Xishan Hao

Polymorphisms in MicroRNA Targets: A Gold Mine for Molecular Epidemiology

MicroRNAs are non-coding small RNAs that regulate gene expression by Watson-Crick base pairing to target messenger RNA (mRNA). They are involved in most biological and pathological processes, including tumorigenesis. The binding of microRNA to mRNA is critical for regulating the mRNA level and protein expression. However, this binding can be affected by single-nucleotide polymorphisms that can reside in the microRNA target site, which can either abolish existing binding sites or create illegitimate binding sites. Therefore, polymorphisms in microRNA can have a differing effect on gene and protein expression and represent another type of genetic variability that can influence the risk of certain human diseases. Different approaches have been used to predict and identify functional polymorphisms within microRNA-binding sites. The biological relevance of these polymorphisms in predicted microRNA-binding sites is beginning to be examined in large case-control studies.

Reduced mitochondrial DNA copy number is correlated with tumor progression and prognosis in Chinese breast cancer patients.

Somatic mutations and large‐scale depletion in mitochondrial DNA (mtDNA) have been extensively detected in various human cancers. However, it still remains unclear whether the alterations in mtDNA content are related to the clinicopathological parameters and patient prognosis in breast cancer. In the present study, we analyzed the copy number of mtDNA in 59 cases of invasive breast tumors and paired nontumorous tissues using quantitative real‐time PCR. Our data showed that the level of mtDNA was significantly decreased in tumor tissues as compared to the adjacent nontumorous counterparts (P = 0.001). The reduced copy number in mtDNA was associated with an older onset age (≥50 years old, P = 0.035) as well as a higher histological grade (P = 0.012). Survival analysis measured by the Kaplan‐Meier curves and the log‐rank test indicated that patients with reduced mtDNA content had significantly poorer disease‐free survival (DFS, P = 0.0079) and overall survival (OS, P = 0.011) rate. In addition, tumors harboring mutations in displacement (D)‐loop region, particularly at the polycytidine stretch (T/N ratio = 64.3 ± 8.2%) or close to the replication origins of the heavy‐strand (T/N ratio = 68.7 ± 5.5%), had a significantly lower copy number of mtDNA than the ones without D‐loop alterations. Together, our results suggested that reduced copy number of mtDNA may be involved in breast neoplastic transformation or progression and mtDNA content might be potentially used as a tool to predict prognosis. Somatic mutation in the D‐loop region probably is one of key contributing factors leading to decreased mtDNA level in breast tumors. iubmb Life, 59: 450‐457, 2007

Randomized Study of Autologous Cytokine-Induced Killer Cell Immunotherapy in Metastatic Renal Carcinoma

Purpose: The therapeutic benefit of the cytokine-induced killer (CIK) cells was unknown in the renal cell carcinoma (RCC). This prospectively randomized study was conducted to evaluate the effects of autologous CIK cell immunotherapy in patients with metastatic clear cell RCCs. Experimental Design: From June 2005 to June 2008, 148 patients with metastatic clear cell RCC were randomized to autologous CIK cell immunotherapy (arm 1, n = 74), or interleukin-2 treatment combination with IFN-α-2a (arm 2, n = 74). The primary endpoint was overall survival (OS) and secondary endpoint was progression-free survival (PFS) evaluated by Kaplan–Meier analyses and treatment HRs with the Cox proportional hazards model. Results: The 3-year PFS and OS in arm 1 were 18% and 61%, as compared with 12% and 23% in arm 2 (P = 0.031 and <0.001, respectively). The median PFS and OS in arm 1 were significantly longer than those in arm 2 (PFS, 12 vs. 8 months, P = 0.024; OS, 46 vs. 19 months, P < 0.001). Multivariate analyses indicated that the cycle count of CIK cell immunotherapy as a continuous variable was significantly associated with prolonged PFS [HR = 0.88; 95% confidence interval (CI), 0.84-0.93; P < 0.001] and OS (HR = 0.58; 95% CI, 0.48–0.69; P < 0.001) in arm 1. Conclusion: The data suggested that CIK cell immunotherapy could improve the prognosis of metastatic clear cell RCC, and increased cycle count of CIK cell treatment could further enhance the beneficial effects. Clin Cancer Res; 18(6); 1751–9. ©2012 AACR.

