Author Manuscript Published OnlineFirst on August 23, 2011; DOI: 10.1158/0008-5472.CAN-11-1021 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Kogo et al. 1 Long non-coding RNA HOTAIR regulates Polycomb-dependent chromatin modification and is associated with poor prognosis in colorectal cancers. Ryunosuke Kogo1, 4, 6, Teppei Shimamura2, 6, Koshi Mimori1, 6, Kohichi Kawahara3, Seiya Imoto2, Tomoya Sudo1, Fumiaki Tanaka1, Kohei Shibata1, Akira Suzuki3, Shizuo Komune4, Satoru Miyano2, and Masaki Mori5 Authors’ Affiliations 1 Department of Surgery, Medical Institute of Bioregulation, Kyushu University, 4546, Tsurumihara, Beppu 874-0838, Japan. 2 Human Genome Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. 3 Division of Cancer Genetics, Medical Institute Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan. 4 Department of Otorhinolaryngology, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka, 812-8582, Japan. 5 Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2011 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 23, 2011; DOI: 10.1158/0008-5472.CAN-11-1021 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Kogo et al. 2 University, 2-2 Yamadaoka, Suita, 565-0871, Japan 6) These authors contributed equally to this work. Running title: HOTAIR in colorectal cancer Key words: HOTAIR, ncRNA, colorectal cancer, PRC2, liver metastasis Disclosure: There are no potential conflicts of interest to disclose. Corresponding author: Masaki Mori, MD, PhD, FACS Department of Gastroenterological Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, 565-0871, Japan Tel; +81-6-6879-3251, Fax; +81-6-6879-3259 E-mail; [email protected] Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2011 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 23, 2011; DOI: 10.1158/0008-5472.CAN-11-1021 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Kogo et al. 3 Abstract The functional impact of recently discovered long non-coding RNAs (ncRNAs) in human cancer remains to be clarified. One long ncRNA which has attracted attention is the Hox antisense intergenic RNA termed HOTAIR, a long ncRNA expressed from the developmental HOXC locus located on chromosome 12q13.13. In cooperation with Polycomb complex PRC2 the HOTAIR long ncRNA is reported to reprogram chromatin organization and promote breast cancer metastasis. In this study, we examined the status and function of HOTAIR in stage IV colorectal cancer (CRC) patients who have liver metastases and a poor prognosis. HOTAIR expression levels were higher in cancerous tissues than corresponding noncancerous tissues and high HOTAIR expression correlated tightly with the presence of liver metastasis. Moreover, patients with high HOTAIR expression had a relatively poorer prognosis. In a subset of 32 CRC speciments, gene set enrichment analysis using cDNA array data revealed a close correlation between expression of HOTAIR and members of the PRC2 complex (SUZ12, EZH2 and H3K27me3). Our findings suggest that HOTAIR expression is associated with a genome-wide reprogramming of PRC2 function not only in breast cancer but also in CRC, where upregulation of this long ncRNA may be a critical element in metastatic progression. Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2011 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 23, 2011; DOI: 10.1158/0008-5472.CAN-11-1021 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Kogo et al. 4 Introduction Non-coding RNAs (ncRNAs) are found throughout the genome. However, the functions of ncRNAs are only partially understood. The function and clinical significance of short ncRNAs, such as miRNA and siRNA were elucidated first, then, long ncRNAs were reported more recently. Most long ncRNAs work with DNA binding proteins, such as chromatin-modifying complexes, and epigenetically regulate the expression of multiple genes (1-3). Hox transcript antisense intergenic RNA (HOTAIR) is a long ncRNA that was identified from a custom tilling array of the HOXC locus (12q13.13) (2). HOTAIR trimethylates histone H3 lysine-27 (H3K27me3) of the HOXD locus with the polycomb repressive complex 2 (PRC2), which is composed of EZH2, SUZ12 and EED, and inhibits HOXD gene expression (2). Thus, HOTAIR epigenetically regulates HOXD expression, located on a different chromosome. Gupta et al. reported