RESEARCH ARTICLE
DNA-PKcs and Ku70 are Predictive Markers for Poor Prognosis of Patients With Gall Bladder Malignancies Feng Ren, MD, PhD,* Zhu-lin Yang, MD, PhD,w Xingguo Tan, MD,w Dongcai Liu, MD, PhD,* Qiong Zou, MD,z Yuan Yuan, MD,z Jinghe Li, MD,y Lufeng Liang, MD,8 Guixiang Zeng, MD,z and Senlin Chen, MD# Abstract: Gall bladder cancers (GBCs) are highly resistant to radiotherapy and chemotherapy. Unfortunately, the key molecular mechanisms responsible for therapeutic resistance have not been identified. In this study, the expression of DNA-PKcs and Ku70 in 46 squamous cell/adenosquamous carcinomas (SC/ASCs) and 80 adenocarcinomas (ACs) were examined by immunohistochemical analysis. Positive DNA-PKcs and Ku70 expression were significantly associated with less lymph node metastasis, invasion, and low TNM stage of SC/ASCs and ACs. Univariate Kaplan-Meier analysis showed that loss of DNA-PKcs and Ku70 expression significantly correlated with decreased survival in both SC/ASC and AC patients. Multivariate Cox regression analysis showed that loss of DNA-PKcs and Ku70 expression was an independent poor prognostic predictor in both SC/ASC and AC patients. Our study suggested that DNA-PKcs and Ku70 are tumor suppressors, and loss of DNA-PKcs and Ku70 expression is an important biological marker for metastasis, invasion, and prognosis of GBC. Currently, there is no implication of DNA-PKcs and Ku70 expression in chemoresistance or radioresistance in GBC. Key Words: gall bladder cancer, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, DNA-PKcs, Ku70, prognosis, metastasis (Appl Immunohistochem Mol Morphol 2014;22:741–747)
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all bladder cancers (GBCs) are highly aggressive cancers with high mortality rates among gastrointestinal cancers. Over 90% of GBC patients are diagnosed at an advanced clinical stage with severe invasion and metastasis owing to a lack of specific early signs or symptoms of GBC.1 Patients with advanced clinical stage of GBC generally have extremely poor prognosis.2 UnReceived for publication July 8, 2013; accepted September 9, 2013. From the *Department of Surgery; wResearch Laboratory of Hepatobiliary Diseases, Second Xiangya Hospital; zDepartment of Pathology, Third Xiangya Hospital; yDepartment of Pathology, Xiangya Medical School, Central South University; 8Department of Hepatobiliary and Pancreatic Surgery, Hunan Provincial People’s Hospital; #Department of Pathology, Hunan Provincial Tumor Hospital, Changsha; and zDepartment of Pathology, Loudi Central Hospital, Loudi, P.R. China. The authors declare no conflict of interest. Reprints: Zhu-lin Yang, MD, PhD, Research Laboratory of Hepatobiliary Diseases, Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, China (e-mail: yangzhulin8@ sina.com). Copyright r 2014 by Lippincott Williams & Wilkins
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fortunately, palliative chemotherapy and radiation therapy offer little benefit to the survival of GBC patients.3 The most successful clinical trial only extended the survival of biliary tract cancer patients by 3 months using a combination of cisplatin and gemcitabine.4 GBCs have several histologic subtypes. Adenocarcinoma (AC) is a major subtype (90% to 95%),5 whereas squamous cell/adenosquamous carcinoma (SC/ASC) is a rare GBC subtype.2,6 Currently, no study has compared the clinicopathologic and biological differences between SC/ASC and AC subtypes. Importantly, the key molecules responsible for the aggressive nature of GBC have not been identified. DNA double-strand breaks (DSBs) are widely considered to be the most lethal form of DNA damage in mammalian cells. DSBs can be introduced by a variety of exogenous agents including ionizing radiation and radiomimetic drugs (eg, bleomycin and neocarzinostatin).7 Nonhomologous end-joining (NHEJ) is a major repair mechanism for DSBs. The capture of both broken DNA ends together and presenting them together to the DNAprotein complex is an initial and key step of NHEJ.8 The Ku70/80 heterodimer (Ku) is composed of Ku70 and Ku80 subunits. Each subunit contributes to a central DNA-binding core during NHEJ by binding the ends of broken DNA strands and recruiting the catalytic subunit of DNA-protein kinase (DNA-PKcs).9 Inward translocation of Ku allows DNA-PKcs to interact with the terminal ends of the DNA, allowing 2 DNA-PKcs molecules to interact across the DSB.10 It has been demonstrated that silencing Ku or DNAPKcs gene expression can completely shut down NHEJ repair of DSBs.11,12 Therefore, cancer cells with a high expression of DNA repair-associated molecules should be resistant to DSBs. We hypothesize that the high resistance of GBC to radiotherapy and chemotherapy may be associated with high expressions of Ku and DNA-PKcs. In this study, the expression of DNA-PKcs and Ku70 in surgically resected specimens, including AC and SC/ASC, was examined by immunohistochemical analysis. The correlation of DNA-PKcs and Ku70 expression with clinicopathologic characteristics and prognosis of AC and SC/ASC was comparatively evaluated.
