Targeted Next-Generation Sequencing-Based Multiple Gene Mutation Profiling of Patients with Rectal Adenocarcinoma Receiving or Not Receiving Neoadjuvant Chemoradiotherapy.

This study investigated whether oncogenic and tumor-suppressive gene mutations are involved in the differential outcomes of patients with rectal carcinoma receiving neoadjuvant chemoradiotherapy (nCRT). Genomic DNA was obtained from formalin-fixed paraffin-embedded (FFPE) specimens of patients with rectal carcinoma who received a complete nCRT course. Gene mutation status was examined in specimens from patients before and after nCRT by using the AmpliSeq platform. Our data revealed that the nonsynonymous p53, APC, KRAS, CDKN2A, and EGFR mutations were observed in 93.1%, 65.5%, 48.6%, and 31% of the patients with rectal adenocarcinoma, respectively. BRAF, FBXW7, PTEN, and SMAD4 mutations were observed in 20.7% of patients with rectal carcinoma. The following 12 gene mutations were observed more frequently in the patients exhibiting a complete response than in those demonstrating a poor response before nCRT: ATM, BRAF, CDKN2A, EGFR, FLT3, GNA11, KDR, KIT, PIK3CA, PTEN, PTPN11, SMAD4, and TP53. In addition, APC, BRAF, FBXW7, KRAS, SMAD4, and TP53 mutations were retained after nCRT. Our results indicate a complex mutational profile in rectal carcinoma, suggesting the involvement of BRAF, SMAD4, and TP53 genetic variants in the outcomes of patients with nCRT.

Effects of di(2-ethylhexyl)phthalate exposure on 1,2-dimethyhydrazine-induced colon tumor promotion in rats.

Di(2-ethylhexyl)phthalate (DEHP) may cause carcinogenicity in the liver; however, few have detailed on the potential effects of DEHP exposure on colorectal cancer. Male Sprague-Dawley rats received i.p. injections of 1,2-dimethylhydrazine (DMH) once-a-week for the first 4 weeks, and rats in each group were treated with DEHP through oral gavage daily for either 7, 10 or 15 weeks; after which, all rats were euthanized and their colons were assessed (a) morphologically for aberrant crypt foci (ACF) or tumors, (b) cytologically for mitotic index (MI), and (c) immunohistochemically for the expression of ß-catenin, cyclooygenase (COX)-2, vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen (PCNA), cyclin D1, and c-myc. Our results indicated that the mean total ACF, tumor incidence, and MI were significantly higher in the DEHP-treated DMH compared to control and the DEHP-alone groups. The level of ß-catenin and cyclin D1 was increased in DEHP-exposed rats. Expression of ß-catenin, COX-2, VEGF, and cyclin D1 was significantly higher in the combined DMH and DEHP-treated rats by comparison to that of the DMH group. In conclusion, this study indicates that exposure to DEHP may exacerbate DMH-induced colon tumorigenesis and provides impetus to evaluate the effect of DEHP in conjunction with other carcinogens.

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.

Jaw osteoradionecrosis and dental extraction after head and neck radiotherapy: A nationwide population-based retrospective study in Taiwan.

