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Downregulation of TESTIN and Its Association with Cancer History and a Tendency Toward Poor Survival in Head and Neck Squamous Cell Carcinoma

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Downregulation of TESTIN and Its Association with Cancer History and a Tendency Toward Poor Survival in Head and Neck Squamous Cell Carcinoma ORIGINAL ARTICLE Downregulation of TESTIN and Its Association With Cancer History and a Tendency Toward Poor Survival in Head and Neck Squamous Cell Carcinoma Esra Gunduz, DDS, PhD; Mehmet Gunduz, MD, PhD; Levent Beder, MD, PhD; Hitoshi Nagatsuka, DDS, PhD; Kunihiro Fukushima, MD, PhD; Recep Sutcu, MD; Namik Delibas, MD; Noboru Yamanaka, MD, PhD; Kenji Shimizu, PhD; Noriyuki Nagai, DDS, PhD Objective: To examine the role of TESTIN as a candi- ables to mRNA expression status of TESTIN revealed a date tumor suppressor gene in head and neck carcino- significant difference in terms of cancer history (P=.03). genesis. Moreover, a higher smoking ratio and a family cancer history were also associated with downregulation of Design: Mutation and messenger RNA (mRNA) expres- TESTIN, although the difference was not statistically sig- sion analyses. nificant (P=.43 and P=.16, respectively). Kaplan-Meier survival analysis demonstrated a worse survival rate Setting: Academic research. among the patients with low TESTIN expression com- pared with the patients with normal-high TESTIN ex- Patients: Paired normal and tumor samples were ob- pression. tained from 38 patients with primary head and neck squa- mous cell carcinoma. Conclusions: Our findings suggest that inactivation of Main Outcome Measures: Analysis and comparison TESTIN is involved in head and neck carcinogenesis of TESTIN gene mRNA expression and its relationship through its downregulation. Further studies in various to clinicopathologic variables. human cancer tissues using a large sample size and in vitro functional studies as well as clinical comparison Results: Mutation analysis showed a nucleotide and research studies would give us a better evaluation of amino acid change in 6 of the 38 tumor samples (16.0%). TESTIN’s role and its possible future application in mo- Semiquantitative mRNA expression analysis of TESTIN lecular diagnosis and treatment of different cancer types, revealed a decreased expression in approximately 50% including head and neck squamous cell carcinoma. of the tumors compared with their matched normal con- trols. Interestingly, comparison of clinicopathologic vari- Arch Otolaryngol Head Neck Surg. 2009;135(3):254-260 EAD AND NECK SQUAMOUS mutational inactivation allows cells to dis- Author Affiliations: cell carcinoma (HNSCC) play 1 or more phenotypes of neoplastic Departments of Oral Pathology is one of the most fre- growth. Loss of heterozygosity (LOH) is and Medicine (Drs E. Gunduz, quent cancers that lead to considered an indication of a TSG pres- M. Gunduz, Nagatsuka, and death, making it a major ence, whereby inactivation contributes to Nagai), Molecular Genetics health problem in the world. It includes oral, the development and/or progression of tu- (Drs E. Gunduz and Shimizu), H 2 and Otolaryngology–Head and oropharyngeal, nasopharyngeal, hypopha- mor. Detailed LOH analysis of polymor- Neck Surgery (Dr Fukushima), ryngeal, and laryngeal cancers and ac- phic loci distributed along a chromo- Graduate School of Medicine, counts for more than 644 000 new cases some can reveal a common minimal Dentistry, and Pharmaceutical worldwide each year, with a mortality rate deleted region where putative TSGs may Sciences, Okayama University, of 53% and a male predominance of 3:1.1 reside. Okayama, Japan; Department of Despite advanced technology in its detec- Previous cytogenetic studies as well as Otolaryngology–Head and Neck tion and treatment, HNSCC continues to microdeletion analysis have shown fre- Surgery, Wakayama Medical pose a great threat to human life. Recent ad- quent abnormalities of chromosome 7 in University, Wakayama, Japan vances in the technology and molecular bi- various cancer types, including HNSCC.3-10 (Drs M. Gunduz, Beder, and Yamanaka); and Department of ology of human cancer, including head and Moreover, microcell-mediated transfer of Medical Biochemistry, Suleyman neck carcinoma, have provided possible human chromosome 7 into a murine squa- Demirel University, Faculty of novel diagnostic and prognostic markers. mous cell carcinoma cell line was found Medicine, Isparta, Turkey Tumor suppressor genes (TSGs) are de- to inhibit tumorigenicity of the cell line.11 (Drs Sutcu and Delibasi). fined as genetic elements whose loss or In another study, introduction of a single (REPRINTED) ARCH OTOLARYNGOL HEAD NECK SURG/ VOL 135 (NO. 3), MAR 2009 WWW.ARCHOTO.COM 254 ©2009 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/26/2021 copy of human chromosome 7 into a highly aggressive human prostate carcinoma cell line increased tumor la- Table 1. Clinicopathologic Characteristics of Patients tency by at least 2-fold.12 Similarly, insertion of an in- a tact human chromosome 