MATERIALS AND METHODS Case Selection A total of 46 SC/ASCs from GBC patients that underwent surgical resection or biopsy were collected from January 1995 to December 2009. A total of 1060
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patients with all types of GBCs were diagnosed from January 2001 to December 2009, and tumor tissues of 80 ACs were randomly selected for this study. All diagnoses were based on clinical findings, morphologic criteria, and immunohistochemical stainings. A tumor was diagnosed as ASC when it contains both SCs and AC cells, but the tumor must contain at least 10% AC or SC carcinoma cells. A tumor was diagnosed as SC when most malignant cells are SCs and 3 TNM stage I+II II III Lymph metastasis No Yes Invasion No Yes AC Differentiation Well Moderately Poorly Tumor mass size (cm) r3 >3 TNM stage I+II III IV Lymph metastasis No Yes Invasion No Yes
v2
Ku70 P
Total N
Positive N (%)
46
20 (43.5)
16 24 6
9 (56.3) 10 (41.7) 1 (16.7)
2.852
0.262
20 26
13 (65.0) 7 (26.9)
6.669
0.010
12 20 14
9 (83.3) 9 (45.0) 2 (14.3)
9.724
0.008
17 29
12 (70.6) 8 (27.6)
8.065
0.005
16 30 80
13 (81.3) 7 (23.3) 41 (51.3)
14.243
0.000
27 25 28
18 (66.7) 11 (44.9) 12 (42.9)
3.884
0.143
50 30
29 (58.0) 12 (40.0)
2.432
0.119
21 38 21
16 (76.2) 16 (42.1) 9 (42.9)
7.092
0.029
30 50
20 (66.7) 21 (42.0)
4.566
0.038
31 49
21 (67.7) 20 (40.8)
5.510
0.023
v2
P
9 (56.3) 11 (45.8) 1 (16.7)
2.76
0.250
13 (65.0) 8 (30.8)
5.339
0.021
9.872
0.007
12 (70.6) 9 (31.0)
6.758
0.009
11 (68.8) 10 (33.3) 38 (47.5)
5.275
0.022
2.378
0.305
3.863
0.049
10.392
0.006
19 (63.3) 19 (38.0)
4.825
0.031
21 (67.7) 17 (34.7)
8.316
0.004
Positive N (%) 21 (45.7)
9 (83.3) 10 (50.0) 2 (14.3)
16 (59.3) 11 (44.0) 11 (39.3) 28 (56.0) 10 (33.3) 16 (76.2) 16 (42.1) 6 (28.6)
AC indicates adenocarcinoma; SC/ASC, squamous carcinoma/adenosquamous carcinoma.
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observed in the percentage of cases with positive DNAPKcs and Ku70 expression between SC/ASC and AC patients (data not shown). As shown in Table 1, positive DNA-PKcs expression was observed in 43.5% of SC/ ASC and 51.3% of AC patients, whereas positive Ku70 expression was observed in 45.7% of SC/ASC and 47.5% of AC patients. The percentage of cases with positive DNA-PKcs or Ku70 expression was significantly higher in the tumor tissues from SC/ASC patients with lower TMN stage, small tumor size (< 3 cm), low invasion, and lymph node metastasis compared with the tumor tissues from patients with higher TMN stage, larger tumor size
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(>3 cm), high invasion, and lymph metastasis (P < 0.05, 0.01, or P < 0.001). DNA-PKcs and Ku70 exhibited no significant association with differentiation in SC/ASC. The percentage of cases with positive DNA-PKcs and Ku70 expression in AC tumors was significantly higher in cases with lower TMN stage, no invasion, and lymph node metastasis compared with the cases with higher TMN stage with lymph metastasis and invasion (P < 0.05 or 0.01; Table 1). Ku70, but not DNA-PKcs expression, was observed more in patients with small tumor size. Both DNA-PKcs and Ku70 exhibited no significant association with differentiation of AC (Table 1).