that HOTAIR induced genome-wide re-targeting of PRC2, leading to H3K27me3, and promoted metastasis of breast cancer by silencing multiple metastasis suppressor genes (4). In particular, they concluded that HOTAIR suppressed tumor suppressor genes such as HOXD10, PGR, and the Protocadherin gene family in breast cancer cells. HOTAIR expression was low in normal breast epithelia, but high in primary breast cancer as well as metastatic lesions. Moreover, breast cancer patients Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2011 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 23, 2011; DOI: 10.1158/0008-5472.CAN-11-1021 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Kogo et al. 5 with high HOTAIR expression had a poorer prognosis for overall survival and for metastasis-free survival than did those with low HOTAIR expression. Colorectal cancer (CRC) is the one of the most common cancers in the world. However, the existence of multiple known carcinogens and varying genetic backgrounds makes it difficult to determine which factors are most important in the development of CRC. Therefore, the identification of a bona-fide molecule involved in progression of CRC has been greatly sought after. In the current study, we clarified the clinical significance of HOTAIR expression in CRC. Moreover, to determine the function of HOTAIR in CRC, we used cDNA microarray data from another subset of 32 CRC samples obtained by laser micro dissection (LMD). We performed Gene Set Enrichment Analysis (GSEA) and investigated whether HOTAIR expression was highly correlated with previously curated gene expression signatures of PRC2 (4). Materials and Methods Clinical samples and cell lines Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2011 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 23, 2011; DOI: 10.1158/0008-5472.CAN-11-1021 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Kogo et al. 6 One hundred CRC samples (bulk samples) were obtained from patients who underwent surgery at the Medical Institute of Bioregulation Hospital, Kyushu University between 1993 and 2000. Another 32 CRC samples (LMD samples) were obtained from Medical Institute of Bioregulation Hospital, Kyushu University, Tokyo Medical and Dental University Hospital, Kitazato University Hospital, National Cancer center, and National Defense Medical College Hospital. All specimens were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Written informed consent was obtained from all patients. No patient received chemotherapy or radiotherapy before surgery. The follow-up periods ranged from two months to 11 years, with a mean of three years. HEK293T, HCT116 and SW480 cell lines were provided by the American Type Culture Collection (ATCC) and were maintained in DMEM, McCoy 5A, or RPMI1640, respectively, containing 10% fetal bovine serum with 100 units / mL penicillin and 100 μg / mL streptomycin and cultured in a humidified 5% CO2 incubator at 37°C. RNA preparation, reverse transcription and quantitative real time PCR One hundred bulk samples Total RNAs from frozen CRC samples were extracted using ISOGEN (Nippongene) following the manufacturer’s protocol. 32 LMD samples Total RNAs were extracted using QIAamp DNA Micro Kit (Qiagen) following the manufacturer’s protocol. As previously reported, cDNAs from all samples were synthesized from 8.0 μg of total RNA (5). HOTAIR levels were quantified using LightCyclerTM 480 Probes Master kit Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2011 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 23, 2011; DOI: 10.1158/0008-5472.CAN-11-1021 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Kogo et al. 7 (Roche Applied Science) following the manufacturer’s protocol with the following specific HOTAIR primers (forward, 5’-CAGTGGGGAACTCTGACTCG-3’; reverse, 5’-GTGCCTGGTGCTCTCTTACC-3’). HOTAIR levels were normalized to GAPDH (forward, 5'-GTCAACGGATTTGGTCTGTATT-3'; reverse, 5'-AGTCTTCTGGGTGGCAGTGAT-3'). Laser microdissection RNAs from another 32 CRC tissues were collected for laser microdissection (LMD). CRC tissues were microdissected using the LMD system (Leica Microsystems) as previously described (6). Gene set enrichment analysis of CRC with HOTAIR expression HOTAIR/GAPDH levels in 32 CRC tissues (LMD samples) were measured by quantitative