Functional SNP in the microRNA-367 binding site in the 3′UTR of the calcium channel ryanodine receptor gene 3 (RYR3) affects breast cancer risk and calcification

We have evaluated and provided evidence that the ryanodine receptor 3 gene (RYR3), which encodes a large protein that forms a calcium channel, is important for the growth, morphology, and migration of breast cancer cells. A putative binding site for microRNA-367 (miR-367) exists in the 3′UTR of RYR3, and a genetic variant, rs1044129 A→G, is present in this binding region. We confirmed that miR-367 regulates the expression of a reporter gene driven by the RYR3 3′UTR and that the regulation was affected by the RYR3 genotype. A thermodynamic model based on base pairing and the secondary structure of the RYR3 mRNA and miR-367 miRNA showed that miR-367 had a higher binding affinity for the A genotype than for the G genotype. The rs1044129 SNP was genotyped in 1,532 breast cancer cases and 1,600 healthy Chinese women. The results showed that compared with the AA genotype, G was a risk genotype for breast cancer development and was also associated with breast cancer calcification and poor survival. Thus, rs1044129 is a unique SNP that resides in a miRNA-gene regulatory loop that affects breast cancer risk, calcification, and survival.

The hOGG1 Ser326Cys Polymorphism and Lung Cancer Risk: A Meta-analysis

The potentially functional polymorphism Ser326Cys in the human 8-oxoguanine DNA glycosylase (hOGG1) gene has been implicated in lung cancer risk, but published studies have mixed findings. To summarize published data, we did a comprehensive meta-analysis. Two investigators extracted data independently from 17 case control studies published in the PubMed using the search phrases “hOGG1/OGG1/OGG and polymorphism/genetic variation and lung cancer.” The meta-analysis included 6,375 cancer cases and 6,406 control subjects. The results showed that individuals carrying the hOGG1 Cys/Cys genotype did not have significantly increased risk of lung cancer [odds ratios (OR), 1.15; 95% (confidence interval) CI, 0.94-1.41] compared with those with the Ser/Ser genotype; similarly, no significant association with lung cancer risk was found either in the recessive (OR, 1.09; 95% CI, 0.90-1.32 for Cys/Cys versus Ser/Cys+Ser/Ser) or dominant model of the Ser326 allele (OR, 1.06; 95% CI, 0.93-1.21 for Cys/Cys+Ser/Cys versus Ser/Ser). However, significantly increased risks were found among Asian subjects (OR, 1.18; 95% CI, 1.01-1.38 for Cys/Cys+Ser/Cys versus Ser/Ser) in a dominant model. In stratified analyses by control source, compared with the Ser/Ser genotype, lung cancer risk associated with the hOGG1 Cys/Cys genotype was significantly increased in population-based studies (OR, 1.32; 95% CI, 1.04-1.67) but not in hospital-based studies (OR, 1.18; 95% CI, 0.98-1.42); in stratified analyses by the smoking status, however, the increased risk was observed only among nonsmokers in a dominant model (OR, 1.32; 95% CI, 1.04-1.67). The meta-analysis suggested that a careful matching should be considered in future larger genetic association studies including multiple ethnic groups. (Cancer Epidemiol Biomarkers Prev 2008;17(7):1739–45)

mTOR Complex Component Rictor Interacts with PKCζ and Regulates Cancer Cell Metastasis

Epidermal growth factor (EGF) mediates breast cancer cell chemotaxis and metastasis through mechanisms that involve the growth-regulatory mammalian target of rapamycin (mTOR) complex mTORC2, but the mechanisms involved remain obscure. Here, we report that the rapamycin-insensitive mTORC2 component protein Rictor is a critical mediator of metastasis in breast cancer cells. In patients with ductal carcinoma, Rictor expression was associated with increased lymph node metastasis. EGF induced translocation and colocalization of Rictor with protein kinase Cζ (PKCζ), a pivotal molecule in chemotaxis signaling. Further, Rictor coimmunoprecipitated with PKCζ in the absence of the mTORC2 complex. Small interfering RNA-mediated knockdown of Rictor inhibited EGF-induced PKCζ phosphorylation and translocation along with phosphorylation of the key F-actin binding protein cofilin. In parallel, Rictor knockdown reduced cellular chemotactic capacity and ablated pulmonary metastasis in a xenograft mouse model of breast cancer. Our findings identify Rictor as an important mediator of chemotaxis and metastasis in breast cancer cells.

An miR-502–Binding Site Single-Nucleotide Polymorphism in the 3′-Untranslated Region of the SET8 Gene Is Associated with Early Age of Breast Cancer Onset