TABLE 2. Relationship Between DNA-PKcs and Ku70 Expression, Clinicopathologic Characteristics, and Average Survival of SC/ASC and AC Patients Clinicopathologic Characteristics SC/ASC Differentiation Well Moderately Poorly Tumor mass size (cm) r3 >3 TNM stage I+II III IV Lymph metastasis No Yes Invasion No Yes DNA-PKcs Negative Positive Ku70 Negative Positive AC Differentiation Well Moderately Poorly Tumor mass size (cm) r3 >3 TNM stage I+II III IV Lymph metastasis No Yes Invasion No Yes DNA-PKcs Negative Positive Ku70 Negative Positive
Samples (n)
Average Survival (mo)
v2
P
16 24 6
13.81 (5-24) 8.92 (4-18) 5.83 (4-9)
19.125
0.000
20 26
14.35 (7-24) 7.04 (4-11)
31.337
0.000
12 20 14
17.00 (9-24) 9.20 (7-15) 5.86 (4-8)
51.139
0.000
17 29
14.24 (4-24) 7.86 (4-15)
16.219
0.000
16 30
15.75 (9-24) 7.27 (4-12)
32.271
0.000
26 20
7.73 (4-24) 13.45 (7-24)
15.243
0.000
25 21
8.48 (4-24) 12.29 (7-24)
5.920
0.015
27 25 28
15.07 (5-24) 10.60 (4-24) 6.68 (3-14)
32.501
0.000
50 30
13.70 (6-24) 5.80 (3-10)
68.283
0.000
21 38 21
18.96 (5-24) 9.29 (6-15) 5.14 (3-7)
105.825
0.000
30 50
16.27 (4-24) 7.42 (3-14)
42.372
0.000
31 49
16.68 (7-24) 6.98 (3-11)
55.535
0.000
39 41
8.59 (3-24) 12.78 (3-24)
8.950
0.003
42 38
8.69 (3-24) 13.00 (3-24)
8.832
0.003
AC indicates adenocarcinoma; SC/ASC, squamous carcinoma/adenosquamous carcinoma.
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The Correlation Between DNA-PKcs and Ku70 Expression With Survival in Patients With SC/ASC and AC
Biomarkers in GBC
Survival information of patients was obtained through letters and phone calls. The follow-up time was 2 years, and patients who survived longer than 2 years were included in the analysis as censored cases. Of the 46 SC/ASCs patients, 33 patients survived 3 cm I+II/III/IV No/yes No/yes /+ /+
0.300 2.733 1.142 1.533 2.944 0.891 0.878
0.366 0.835 0.532 0.623 0.864 0.364 0.350
0.672 10.713 4.608 6.055 11.610 5.992 6.293
0.412 0.001 0.032 0.014 0.001 0.014 0.012
1.350 15.379 3.133 4.632 18.992 0.410 0.416
0.659 2.993 1.104 1.366 3.492 0.201 0.209
2.766 79.012 8.888 15.707 103.280 0.837 0.825
Well/moderately/poorly r3 cm/ >3 cm I+II/III/IV No/yes No/yes /+ /+
1.415 0.906 1.219 0.972 1.523 0.949 1.117
0.547 0.417 0.448 0.457 0.498 0.437 0.446
6.692 4.720 7.404 4.524 9.353 4.716 6.272
0.010 0.030 0.007 0.033 0.002 0.030 0.012
4.116 2.474 3.384 2.643 4.586 0.387 0.327
1.409 1.093 1.406 1.079 1.728 0.164 0.137
12.027 5.603 8.142 6.474 12.171 0.912 0.784
AC indicates adenocarcinoma; CI, confidence interval; RC, regression coefficients; RR, relative risk; SC/ASC, squamous carcinoma/adenosquamous carcinoma.