7 into an immortalized hu- Characteristic Values man fibroblast cell line with LOH in the 7q31-32 region Tumor site suppressed immortality of the cells and restored their se- Oral cavity 17 (44.7) nescent ability.13 All these studies have provided strong Oropharynx 8 (21.1) Larynx 7 (18.4) evidence for the existence of a TSG(s) in chromosome Hypopharynx 1 (2.6) 7q31 region. Maxilla 5 (13.2) Based on these studies, we previously examined the Age, mean (SD), y 64.2 (9.3) chromosome region 7q22-31 using a set of highly poly- Sex morphic microsatellite markers to evaluate allele loss ra- Male 30 (78.9) tios and candidate TSGs in HNSCC.14 The study indi- Female 8 (21.1) cated 2 differentially deleted chromosomal areas at 7q31 T category T1 3 (7.9) around the markers D7S486 and D7S643. The presence T2 15 (39.4) of microdeletion in both loci but retention of the chro- T3 8 (21.1) mosomal region between these 2 peaks in some cases sug- T4 12 (31.6) gested that chromosome 7q31 included 2 different TSGs, N category at least in HNSCC. We have already shown that ING3 N0 19 (50.0) located at one of these regions (D7S643) has been in- N1 7 (18.4) 14 N2 12 (31.6) volved as a TSG in HNSCC. However, the role of the TNM stage other possible candidate gene in HNSCC remained un- I 1 (2.6) known. Therefore, in the current study, we focused on II 10 (26.3) the other highly deleted chromosomal area around the III 9 (23.7) marker D7S486, which is located in intron 6 of the IV 18 (47.4) b TESTIN gene, confirming its high ratio of deletion. In Differentiation Well 15 (40.6) fact, missense mutations and decreased expression of Moderate 17 (45.9) TESTIN were detected in various cancer cell lines, in- Poor 5 (13.5) cluding breast cancer, pancreatic cancer, and hemato- 15 logic malignant neoplasms. Moreover, adenoviral trans- a Values other than age are expressed as number (percentage). fection of TESTIN into breast and uterine cancer cell lines b One case with an unknown situation was not included. reduced tumor growth in mice.16 A recent study also dem- onstrated tumor suppressor function of TESTIN in a tion approved the study. The clinicopathologic characteris- knockout mouse model.17 Thus, we analyzed the expres- tics of the patients were shown in Table 1. sion level and mutation status of TESTIN to clarify its role in HNSCC. We also compared the messenger RNA ISOLATION, COMPLEMENTARY DNA RNA (mRNA) status of TESTIN with clinicopathologic PREPARATION, AND RT-PCR ANALYSIS variables. Total RNA samples were prepared using a modified acid METHODS guanidinium phenol chloroform method (ISOGEN; Nippon Gene Co Ltd, Tokyo, Japan). Total RNA was reverse- transcribed with a preamplification system (ReverTra Ace PATIENTS AND SAMPLES Kit; Toyobo Co Ltd, Osaka, Japan) starting with 2 µg of total RNA from each sample, according to the procedures pro- Paired normal and tumor samples were obtained from 38 vided by the supplier. TESTIN (GenBank NM_015641) mRNA patients with primary HNSCC between 1999 and 2005 at the expression in paired tumor and normal tissues was examined Department of Otolaryngology–Head and Neck Surgery, by duplex reverse transcriptase–polymerase chain reaction (RT- Okayama University Hospital, Okayama, Japan, after PCR). One microliter of each RT reaction was amplified in 50 informed consent was obtained from each patient. Normal µL of mixture containing 1.2mM magnesium chloride, 1X PCR control samples were obtained from grossly normal tissue as buffer, 200 µmol/L of each deoxynucleotide triphosphate, 20 far as possible from the tumor tissue. Although surgical mar- pmol of each primer, and1Uofrecombinant Thermus ther- gins were examined during surgery, we also confirmed the mophilus (rTth) DNA polymerase XL (Applied Biosystems, Fos- histopathologic appearance of the normal tissues by ter City, California). Thirty PCR cycles of TESTIN primers, RT-S hematoxylin-eosin staining. All tissues were frozen in liquid (5Ј- GAA TGA GAA GCT ATA CTG TGG C) and RT-AS (5Ј- nitrogen immediately after surgery and stored at −80°C until ATG GCT CGA TAC TTC TGG GTG), and 25 cycles of ␤- the extraction of RNA. The study included 30 men and 8 actin primers, S1 (5Ј-GGC CAA CCG CGA GAA GAT GAC) women (mean age, 64 years; age range, 40-81 years). The and AS1 (5Ј-GCT CGT AGC TCT TCT CCA GGG), were used histologic diagnosis in all cases was squamous cell carci- for amplification (the primers were designed with noma. None of the patients received preoperative chemo- GENETYX-MAC 10.1; Software Development Co Ltd, Tokyo, therapy or radiotherapy. The differentiation and diagnosis of Japan). An initial denaturation step at 94°C for 3 minutes was the tumor was based on the surgical pathology reports of the followed by 30 cycles of a denaturation step at 94°C for 30 sec- hospital. All clinical information was obtained from the onds, an annealing step at 60°C for 1 minute, and an exten- patient files, which included the initial diagnosis, treatment, sion step at 72°C for 1 minute. A final extension step at 72°C and follow-up data.
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