Vol. 7, 2815–2820, September 2001 Clinical Cancer Research 2815 Overexpression of HRad17 mRNA in Human Breast Cancer: Correlation with Lymph Node Metastasis Akemi Kataoka, Noriaki Sadanaga, tasis in human breast cancers. Although its function still Koshi Mimori, Hiroaki Ueo, Graham F. Barnard, remains unclear, the expression of HRad17 mRNA could Keizo Sugimachi, Daniel Auclair, Lan Bo Chen,1 open up a new window for the diagnostic staging and treat- ment of human breast cancers. and Masaki Mori1 Department of Surgery, Medical Institute of Bioregulation, Kyushu INTRODUCTION University, Beppu 874-0838, Japan [A. K., N. S., M. M.]; Kimmel Cancer Institute, Jefferson Medical College, Philadelphia, In Schizosaccharomyces pombe, the products of six genes, Pennsylvania 19107 [K. M.]; Department of Surgery, Oita Prefectural Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1, have been iden- Hospital, Oita 870-8511, Japan [H. U.]; Division of Digestive Disease tified as essential components of checkpoint pathways. Several and Nutrition, University of Massachusetts Medical Center, of these genes have structural homologues in the budding yeasts, Worcester, Massachusetts 01655 [G. F. B.]; Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 and additional conservation across eukaryotes has been demon- [D. A., L. B. C.]; and Department of Surgery II, Faculty of Medicine, strated, e.g., human homologues of S. pombe Rad3, ataxia Kyushu University, Fukuoka 812-8582, Japan [K. S.] telangiectasia mutated (1), and ataxia telangiectasia- and Rad3- related; 2, 3); a human homologue of S. pombe Rad9, HRad9 (4); and a human homologue of S. pombe Rad1, HRad1 (5). ABSTRACT Recently, a human homologue of the S. pombe Rad17 check- Purpose: A novel human gene, designated HRad17, was point gene named HRad17 was identified (6–8). S. pombe identified as the human homologue of the Rad17 of Schizos- Rad17 is known to play critical roles in maintaining genomic accharomyces pombe and Rad24 of Saccharomyces cerevi- stability and integrity, as a G2-M checkpoint protein in the cell siae. In yeast, these genes play a critical role in maintaining cycle, and to prevent the development of cancer and hereditary genomic stability. The aim of this study was to evaluate the diseases; however, the function of HRad17 is still unclear. expression of HRad17 in human breast cancer. It was reported independently that HRad17 could be local- Experimental Design: We investigated HRad17 mRNA ized on human chromosome 4q or 5q by fluorescence in situ expression in 64 cases of human breast cancer by means of hybridization analysis (6, 7, 9). These regions are implicated in reverse-transcription-PCR, in situ hybridization, and immu- the etiology of a variety of human cancers, including breast nohistochemistry. cancer, hepatocellular carcinoma, small cell lung cancer, non- Results: The HRad17 mRNA was overexpressed in 35 small cell lung cancer, duodenal adenocarcinoma, and head and cases (54.7%). Twenty-four (68.6%) of 35 cases with neck squamous cell carcinoma (1, 10–13). IHC2 and RT-PCR HRad17 overexpression in cancer tissues were node-posi- analysis indicates elevated levels of expression of HRad17 in tive, whereas only 8 (27.6%) of 29 cases without HRad17 human testis and colon cancer (6). But there are no previous overexpressions were node-positive. The expression of reports studying the clinical significance of HRad17 expression HRad17 mRNA correlated with both lymph node metastasis in human cancer. Thus, we investigated its expression in human ؍ ؍ (P 0.001) and high Ki67 labeling index (P 0.006). breast cancer cell lines and in clinical breast cancers by means Although not significantly different, expression of HRad17 of RT-PCR and IHC using a monoclonal antibody to evaluate its ؍ mRNA tended to correlate with tumor size (P 0.06) and clinical significance. We also determined whether expression of ؍ expression of mutant p53 protein (P 0.10). Furthermore, HRad17 mRNA correlates with p53 mutation, overexpression of expression of HRad17 mRNA was an independent predictor oncoprotein c-erbB-2 (also known as HER2/neu), or Ki67 la- of axillary lymph node metastasis as well as of lymphatic beling index as a marker of proliferation. permeation by multivariate analysis (P < 0.0001). Conclusions: Our study demonstrates that HRad17 MATERIALS AND METHODS might be related to the development of lymph node metas- Tissue Specimens and Cell Lines. Sixty-four matched normal breast and breast cancer tissue samples were obtained from radical mastectomy performed at the Oita Prefectural Hos- pital (Beppu, Japan), the National Beppu Hospital (Beppu, Ja- Received 7/5/00; revised 5/22/01; accepted 5/31/01. pan), the Matsuyama Red Cross