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breast cancer.22 Consistent with previous findings in various tumors, loss of DNA-PKcs and Ku70 expression was associated with high TMN stage, invasion, and metastasis in SC/ASC and AC tumors. Interestingly, we found that negative DNA-PKcs and Ku70 expression correlated with shorter survival in both SC/ASC and AC patients, suggesting that DNA-PKcs and Ku70 expression are poor prognostic factors in GBC. These observations seem to conflict with the functions of DNA-PK complex in DSBs repair. Besides the crucial role of DNA-PKcs in DNA repair, the activated DNA-PKcs can phosphorylate proteins involved in the control of cell cycle checkpoints and different cell-death pathways.25 For example, DNA-PKcs can phosphorylate tumor suppressor p53, which stabilizes p53 and subsequently causes increased expression of the cell cycle regulator p21 or the proapoptotic protein Bax.26 Besides its vital role in DNA repair, Ku is also involved in numerous other cellular processes, including telomere maintenance, regulation of specific gene transcription, apoptosis, and regulation of cell cycle.27 For example, hypoxia inhibits Ku70/80 expression, leading to an increase in angiogenesis and altered p53 expression.28 Therefore, negative DNA-PKcs and Ku70 expression may implicate an increase in tumor cell survival. In conclusion, DNA-PKcs and Ku70 expression may be a malignant sign of tumor in GBC and is associated with the aggressive nature of GBC, such as invasion, metastasis, and poor prognosis. In contrast, DNA-PKcs and Ku70 expression may not be associated with the resistance of GBC to radiotherapy and chemotherapy. REFERENCES 1. Hawkins WG, DeMatteo RP, Jarnagin WR, et al. Jaundice predicts advanced disease and early mortality in patients with gallbladder cancer. Ann Surg Oncol. 2004;11:310–315. 2. Jayaraman S, Jarnagin WR. Management of gallbladder cancer. Gastroenterol Clin North Am. 2010;39:331–342. 3. de Aretxabala X, Roa I, Burgos L, et al. Gallbladder cancer: an analysis of a series of 139 patients with restricted to the subserosal layer. J Gastrointest Surg. 2006;10:186–192. 4. Valle J, Wasan H, Palmer DH, et al. ABC-02 Trial Investigators. Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer. N Engl J Med. 2010;362:1273–1281. 5. Ootani T, Shirai Y, Tsukada K, et al. Relationship between gallbladder carcinoma and the segmental type of adenomyomatosis of the gallbladder. Cancer. 1992;69:2647–2652. 6. Roa JC, Tapia O, Cakir A, et al. Squamous cell and adenosquamous carcinomas of the gallbladder: clinicopathological analysis of 34 cases identified in 606 carcinomas. Mod Pathol. 2011;24:1069–1078. 7. Mahaney BL, Meek K, Lees-Miller SP. Repair of ionizing radiationinduced DNA double-strand breaks by non-homologous endjoining. Biochem J. 2009;417:639–650. 8. Bouchaert P, Guerif S, Debiais C, et al. DNA-PKcs expression predicts response to radiotherapy in prostate cancer. Int J Radiat Oncol Biol Phys. 2012;84:1179–1185.