OBJECTIVES: Osteoradionecrosis of the jaws (ORNJ) is painful for patients and relatively difficult to treat clinically. The high risk of ORNJ for post radiotherapy R/T dental extraction is known; however, many patients still have to have teeth extracted after head and neck R/T. The objective of the present study is to review post R/T dental extraction and determine the ORNJ risk. MATERIALS AND METHODS: We preformed a retrospective cohort study of 1759 patients with head and neck cancer s/p R/T from a random sample of 1,000,000 insurants in the National Health Insurance Research Database during 2000-2013 in Taiwan. Statistical methods included two-proportion Z-test. RESULTS: We evaluated two cohorts: 522 patients with post R/T dental extraction and 1237 patients without post R/T extraction. Overall moderate-to-severe ORNJ after R/T was 2.22% (39/1759), and a total of 39 ORNJ cases were noted during an average of 3.02years (range: 0.62-8.89years, ±2.07). ORNJ prevalence in the overall post R/T extraction-exposed cohort (5.17%, 27/522) was significantly greater than that in the unexposed cohort (0.97%, 12/1237). In a group of patients with ⩽5 post R/T dental extractions (n=373), the ORNJ risk was 2.4% (ORNJ case n=9); in a group of patients with >5 dental extractions (n=149), the ORNJ risk was 12.1% (ORNJ case n=18) (Z-score=4.5062; p-value<0.0001). In the extraction-exposed cohort, the ORNJ risk is higher if the index day to first extraction day was ⩽0.5year (n=103) compared with the group with the index day to first extraction day >0.5year (n=419) (Z-score=-2.1506; p-value=0.0315). CONCLUSION: A tooth extraction time less than half a year after R/T or during the head and neck R/T period, and extraction tooth number ⩽5 would significant lower the ORNJ prevalence.

Indium acetate toxicity in male reproductive system in rats.

Indium, a rare earth metal characterized by high plasticity, corrosion resistance, and a low melting point, is widely used in the electronics industry, but has been reported to be an environmental pollutant and a health hazard. We designed a study to investigate the effects of subacute exposure of indium compounds on male reproductive function. Twelve-week old male Sprague-Dawley rats were randomly divided into test and control groups, and received weekly intraperitoneal injections of indium acetate (1.5 mg/kg body weight) and normal saline, respectively, for 8 weeks. Serum indium levels, cauda epididymal sperm count, motility, morphology, chromatin DNA structure, mitochondrial membrane potential, oxidative stress, and testis DNA content were investigated. The indium acetate-treated group showed significant reproductive toxicity, as well as an increased percentage of sperm morphology abnormality, chromatin integrity damage, and superoxide anion generation. Furthermore, positive correlations among sperm morphology abnormalities, chromatin DNA damage, and superoxide anion generation were also noted. The results of this study demonstrated the toxic effect of subacute low-dose indium exposure during the period of sexual maturation on male reproductive function in adulthood, through an increase in oxidative stress and sperm chromatin DNA damage during spermiogenesis, in a rodent model.

Effects of indium chloride exposure on sperm morphology and DNA integrity in rats.

Indium, a Group IIIA element of the periodic chart and a rare earth metal characterized by high plasticity, corrosion resistance, and a low melting point, is widely used in the electronics industry where released streams can contaminate the environment. Consequently, indium can reach humans mainly by natural ways, which could result in a health hazard. Although reproductive toxicities have been surveyed in some studies in animal models, the infertility effects of sperm function induced by indium compounds have not been greatly investigated. We designed a study to investigate the toxicities of subacute exposure to indium compounds on male sperm function and the process of spermatogenesis in a rodent model. Fourteen Sprague-Dawley rats on postnatal Day (PND) 84 were randomly divided into exposure and control groups, and weekly received intraperitoneal injections of indium chloride (1.5 mg/kg body weight) and normal saline, respectively, for 8 weeks. Cauda epididymal sperm count, motility, morphology, chromatin DNA integrity, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) generation, and testis DNA content were investigated. The indium chloride exposed group showed significant toxicity to sperm function, as well as an increased percentage of sperm morphological abnormality and chromatin DNA damage. Furthermore, positive correlations between abnormal sperm morphology, chromatin DNA damage, and superoxide anion generation were also noted. The results of this study demonstrated the toxic effect of subacute low dose indium exposure during sexual maturation on sperm function, resulting in sperm chromatin DNA damage through an increase in sperm ROS generation in a rodent model.

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 G 1/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 G 1/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 G 1/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.

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 (>1.5­fold) 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-ß (TGF-ß), 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.

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-ß (TGF-ß) 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.

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

BACKGROUND: MicroRNAs (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. RESULTS: The 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). CONCLUSION: Fifteen 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.

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.

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.