"MicroRNA Expression, Survival, and Response to Interferon in Liver Cancer".

Junfang Ji, Ph.D., Jiong Shi, M.D., Anuradha Budhu, Ph.D., Zhipeng Yu, B.S., Marshonna Forgues, B.S., Stephanie Roessler, Ph.D., Stefan Ambs, Ph.D., M.P.H., Yidong Chen, Ph.D., Paul S. Meltzer, M.D., Carlo M. Croce, M.D., Lun-Xiu Qin, M.D., Ph.D., Kwan Man, M.D., Ph.D., Chung-Mau Lo, M.D., Joyce Lee, B.S., Irene O.L. Ng, M.D., Jia Fan, M.D., Ph.D., Zhao-You Tang, M.D., Hui-Chuan Sun, M.D., Ph.D., and Xin Wei Wang, Ph.D.

ABSTRACT:

Background
Hepatocellular carcinoma is a common and aggressive cancer that occurs mainly in men. We examined microRNA expression patterns, survival, and response to interferon alfa in both men and women with the disease.

Methods
We analyzed three independent cohorts that included a total of 455 patients with hepatocellular carcinoma who had undergone radical tumor resection between 1999 and 2003. MicroRNA-expression profiling was performed in a cohort of 241 patients with hepatocellular carcinoma to identify tumor-related microRNAs and determine their association with survival in men and women. In addition, to validate our findings, we used quantitative reverse-transcriptase–polymerase-chain-reaction assays to measure microRNAs and assess their association with survival and response to therapy with interferon alfa in 214 patients from two independent, prospective, randomized, controlled trials of adjuvant interferon therapy.

Results
In patients with hepatocellular carcinoma, the expression of miR-26a and miR-26b in nontumor liver tissue was higher in women than in men. Tumors had reduced levels of miR-26 expression, as compared with paired noncancerous tissues, which indicated that the level of miR-26 expression was also associated with hepatocellular carcinoma. Moreover, tumors with reduced miR-26 expression had a distinct transcriptomic pattern, and analyses of gene networks revealed that activation of signaling pathways between nuclear factor B and interleukin-6 might play a role in tumor development. Patients whose tumors had low miR-26 expression had shorter overall survival but a better response to interferon therapy than did patients whose tumors had high expression of the microRNA.

Conclusions
The expression patterns of microRNAs in liver tissue differ between men and women with hepatocellular carcinoma. The miR-26 expression status of such patients is associated with survival and response to adjuvant therapy with interferon alfa.

Source Information

From the Liver Carcinogenesis Section (J.J., J.S., A.B., Z.Y., M.F., S.R., X.W.W.), Breast and Prostate Unit, Laboratory of Human Carcinogenesis (S.A.), and Genetics Branch (Y.C., P.S.M.), Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD; the Liver Cancer Institute and Zhongshan Hospital, Fudan University, Shanghai, China (J.S., L.-X.Q., J.F., Z.-Y.T., H.-C.S.); the Comprehensive Cancer Center, Ohio State University, Columbus (C.M.C.); and the Departments of Surgery (K.M., C.-M.L.) and Pathology (J.L., I.O.L.N.), Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong.

Supported in part by grants (Z01-BC 010313 and Z01-BC 010876) from the Intramural Research Program of the Center for Cancer Research of the National Cancer Institute.

Address reprint requests to Dr. Wang at the National Cancer Institute, 37 Convent Dr., Bldg. 37, Rm. 3044A, Bethesda, MD 20892, or at xw3u@nih.gov;
or to Dr. Sun at the Liver Cancer Institute and Zhongshan Hospital, Fudan University, Shanghai, China, or at sun.huichuan@zs-hospital.sh.cn.

Figure 2. Distinct Transcriptional Activities in Hepatocellular Carcinomas with Low miR-26 Expression.

Panel A shows a multidimensional scaling plot, based on the expression of 11,580 genes, for 224 patients with hepatocellular carcinoma. The plots are placed in three-dimensional space, spanned by the first three principal components of these genes. The level of miR-26 expression was dichotomized on the basis of the median value as low (blue) or high (red).

Panel B shows the activation of gene networks of signaling between nuclear factor B (NF-B) and interleukin-6 (IL6) in tumors with low miR-26 expression. Shaded ovals represent up-regulated genes in tumors with low miR-26 expression, and open ovals represent genes that are not on the list of significant genes but are reported to be associated with the network. The open ovals that are labeled as NF-B, Ib, and IKK represent molecular nodes related to protein complexes of NF-B, inhibitor of NF-B, and Ib kinase, respectively. The open oval labeled as 5-HT (5-hydroxytryptamine) represents a chemical node related to serotonin receptor signaling, as categorized by pathway analysis software (Ingenuity). Arrows represent positive regulation of gene expression, with solid arrows indicating direct regulation and broken arrows indirect regulation.