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9. Lee HS, Yang HK, Kim WH, et al. Loss of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) expression in gastric cancers. Cancer Res Treat. 2005;37:98–102. 10. Hande MP. DNA repair factors and telomere-chromosome integrity in mammalian cells. Cytogenet Genome Res. 2004;104:116–122. 11. Kon T, Zhang X, Huang Q, et al. Oncolytic virus-mediated tumor radiosensitization in mice through DNA-PKcs-specific shRNA. Transl Cancer Res. 2012;1:4–14. 12. Vandersickel V, Mancini M, Marras E, et al. Lentivirus-mediated RNA interference of Ku70 to enhance radiosensitivity of human mammary epithelial cells. Int J Radiat Biol. 2010;86:114–124. 13. Nishihara K, Nagai E, Izumi Y, et al. Adenosquamous carcinoma of the gallbladder: a clinicopathological, immunohistochemical and flow-cytometric study of twenty cases. Jpn J Cancer Res. 1994; 85:389–399. 14. Kondo M, Dono K, Sakon M, et al. Adenosquamous carcinoma of the gallbladder. Hepatogastroenterology. 2002;49:1230–1234. 15. Oohashi Y, Shirai Y, Wakai T, et al. Adenosquamous carcinoma of the gallbladder warrants resection only if curative resection is feasible. Cancer. 2002;94:3000–3005. 16. Beskow C, Skikuniene J, Holgersson A, et al. Radioresistant cervical cancer shows upregulation of the NHEJ proteins DNA-PKcs, Ku70 and Ku86. Br J Cancer. 2009;101:816–821. 17. Wilson CR, Davidson SE, Margison GP, et al. Expression of Ku70 correlates with survival in carcinoma of the cervix. Br J Cancer. 2000;83:1702–1706. 18. Kienker LJ, Shin EK, Meek K. Both V(D)J recombination and radioresistance require DNA-PK kinase activity, though minimal levels suffice for V(D)J recombination. Nucleic Acids Res. 2000;28:2752–2761. 19. Lee HS, Choe G, Park KU, et al. Altered expression of DNAdependent protein kinase catalytic subunit (DNA-PKcs) during gastric carcinogenesis and its clinical implications on gastric cancer. Int J Oncol. 2007;31:859–866. 20. Rigas B, Borgo S, Elhosseining A, et al. Decreased expression of DNA-dependent protein kinase, a DNA repair protein, during human colon carcinogenesis. Cancer Res. 2001;61:8381–8384. 21. Treilleux I, Chapot B, Goddard S, et al. The molecular causes of low ATM protein expression in breast carcinoma; promoter methylation and levels of the catalytic subunit of DNA-dependent protein kinase. Histopathology. 2007;51:63–69. 22. Someva M, Sakata K, Matsumoto Y, et al. Immunohistochemical analysis of Ku70/86 expression of breast cancer tissues. Oncol Rep. 2007;17:1483–1487. 23. Yang MD, Wang HC, Chang WS, et al. Genetic polymorphisms of DNA double strand break gene Ku70 and gastric cancer in Taiwan. BMC Cancer. 2011;11:174. 24. Persson O, Salford LG, Fransson J, et al. stribution, cellular localization, and therapeutic potential of the tumor-associated antigen Ku70/80 in glioblastoma multiforme. J Neuro Oncol. 2010; 97:207–215. 25. Lees-Miller SP, Sakaguchi K, Ullrich SJ, et al. Human DNAactivated protein kinase phosphorylates serines 15 and 37 in the amino-terminal transactivation domain of human p53. Mol Cell Biol. 1992;12:5041–5049. 26. Bernstein C, Bernstein H, Payne CM, et al. DNA repair/proapoptotic dual-role proteins in five major DNA repair pathways: fail-safe protection against carcinogenesis. Mutat Res. 2002;511:145–178. 27. Gullo C, Au M, Feng G, et al. The biology of Ku and its potential oncogenic role in cancer. Biochim Biophys Acta. 2006;1765:223–234. 28. Lara PC, Lloret M, Clavo B, et al. Hypoxia downregulates Ku70/80 expression in cervical carcinoma tumors. Radiother Oncol. 2008; 89:222–226.
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DNA-PKcs and Ku70 are Predictive Markers for Poor Prognosis of Patients With Gall Bladder Malignancies Feng Ren, MD, PhD,* Zhu-lin Yang, MD, PhD,w Xingguo Tan, MD,w Dongcai Liu, MD, PhD,* Qiong Zou, MD,z Yuan Yuan, MD,z Jinghe Li, MD,y Lufeng Liang, MD,8 Guixiang Zeng, MD,z and Senlin Chen, MD# Abstract: Gall bladder cancers (GBCs) are highly resistant to radiotherapy and chemotherapy. Unfortunately, the key molecular mechanisms responsible for therapeutic resistance have not been identified. In this study, the expression of DNA-PKcs and Ku70 in 46 squamous cell/adenosquamous carcinomas (SC/ASCs) and 80 adenocarcinomas (ACs) were examined by immunohistochemical analysis. Positive DNA-PKcs and Ku70 expression were significantly associated with less