwan. The patients were subcategorized into those who had limited intracranial involvement (primary nasopharyngeal tumors with involvement of the unilateral cavernous sinus or the parasellar region only) or extensive involvement (extension of the tumors to the bilateral cavernous sinus or the parasellar region, into the orbit and the ethmoid sinus anteriorly, or to the prepontine region and the posterior cranial fossa). RESULTS Extensive intracranial involvement was found in 51.2% of the patients. Among these patients, the 5-year rate of overall survival after conventional radiotherapy was only 3.4%. In contrast, the 5-year survival among patients with limited intracranial involvement was 42.9%. This difference was significant (P < 0.001). In the multivariate analysis, extensive intracranial involvement, advanced age and a nodal status of N3 correlated with poor overall survival (all P < 0.05). CONCLUSIONS Among patients with T4 nasopharyngeal carcinoma, better treatment outcomes were associated with limited intracranial involvement. We conclude that a subdivision of Stage T4 nasopharyngeal carcinoma disease based on the extent of intracranial involvement would provide better prognostic information.

DNA Methylation in Aggressive Gastric Carcinoma

Gastric cancer remains a common cancer type in humans to dates, especially in the Andean region of South America and in the Far East. Various factors contribute to cause of stomach cancer, including Helicobacter pylori, smoking and diet. Most patients are diagnosed with ad‐ vanced gastric cancer, therefore, detailed elucidating mechanisms mediate gastric cancer progression and improving gastric cancer clinic strategies are helpful.

A Case of Squamous Cell Carcinoma Arising from Presacral (Retrorectal) Epidermoid Cyst

Epidermoid cyst of the presacral space is a rare congenital lesion. Cases of malignance arising from the lesion are extremely rare. After searching the literature, there are only 1 image report in English literature (PubMed) and 4 cases reports in Japanese literature of the disease. Here we report a 62-year-old female who was diagnosed with presacral cyst before surgery. Wide excision of the cystic tumor by Kraskes operation was performed. The pathology showed squamous cell carcinoma. Because of tumor rapture during operation, adjuvant radiotherapy 50 Gray/ 25 fractions was performed. After 1 year follow up, there is no evidence of disease.

Solitary Extramedullary Plasmacytoma of the Head and Neck: Treatment Outcome in 6 Patients and a Review of Literature

Purpose: Solitary extramedullary plasmacytoma of the head and neck (EMPHN) is a rare disease. The objective of this study was to review the treatment results of EMPHN patients in a single institution in TaiwanMethods and Materials: The cases of 6 patients (4 males and 2 females) diagnosed at our hospital with EMPHN between 1980 and 2010 were reviewed. The median age of the patients was 64 years (range, 54-79 years). Two patients underwent surgery alone, 2 patients underwent radiotherapy alone, and 2 patients underwent surgery and adjuvant radiotherapy. The dose of radiotherapy was 50-50.4 grays (Gy) in 25-28 fractions.Results: The median follow-up duration was 5 years. The primary tumor sites were the nasal cavity (2), paranasal sinuses (2), oropharynx (1), and nasopharynx (1). Local recurrence was observed in 2 patients who underwent surgery alone and local control was achieved in 1 after salvage surgery (no evidence of disease after salvage surgery in the follow up 3 years period). Local control was achieved in 2 patients treated with surgery and adjuvant radiotherapy. Distant metastasis to the skin and bone without evidence of local recurrence was observed in 2 patients treated with radiotherapy alone. None of the patients progressed to multiple myeloma during follow-up evaluations and no severe radiotherapy-related morbidity was observed. One patient who treated with surgery alone died from recurrent EMPHN within the follow-up duration.Conclusions: Local control of the tumor in the primary site was achieved in 4 patients with EMPHN who underwent radiotherapy alone or adjuvant, although 2 of these patients presented with distant metastasis. According to the review of literature and our results, radiotherapy might be safe and effective for local control of EMPHN.