Teng Xu

A functional polymorphism in the miR-146a gene is associated with the risk for hepatocellular carcinoma

A G > C polymorphism (rs2910164) is located in the stem region opposite to the mature miR-146a sequence, which results in a change from G:U pair to C:U mismatch in the stem structure of miR-146a precursor. Here, we elucidated the biological significance of this polymorphism, based on cancer association study and cell model system. The cancer association study included 479 hepatocellular carcinoma (HCC) and 504 control subjects. We found that the genotype distribution of this polymorphism in HCC cases was significantly different from that in control subjects (P = 0.026). The association between the genotype and the risk of HCC was further analyzed using multivariate unconditional logistic regression, with adjustment for sex, age and hepatitis B virus status. The results revealed that male individuals with GG genotype were 2-fold more susceptible to HCC (odds ratio = 2.016, 95% confidence interval = 1.056-3.848, P = 0.034) compared with those with CC genotype. We next examined the influence of this polymorphism on the production of mature miR-146a and found that G-allelic miR-146a precursor displayed increased production of mature miR-146a compared with C-allelic one. Further investigations disclosed that miR-146a could obviously promote cell proliferation and colony formation in NIH/3T3, an immortalized but non-transformed cell line. These data suggest that the G > C polymorphism in miR-146a precursor may result in important phenotypic traits that have biomedical implications. Our findings warrant further investigations on the relation between microRNA polymorphism and human diseases.

MicroRNA‐195 suppresses tumorigenicity and regulates G1/S transition of human hepatocellular carcinoma cells

Growing evidence indicates that deregulation of microRNAs (miRNAs) contributes to tumorigenesis. Down‐regulation of miR‐195 has been observed in various types of cancers. However, the biological function of miR‐195 is still largely unknown. In this study we aimed to elucidate the pathophysiologic role of miR‐195. Our results showed that miR‐195 expression was significantly reduced in as high as 85.7% of hepatocellular carcinoma (HCC) tissues and in all of the five HCC cell lines examined. Moreover, introduction of miR‐195 dramatically suppressed the ability of HCC and colorectal carcinoma cells to form colonies in vitro and to develop tumors in nude mice. Furthermore, ectopic expression of miR‐195 blocked G1/S transition, whereas inhibition of miR‐195 promoted cell cycle progression. Subsequent investigation characterized multiple G1/S transition‐related molecules, including cyclin D1, CDK6, and E2F3, as direct targets of miR‐195. Silencing of cyclin D1, CDK6, or E2F3 phenocopied the effect of miR‐195, whereas overexpression of these proteins attenuated miR‐195‐induced G1 arrest. In addition, miR‐195 significantly repressed the phosphorylation of Rb as well as the transactivation of downstream target genes of E2F. These results imply that miR‐195 may block the G1/S transition by repressing Rb‐E2F signaling through targeting multiple molecules, including cyclin D1, CDK6, and E2F3. Conclusion: Our data highlight an important role of miR‐195 in cell cycle control and in the molecular etiology of HCC, and implicate the potential application of miR‐195 in cancer therapy. (HEPATOLOGY 2009.)

Elevated snoRNA biogenesis is essential in breast cancer

Hyperactive ribosomal biogenesis is widely observed in cancer, which has been partly attributed to the increased rDNA transcription by Pol I in cancer. However, whether small nucleolar RNAs (snoRNAs), a class of non-coding RNAs crucial in ribosomal RNA (rRNA) maturation and functionality, are involved in cancer remains elusive. We report that snoRNAs and fibrillarin (FBL, an enzymatic small nucleolar ribonucleoprotein, snoRNP) are frequently overexpressed in both murine and human breast cancer as well as in prostate cancers, and significantly, that this overexpression is essential for tumorigenicity in vitro and in vivo. We demonstrate that when the elevated snoRNA pathway is suppressed, the tumor suppressor p53 can act as a sentinel of snoRNP perturbation, the activation of which mediates the growth inhibitory effect. On the other hand, high level of FBL interferes with the activation of p53 by stress. We further show that p53 activation by FBL knockdown is not only regulated by the ribosomal protein-MDM2-mediated protein stabilization pathway, but also by enhanced PTB-dependent, cap-independent translation. Together, our data uncover an essential role of deregulated snoRNA biogenesis in tumors and a new mechanism of nucleolar modulation of p53.