lymph node metastasis, invasion, and low TNM stage of SC/ASCs and ACs. Univariate Kaplan-Meier analysis showed that loss of DNA-PKcs and Ku70 expression significantly correlated with decreased survival in both SC/ASC and AC patients. Multivariate Cox regression analysis showed that loss of DNA-PKcs and Ku70 expression was an independent poor prognostic predictor in both SC/ASC and AC patients. Our study suggested that DNA-PKcs and Ku70 are tumor suppressors, and loss of DNA-PKcs and Ku70 expression is an important biological marker for metastasis, invasion, and prognosis of GBC. Currently, there is no implication of DNA-PKcs and Ku70 expression in chemoresistance or radioresistance in GBC. Key Words: gall bladder cancer, adenocarcinoma, squamous cell carcinoma, adenosquamous carcinoma, DNA-PKcs, Ku70, prognosis, metastasis (Appl Immunohistochem Mol Morphol 2014;22:741–747)
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all bladder cancers (GBCs) are highly aggressive cancers with high mortality rates among gastrointestinal cancers. Over 90% of GBC patients are diagnosed at an advanced clinical stage with severe invasion and metastasis owing to a lack of specific early signs or symptoms of GBC.1 Patients with advanced clinical stage of GBC generally have extremely poor prognosis.2 UnReceived for publication July 8, 2013; accepted September 9, 2013. From the *Department of Surgery; wResearch Laboratory of Hepatobiliary Diseases, Second Xiangya Hospital; zDepartment of Pathology, Third Xiangya Hospital; yDepartment of Pathology, Xiangya Medical School, Central South University; 8Department of Hepatobiliary and Pancreatic Surgery, Hunan Provincial People’s Hospital; #Department of Pathology, Hunan Provincial Tumor Hospital, Changsha; and zDepartment of Pathology, Loudi Central Hospital, Loudi, P.R. China. The authors declare no conflict of interest. Reprints: Zhu-lin Yang, MD, PhD, Research Laboratory of Hepatobiliary Diseases, Second Xiangya Hospital, Central South University, Changsha, Hunan 410011, China (e-mail: yangzhulin8@ sina.com). Copyright r 2014 by Lippincott Williams & Wilkins
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fortunately, palliative chemotherapy and radiation therapy offer little benefit to the survival of GBC patients.3 The most successful clinical trial only extended the survival of biliary tract cancer patients by 3 months using a combination of cisplatin and gemcitabine.4 GBCs have several histologic subtypes. Adenocarcinoma (AC) is a major subtype (90% to 95%),5 whereas squamous cell/adenosquamous carcinoma (SC/ASC) is a rare GBC subtype.2,6 Currently, no study has compared the clinicopathologic and biological differences between SC/ASC and AC subtypes. Importantly, the key molecules responsible for the aggressive nature of GBC have not been identified. DNA double-strand breaks (DSBs) are widely considered to be the most lethal form of DNA damage in mammalian cells. DSBs can be introduced by a variety of exogenous agents including ionizing radiation and radiomimetic drugs (eg, bleomycin and neocarzinostatin).7 Nonhomologous end-joining (NHEJ) is a major repair mechanism for DSBs. The capture of both broken DNA ends together and presenting them together to the DNAprotein complex is an initial and key step of NHEJ.8 The Ku70/80 heterodimer (Ku) is composed of Ku70 and Ku80 subunits. Each subunit contributes to a central DNA-binding core during NHEJ by binding the ends of broken DNA strands and recruiting the catalytic subunit of DNA-protein kinase (DNA-PKcs).9 Inward translocation of Ku allows DNA-PKcs to interact with the terminal ends of the DNA, allowing 2 DNA-PKcs molecules to interact across the DSB.10 It has been demonstrated that silencing Ku or DNAPKcs gene expression can completely shut down NHEJ repair of DSBs.11,12 Therefore, cancer cells with a high expression of DNA repair-associated molecules should be resistant to DSBs. We hypothesize that the high resistance of GBC to radiotherapy and chemotherapy may be associated with high expressions of Ku and DNA-PKcs. In this study, the expression of DNA-PKcs and Ku70 in surgically resected specimens, including AC and SC/ASC, was examined by immunohistochemical analysis. The correlation of DNA-PKcs and Ku70 expression with clinicopathologic characteristics and prognosis of AC and SC/ASC was comparatively evaluated.