Purpose: MicroRNAs regulate gene expression by binding to the 3′-untranslated region (UTR) of target genes. Single-nucleotide polymorphisms of critical genes may affect their regulation by microRNAs. We have identified a single-nucleotide polymorphism within the miR-502 seed binding region in the 3′-UTR of the SET8 gene. SET8 methylates TP53 and regulates genome stability. We investigated the role of this SET8 single-nucleotide polymorphism and in concert with the TP53 codon 72 single-nucleotide polymorphism in the propensity for onset of breast cancer. Experimental Design: We measured the SET8 single-nucleotide polymorphisms in a case-control study on 1,110 breast cancer cases and 1,097 controls. Results: The SET8 CC and TP53 GG genotypes were independently associated with an earlier age of breast cancer onset in an allele-dose-dependent manner (for SET8, 52.2 years for TT, 51.4 for TC, and 49.5 for CC; and for TP53, 53.1 years for CC, 51.5 for GC, 50.7 for GG). Individuals with combined SET8 CC and TP53 GG genotypes developed cancer at a median age of 47.7 years as compared with 54.6 years for individuals with combined SET8 TT and TP53 CC genotypes. In the 51 breast cancer tissue samples tested, the SET8 CC genotype was associated with reduced SET8, but not miR-502, transcript levels. Conclusions: These data suggest that the miR-502–binding site single-nucleotide polymorphism in the 3′-UTR of SET8 modulates SET8 expression and contributes to the early development of breast cancer, either independently or together with the TP53 codon 72 single-nucleotide polymorphism. Larger studies with multiethnic groups are warranted to validate our findings. (Clin Cancer Res 2009;15(19):6292–300)

Long non-coding RNA HOTAIR promotes tumor cell invasion and metastasis by recruiting EZH2 and repressing E-cadherin in oral squamous cell carcinoma

HOX transcript antisense RNA (HOTAIR), a long intergenic non-coding RNA (lncRNA), functions as a molecular scaffold to link and target the histone modification complexes PRC2 and LSD1, then reprograms chromatin states by coupling histone H3K27 methylation and H3K4 demethylation for epigenetic gene silencing to promote cancer metastasis. It is associated with poor survival in several solid cancers. In this study, we show that HOTAIR expression increased in oral squamous cell carcinoma (OSCC) compared with non-tumor tissue and is associated with metastasis, the stage and histological differentiation. In addition, overexpression of HOTAIR indicated poor overall survival (OS) and disease-free survival (DFS) in OSCC patients. Knockdown of HOTAIR by siRNA in OSCC cells decreased cell proliferation and colony formation, increased cell invasion and migration, and induced apoptosis in vitro. Furthermore, significant negative correlation between HOTAIR levels and E-cadherin levels was found in OSCC tissues and cell lines, and HOTAIR contributed to the regulation of E-cadherin through binding to EZH2 and H3K27me3 with the E-cadherin promoter. Our findings suggest that HOTAIR expression is associated with OSCC and may be one of critical targets in progression and metastasis, and an indicator of poor survival in OSCC.

Long noncoding RNA HOTAIR involvement in cancer

Evidences have been provided that long noncoding RNAs (lncRNAs) act as key molecules in epigenetic regulation and are involved in the development process of cancer in recent studies. HOX transcript antisense RNA (HOTAIR), a long intergenic noncoding RNA (lincRNA), functions as a molecular scaffold to link and target two histone modification complexes PRC2 and LSD1, then reprograms chromatin states by couples histone H3K27 methylation and H3K4 demethylation for epigenetic gene silencing to promote cancer metastasis. HOTAIR, regarded as an oncogene, is pervasively overexpressed in most solid cancers and correlated with tumor invasion, progression, metastasis, and poor prognosis, and HOTAIR has been proven to play a critical role in most biological process of cancer and would be a potential new target in cancer therapy.

Genetic aberrations of gastrointestinal stromal tumors

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal neoplasm in the gastrointestinal tract and is associated with mutations of the KIT or PDGFRA gene. In addition, other genetic events are believed to be involved in GIST tumorigenesis. Cytogenetic aberrations associated with these tumors thus far described include loss of 1p, 13q, 14q, or 15q, loss of heterozygosity of 22q, numeric chromosomal imbalances, and nuclear/mitochondrial microsatellite instability. Molecular genetic aberrations include loss of heterozygosity of p16(INK4A) and p14(ARF), methylation of p15(INK4B), homozygous loss of the Hox11L1 gene, and amplification of C‐MYC, MDM2, EGFR1, and CCND1. GISTs in patients with neurofibromatosis type 1 appear to lack the KIT and PDGFRA mutations characteristic of GISTs and may have a different pathogenetic mechanism. Gene mutations of KIT or PDGFRA are critical in GISTs, because the aberrant versions not only are correlated with the specific cell morphology, histologic phenotype, metastasis, and prognosis, but also are the targets of therapy with imatinib and other agents. Furthermore, specific mutations in KIT and PDGFR appear to lead to differential drug sensitivity and may in the future guide selection of tyrosine kinase inhibitors. Activation of the receptor tyrosine kinases involves a signal transduction pathway whose components (mitogen‐activated protein kinase, AKT, phosphoinositide 3‐kinase, mammalian target of rapamycin, and RAS) are also possible targets of inhibition. A new paradigm of classification, integrating the standard clinical and pathological criteria with molecular aberrations, may permit personalized prognosis and treatment. Cancer 2008.