Large-scale analysis of the genetic and epigenetic alterations in hepatocellular carcinoma from Southeast China

Our knowledge about molecular alterations during hepatocarcinogenesis is still fragmentary, due to lack of comprehensive genetic and epigenetic analyses in the same set of hepatocellular carcinomas (HCCs). In this study, we conducted a large-scale analysis, including mutation screening in 50 genes and methylation assays in three genes in 54 pairs of HCCs and their neighboring non-cancerous tissues. All samples were collected from the residents in Southeast China. We found HBV infection and chronic hepatitis/cirrhosis in 83.3% and 98.1% of the cases, respectively. Mutations were identified in 18 out of 54 (33.3%) samples, with p53 alterations in 14 cases and beta-catenin mutations in four tumors. No mutations were identified in the neighboring tissues. Interestingly, 9 out of 14 (64.3%) tumors carrying p53 mutations displayed substitution of serine by arginine at codon 249, a characteristic change believed to be induced by aflatoxin-B1. Furthermore, p53 mutation was significantly associated with shorter recurrence-free survival (P=0.004). The results also revealed aberrant methylation in two or more genes in as high as 90% of tumors and 40% of adjacent tissues. The frequency of RASSF1A hypermethylation was much higher than that of p16INK4a and HAI2 in both HCC and neighboring tissues, indicating that deregulation of RASSF1A may precede the other two genes. These data suggest that aberrant methylation occurs before mutation and is an early event in the development of this set of HCC. Our findings highlight p53 as a prognostic factor of HCC and RASSF1A as a potential target in preventing malignant transformation of hepatocytes.

Low-dose radiation exposure induces a HIF-1-mediated adaptive and protective metabolic response.

Because of insufficient understanding of the molecular effects of low levels of radiation exposure, there is a great uncertainty regarding its health risks. We report here that treatment of normal human cells with low-dose radiation induces a metabolic shift from oxidative phosphorylation to aerobic glycolysis resulting in increased radiation resistance. This metabolic change is highlighted by upregulation of genes encoding glucose transporters and enzymes of glycolysis and the oxidative pentose phosphate pathway, concomitant with downregulation of mitochondrial genes, with corresponding changes in metabolic flux through these pathways. Mechanistically, the metabolic reprogramming depends on HIF1α, which is induced specifically by low-dose irradiation linking the metabolic pathway with cellular radiation dose response. Increased glucose flux and radiation resistance from low-dose irradiation are also observed systemically in mice. This highly sensitive metabolic response to low-dose radiation has important implications in understanding and assessing the health risks of radiation exposure.

Modulation of autophagic activity by extracellular pH.

Reprogramming energy metabolism from oxidative phosphorylation to aerobic glycolysis, a common feature of human cancer, is associated with a relative acidic tumor microenvironment which can sometimes be further accentuated by hypoxia operating within most solid tumors. We found that alteration of extracellular pH induces marked and rapid changes of autophagic activity. Interestingly, acidic and basic conditions induced completely opposite effect on autophagy, with its activity suppressed at lower pH whereas stimulated at higher pH. Gene knockdown experiments indicated that pH induced-autophagy requires Beclin 1, Vps34 and Atg5, key components of the autophagy pathway. Of note, an acidic condition not only inhibits the basal but also blocks the starvation-induced autophagy activity. Significantly, examination of different areas of tumor mass revealed a lower autophagic activity within the inner region than the outer region. These findings have important implications on the connections between autophagy and cancer as well as a wide range of other physiological and pathological processes.

Low-dose arsenic induces chemotherapy protection via p53/NF-κB- mediated metabolic regulation

Most chemotherapeutical drugs kill cancer cells chiefly by inducing DNA damage, which unfortunately also causes undesirable injuries to normal tissues, mainly due to p53 activation. We report a novel strategy of normal tissue protection that involves p53/NF-κB coordinated metabolic regulation. Pretreatment of untransformed cells with low doses of arsenic induced concerted p53 suppression and NF-κB activation, which elicited a marked induction of glycolysis. Significantly, this metabolic shift provided cells effective protection against cytotoxic chemotherapy, coupling the metabolic pathway to cellular resistance. Using both in vitro and in vivo models, we demonstrated an absolute requirement of functional p53 in arsenic-mediated protection. Consistently, a brief arsenic pretreatment selectively protected only normal tissues, but not tumors, from toxicity of chemotherapy. An indispensable role of glycolysis in protecting normal tissues was demonstrated by using an inhibitor of glycolysis, 2-deoxyglucose, which almost totally abolished low-dose arsenic-mediated protection. Together, our work demonstrates that low-dose arsenic renders normal cells and tissues resistant to chemotherapy-induced toxicity by inducting glycolysis.