MATERIALS AND METHODS Case Selection A total of 46 SC/ASCs from GBC patients that underwent surgical resection or biopsy were collected from January 1995 to December 2009. A total of 1060
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patients with all types of GBCs were diagnosed from January 2001 to December 2009, and tumor tissues of 80 ACs were randomly selected for this study. All diagnoses were based on clinical findings, morphologic criteria, and immunohistochemical stainings. A tumor was diagnosed as ASC when it contains both SCs and AC cells, but the tumor must contain at least 10% AC or SC carcinoma cells. A tumor was diagnosed as SC when most malignant cells are SCs and 3 TNM stage I+II II III Lymph metastasis No Yes Invasion No Yes AC Differentiation Well Moderately Poorly Tumor mass size (cm) r3 >3 TNM stage I+II III IV Lymph metastasis No Yes Invasion No Yes
v2
Ku70 P
Total N
Positive N (%)
46
20 (43.5)
16 24 6
9 (56.3) 10 (41.7) 1 (16.7)
2.852
0.262
20 26
13 (65.0) 7 (26.9)
6.669
0.010
12 20 14
9 (83.3) 9 (45.0) 2 (14.3)
9.724
0.008
17 29
12 (70.6) 8 (27.6)
8.065
0.005
16 30 80
13 (81.3) 7 (23.3) 41 (51.3)
14.243
0.000
27 25 28
18 (66.7) 11 (44.9) 12 (42.9)
3.884
0.143
50 30
29 (58.0) 12 (40.0)
2.432
0.119
21 38 21
16 (76.2) 16 (42.1) 9 (42.9)
7.092
0.029
30 50
20 (66.7) 21 (42.0)
4.566
0.038
31 49
21 (67.7) 20 (40.8)
5.510
0.023
v2
P
9 (56.3) 11 (45.8) 1 (16.7)
2.76
0.250
13 (65.0) 8 (30.8)
5.339
0.021
9.872
0.007
12 (70.6) 9 (31.0)
6.758
0.009
11 (68.8) 10 (33.3) 38 (47.5)
5.275
0.022
2.378
0.305
3.863
0.049
10.392
0.006
19 (63.3) 19 (38.0)
4.825
0.031
21 (67.7) 17 (34.7)
8.316
0.004
Positive N (%) 21 (45.7)
9 (83.3) 10 (50.0) 2 (14.3)
16 (59.3) 11 (44.0) 11 (39.3) 28 (56.0) 10 (33.3) 16 (76.2) 16 (42.1) 6 (28.6)
AC indicates adenocarcinoma; SC/ASC, squamous carcinoma/adenosquamous carcinoma.
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observed in the percentage of cases with positive DNAPKcs and Ku70 expression between SC/ASC and AC patients (data not shown). As shown in Table 1, positive DNA-PKcs expression was observed in 43.5% of SC/ ASC and 51.3% of AC patients, whereas positive Ku70 expression was observed in 45.7% of SC/ASC and 47.5% of AC patients. The percentage of cases with positive DNA-PKcs or Ku70 expression was significantly higher in the tumor tissues from SC/ASC patients with lower TMN stage, small tumor size (< 3 cm), low invasion, and lymph node metastasis compared with the tumor tissues from patients with higher TMN stage, larger tumor size
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(>3 cm), high invasion, and lymph metastasis (P < 0.05, 0.01, or P < 0.001). DNA-PKcs and Ku70 exhibited no significant association with differentiation in SC/ASC. The percentage of cases with positive DNA-PKcs and Ku70 expression in AC tumors was significantly higher in cases with lower TMN stage, no invasion, and lymph node metastasis compared with the cases with higher TMN stage with lymph metastasis and invasion (P < 0.05 or 0.01; Table 1). Ku70, but not DNA-PKcs expression, was observed more in patients with small tumor size. Both DNA-PKcs and Ku70 exhibited no significant association with differentiation of AC (Table 1).