From Supplementary Appendix:

Six class prediction algorithms, i.e., Support Vector Machines (SVM), Compound Covariate Predictor (CCP), Diagonal Linear Discriminant (DLD), 1-Nearest Neighbor (1NN), 3-Nearest Neighbor (3NN) or Nearest Centroid (NC), were also used to determine whether mRNA expression patterns could accurately discriminate low miR-26 HCCs from high miR-26 HCCs. In these analyses, 90% of the samples were randomly chosen to
build a classifier which was then used to predict the remaining 10% of the cases. The accuracy of the prediction was calculated after 1000 repetitions of this random partitioning process to control the number and proportion of false discoveries. Hierarchical clustering analysis was performed using BRBarrayTools with mediancentered correlation and complete linkage. Using an unsupervised approach, we also performed multidimensional scaling analysis using all cohort 1 samples based on the first three principal components of 11,580 genes that passed the filter. The expression levels of these genes were log-transformed, and Euclidean distance was used to determine their positions. Gene Network Analyses were used to identify signaling pathways that were enriched with genes differentially expressed in tumors between miR-26 low and miR-26
high HCCs using Ingenuity Pathways Analysis (Ingenuity®, http://www.ingenuity.com).

http://content.nejm.org/cgi/content/full/361/15/1437/DC1

1. Crunkhorn S, (Aug. 2009)
"Therapy: microRNA suppresses liver cancer"

2.  Kota J, Chivukula RR, O'Donnell KA, Wentzel EA, Montgomery CL, Hwang H-W, Chang T-C, Vivekanandan P, Torbenson M, Clark KR, Mendell JR, and Mendel JT    (June, 2009)
"Therapeutic microRNA Delivery Suppresses Tumorigenesis in a Murine Liver Cancer Model",
Cell, Volume 137, Issue 6, 1005-1017, 12 June 2009,

3.  Rossi JJ,   (August, 2009)
"New Hope for a MicroRNA Therapy for Liver Cancer"
http://www.cell.com/abstract/S0092-8674(09)00643-6

4. Strizzi L, Hardy KM, Seftor EA, Costa FF, Kirschmann DA, Seftor RE, Postovit LM, Hendrix MJ.,
"Development and cancer: at the crossroads of Nodal and Notch signaling."

5. Mishra PJ,  and Merlino G,
"MicroRNA reexpression as differentiation therapy in cancer".

6. Taulli R, Bersani F, Foglizzo V, Linari A, Vigna E, Ladanyi M, Tuschl T, and Ponzetto C,
"The muscle-specific microRNA miR-206 blocks human rhabdomyosarcoma growth in xenotransplanted mice by promoting myogenic differentiation".

7. Nayak RR, Kearns M, Spielman RS, and Cheung VG,
  "Coexpression network based on natural variation in human gene expression reveals gene interactions and functions".

8. Ke Q, Ji J, Cheng C, Zhang Y, Lu M, Wang Y, Zhang L, Li P, Cui X, Chen L, He S, Shen A.
"Expression and prognostic role of Spy1 as a novel cell cycle protein in hepatocellular carcinoma".
Exp Mol Pathol. 2009 Aug 15. [Epub ahead of print]

Department of Pathology, Affiliated Cancer Hospital of Nantong University, Medical College of Nantong University, Nantong, 226001, China.

OBJECTIVES:
Spy1 is a novel cell cycle regulatory gene, which can control cell proliferation and survival through the atypical activation of cyclin-dependent kinases. Recent studies suggested that deregulation of Spy1 expression plays a key role in oncogenesis. To investigate the potential roles of Spy1 in hepatocellular carcinoma (HCC), expression of Spy1 was examined in human HCC samples.

METHODS:
Immunohistochemistry and Western blot analysis was performed for Spy1 in 61 hepatocellular carcinoma samples. The data were correlated with clinicopathological features. The univariate and multivariate survival analyses were also performed to determine their prognostic significance.

RESULTS:
Spy1 was overexpressed in hepatocellular carcinoma as compared with the adjacent normal tissue. High expression of Spy1 was associated with histological grade and the level of alpha fetal protein (AFP) (P=0.009 and 0.003, respectively), and Spy1 was positively correlated with proliferation marker Ki-67 (P<0.001). Univariate analysis showed that Spy1 expression was associated with poor prognosis (P=0.03). Multivariate analysis indicated that Spy1 and Ki-67 protein expression was an independent prognostic marker for HCC (P=0.001 and 0.012, respectively). While in vitro, following release from serum starvation of HuH7 HCC cell, the expression of Spy1 was upregulated.

CONCLUSIONS:
Our results suggested that Spy1 overexpression is involved in the pathogenesis of hepatocellular carcinoma, it may be a favorable independent poor prognostic parameter for hepatocellular carcinoma.

PMID: 19686732 [PubMed - as supplied by publisher]

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