Hsa-miR-24-3p increases nasopharyngeal carcinoma radiosensitivity by targeting both the 3′UTR and 5′UTR of Jab1/CSN5

Radiotherapy is the standard therapy for nasopharyngeal carcinoma (NPC); however, radioresistance can hinder successful treatment. Here we report that microRNA (miR)-24 acts as a tumor suppressor and radiosensitizer in NPC cells and xenografts by targeting Jab1/CSN5. Although accumulating evidence has shown that Jab1/CSN5 functions as an oncoprotein in human cancers, its regulation through miRs has not been described. In this study, we found that Jab1/CSN5 functioned in a manner opposite to that of miR-24 in NPC tumorigenesis and radioresistance. We demonstrated that miR-24 inhibits Jab1/CSN5 translation via direct binding to its 3′ untranslated region (3′UTR) and 5′UTR, leading to tumor growth inhibition, and sensitizes NPC tumors to radiation in vivo. Furthermore, silencing Jab1/CSN5 phenocopied the function of miR-24 in NPC cells after ionizing radiation treatment, resulting in increased apoptosis. Finally, we analyzed 50 paired samples of primary and matched recurrent NPC tissues from 25 NPC patients and subjected them to high-throughput genomic quantitative nuclease protection assay for quantifying simultaneously miR and mRNA levels. Our results showed that miR-24 levels were significantly decreased in recurrent NPC and that levels of Jab1/CSN5, as its target, were higher than those in primary NPC. Together, our findings indicate that miR-24 inhibits NPC tumor growth and increases NPC radiosensitivity by directly regulating Jab1/CSN5 and that both miR-24 and Jab1/CSN5 can serve as prognostic markers for NPC recurrence; this, in turn, may provide a promising therapeutic strategy for reversing NPC radioresistance.

Abstract 5458: Elevated level of Fibrillarin induces chemoresistance by interfering p53 pathway in cancers

Fibrillarin (FBL), an rRNA 2′-O-methyltransferase, has long been recognized as the enzymatic unit of the C/D box small nucleolar ribonucleoprotein (snoRNP) involved in the first step of pre-rRNA processing. It is not until recently that the link between FBL and cancers has been unveiled. As a direct regulatory target of Myc and p53, FBL is frequently upregulated in breast cancers and prostate cancers. High expression of FBL has also been shown to be associated with poor survival in patients with breast cancer. Our recent study found that FBL overexpression contributes to tumorigenesis and confers cellular resistance to chemodrugs. However, the underlying mechanism of chemoresistance elicited by FBL upregulation is still elusive. Here, we studied the molecular roles of FBL on p53 response. The p53 pathway in response to etoposide and actinomycin D were examined in a variety cell lines including U-2OS, MCF7, H1299, ZR75-1, A549 and an FBL inducible cell line. Western blotting and immunofluorescent staining were employed to examine protein level and their cellular localization. We found that ectopic expression of FBL enhanced the protein level of MDM2, resulting in decreased p53 accumulation and activity in response to chemodrugs. Interestingly, MDM2 was partially recruited to the nucleoli under stimuli. FBL depletion abrogated the nucleolar recruitment of MDM2 and subsequently dampened p53 activation. In conclusion, physiological level of FBL sequesters and stabilizes MDM2 in the nucleoli, therefore inhibits p53 degradation. When upregulated in cancers, FBL stabilizes nucleus MDM2 and promotes the degradation of p53 Citation Format: Teng Xu, Suthakar Ganapathy, Meijun Long, Chul Ha, Zhi-Min Yuan, Hang Su. Elevated level of Fibrillarin induces chemoresistance by interfering p53 pathway in cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5458. doi:10.1158/1538-7445.AM2015-5458

P5-07-03: Modulation of Autophagic Activity by Extracellular pH.

Reprogramming energy metabolism from oxidative phosphorylation to aerobic glycolysis, a common feature of human cancer, results in a relative acidic tumor microenvironment which is further accentuated by hypoxia operating within most tumors. We report that alteration of extracellular pH induces marked and rapid changes of autophagic activity in a variety of cell lines including mammary non-transformed cells and breast cancer cells. Interestingly, acidic and basic conditions induce completely opposite effect on autophagy, with its activity suppressed at lower pH whereas stimulated at higher pH. Gene knockdown experiments indicate that pH induced-autophagy requires Beclin 1, Vps34 and ATG5, key components of the autophagy pathway. Of note, acidic condition not only inhibits the basal but also blocks starvation-induced autophagy activity. Significantly, examination of different areas of tumor mass reveals a lower autophagic activity within the inner region than the outer region. These findings have important implications on the connections between autophagy and cancer as well as a wide range of other physiological and pathological processes. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P5-07-03.