Hospital (Beppu, Japan), and The costs of publication of this article were defrayed in part by the the Medical Institute of Bioregulation, Kyushu University payment of page charges. This article must therefore be hereby marked (Beppu, Japan), between June 1998 and May 1999. Patients advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Dr. Lan Bo Chen, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115; Phone: (617) 632-3385; Fax: (617) 632-4470; or at Dr. Masaki 2 The abbreviations used are: IHC, immunohistochemistry; RT-PCR, Mori, Department of Surgery, Medical Institute of Bioregulation, reverse-transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehy- Kyushu University, 4546 Tsurumihara, Beppu 874-0838, Japan. drogenase. Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2001 American Association for Cancer Research. 2816 HRad17 Expression in Breast Cancers were excluded if they had received preoperative neoadjuvant Table 1 Clinico-pathological data of 64 patients chemotherapy or radiotherapy. Specimens were frozen in liquid HRad17 mRNA nitrogen immediately after surgical resection and kept at Ϫ90°C until the extraction of RNA. Human breast cancer cell lines Negative Positive Factors (%) (%) P MCF-7, MRK-nu-1, YMB-1-E, and YMB-1 were used. RNA Extraction and RT-PCR. Total RNA was ex- Mean age (yr) 29 (45.3%) 35 (54.7%) Menopause 54.6 Ϯ 12.6 56.1 Ϯ 12.0 NSa tracted by the acid guanidium thiocyanate-phenol-chloroform Premenopausal 11 (34.5%) 11 (32.8%) NS extraction procedure. The cDNA was synthesized from 2.5 ␮g Postmenopausal 19 (65.5%) 24 (67.2%) of total RNA as described previously (14). HRad17-specific Tumor size (cm) PCR was performed with primers 5Ј-TCCTTAGAACAGATT- Յ2.0 14 (48.3%) 9 (25.7%) 0.06 Ј Ј Ն2.1 15 (51.7%) 26 (74.3%) TATGGTTTA-3 and 5 -ATACTTTACATGAAGTTCTA- Ϯ Ϯ Ј Mean 2.4 1.4 2.8 1.6 AGGA-3 to amplify a 523-bp fragment. To prevent amplifica- Histological subtype tion from eventual contamination genomic DNA, these primers NIDC 1 (3.5%) 0 (0%) NS are located in different exons. PCR was performed for 26 cycles IDC 27 (93.0%) 30 (88.6%) (45sat94°C; 45 s at 60°C; and 45 s at 72°C). Five-␮l aliquots Other 1 (3.5%) 4 (11.4%) Lymph node metastasis of the PCR products were size-fractionated on a 1.5% agarose Negative 21 (72.4%) 11 (31.4%) 0.001 gel and visualized after ethidium bromide staining. The PCR Positive 8 (27.6%) 24 (68.6%) products were subcloned into the pCR II vector (Invitrogen) and Lymphatic permeation verified by sequencing (ABI100 version 3.3; ABI PRISM). Negative 17 (58.6%) 16 (45.7%) NS RNA quality was verified by running RT-PCR reactions for Positive 12 (41.4%) 19 (54.3%) Vascular invasion GAPDH on each RNA sample (14). Primer sequences for Negative 25 (86.2%) 29 (82.9%) NS GAPDH were 5GTCAACGGATTTGGTCTGTATT-3Ј and 5Ј- Positive 4 (13.8%) 6 (17.1%) AGTCTTCTGGGTGGCAGTGAT-3Ј(product size, 560 bp), ER status and PCR was performed for 22 cycles (60 s at 94°C; 60 s at Negative 8 (27.6%) 15 (42.9%) NS Positive 20 (69.0%) 19 (54.3%) 56°C; and 60 s at 72°C). Each series of RT-PCR reactions had Unknown 1 (3.5%) 1 (2.9%) a sample without RNA as a negative control, and all specimens PR status were analyzed at least twice. Negative 10 (34.5%) 17 (48.6%) NS In Situ RNA Hybridization. In situ RNA hybridization Positive 18 (62.1%) 17 (48.6%) was performed using a nonradioactive RNA color kit Unknown 1 (3.5%) 1 (2.9%) p53 proteinb (RPN3300; Amersham Pharmacia). A 523-bp PCR product was Negative 20 (69.0%) 17 (48.6%) 0.10 subcloned into TA vectors (pCR II; Invitrogen). The plasmid Positive 9 (31.0%) 18 (51.4%) DNA was isolated and purified using the RPM kit (BIO 101; Overexpression of c-erbB-2c Biotechnologies, Inc.), and fragments corresponding to the Negative 26 (89.7%) 27 (77.1%) NS Ј Positive 3 (10.3%) 8 (22.9%) COOH terminus and 3 region of Hrad17 were excised using Ki67 labeling index (%)d XhoI (Takara Co., Kyoto, Japan). Riboprobes were generated Ͻ8.3 20 (69.0%) 12 (34.2%) 0.006 with T7 and SP6 RNA polymerase. Ն8.3 9 (31.0%) 23 (65.8%) IHC. We used mouse monoclonal antibodies 31E9 for a NS, not significant; NIDC, non-invasive ductal carcinoma; IDC, HRad17 protein (6, 15), DO-7 (Dako Co.) for p53, and NCL- invasive ductal carcinoma; ER, estrogen receptor; PR, progesterone Ki67-MM1 (NovoCastra Laboratories, Ltd.) for Ki67. Rabbit receptor. b Ͼ polyclonal antibody (Nichirei Co., Tokyo, Japan) was used for p53 protein was judged as positive when 10% of cancer cells ␮ were stained with DO-7 by IHC. the detection of overexpression of c-erbB-2. Five- m-thick c Overexpression of c-erbB-2, judged as positive when Ͼ10% of sections of formalin-fixed and paraffin-embedded tissue sam- cancer cells were stained membrane with polyclonal antibody.