TABLE 2. Relationship Between DNA-PKcs and Ku70 Expression, Clinicopathologic Characteristics, and Average Survival of SC/ASC and AC Patients Clinicopathologic Characteristics SC/ASC Differentiation Well Moderately Poorly Tumor mass size (cm) r3 >3 TNM stage I+II III IV Lymph metastasis No Yes Invasion No Yes DNA-PKcs Negative Positive Ku70 Negative Positive AC Differentiation Well Moderately Poorly Tumor mass size (cm) r3 >3 TNM stage I+II III IV Lymph metastasis No Yes Invasion No Yes DNA-PKcs Negative Positive Ku70 Negative Positive
Samples (n)
Average Survival (mo)
v2
P
16 24 6
13.81 (5-24) 8.92 (4-18) 5.83 (4-9)
19.125
0.000
20 26
14.35 (7-24) 7.04 (4-11)
31.337
0.000
12 20 14
17.00 (9-24) 9.20 (7-15) 5.86 (4-8)
51.139
0.000
17 29
14.24 (4-24) 7.86 (4-15)
16.219
0.000
16 30
15.75 (9-24) 7.27 (4-12)
32.271
0.000
26 20
7.73 (4-24) 13.45 (7-24)
15.243
0.000
25 21
8.48 (4-24) 12.29 (7-24)
5.920
0.015
27 25 28
15.07 (5-24) 10.60 (4-24) 6.68 (3-14)
32.501
0.000
50 30
13.70 (6-24) 5.80 (3-10)
68.283
0.000
21 38 21
18.96 (5-24) 9.29 (6-15) 5.14 (3-7)
105.825
0.000
30 50
16.27 (4-24) 7.42 (3-14)
42.372
0.000
31 49
16.68 (7-24) 6.98 (3-11)
55.535
0.000
39 41
8.59 (3-24) 12.78 (3-24)
8.950
0.003
42 38
8.69 (3-24) 13.00 (3-24)
8.832
0.003
AC indicates adenocarcinoma; SC/ASC, squamous carcinoma/adenosquamous carcinoma.
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Appl Immunohistochem Mol Morphol
Volume 22, Number 10, November/December 2014
The Correlation Between DNA-PKcs and Ku70 Expression With Survival in Patients With SC/ASC and AC
Biomarkers in GBC
Survival information of patients was obtained through letters and phone calls. The follow-up time was 2 years, and patients who survived longer than 2 years were included in the analysis as censored cases. Of the 46 SC/ASCs patients, 33 patients survived 3 cm I+II/III/IV No/yes No/yes /+ /+
0.300 2.733 1.142 1.533 2.944 0.891 0.878
0.366 0.835 0.532 0.623 0.864 0.364 0.350
0.672 10.713 4.608 6.055 11.610 5.992 6.293
0.412 0.001 0.032 0.014 0.001 0.014 0.012
1.350 15.379 3.133 4.632 18.992 0.410 0.416
0.659 2.993 1.104 1.366 3.492 0.201 0.209
2.766 79.012 8.888 15.707 103.280 0.837 0.825
Well/moderately/poorly r3 cm/ >3 cm I+II/III/IV No/yes No/yes /+ /+
1.415 0.906 1.219 0.972 1.523 0.949 1.117
0.547 0.417 0.448 0.457 0.498 0.437 0.446
6.692 4.720 7.404 4.524 9.353 4.716 6.272
0.010 0.030 0.007 0.033 0.002 0.030 0.012
4.116 2.474 3.384 2.643 4.586 0.387 0.327
1.409 1.093 1.406 1.079 1.728 0.164 0.137
12.027 5.603 8.142 6.474 12.171 0.912 0.784
AC indicates adenocarcinoma; CI, confidence interval; RC, regression coefficients; RR, relative risk; SC/ASC, squamous carcinoma/adenosquamous carcinoma.
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Appl Immunohistochem Mol Morphol
Volume 22, Number 10, November/December 2014
breast cancer.22 Consistent with previous findings in various tumors, loss of DNA-PKcs and Ku70 expression was associated with high TMN stage, invasion, and metastasis in SC/ASC and AC tumors. Interestingly, we found that negative DNA-PKcs and Ku70 expression correlated with shorter survival in both SC/ASC and AC patients, suggesting that DNA-PKcs and Ku70 expression are poor prognostic factors in GBC. These observations seem to conflict with the functions of DNA-PK complex in DSBs repair. Besides the crucial role of DNA-PKcs in DNA repair, the activated DNA-PKcs can phosphorylate proteins involved in the control of cell cycle checkpoints and different cell-death pathways.25 For example, DNA-PKcs can phosphorylate tumor suppressor p53, which stabilizes p53 and subsequently causes increased expression of the cell cycle regulator p21 or the proapoptotic protein Bax.26 Besides its vital role in DNA repair, Ku is also involved in numerous other cellular processes, including telomere maintenance, regulation of specific gene transcription, apoptosis, and regulation of cell cycle.27 For example, hypoxia inhibits Ku70/80 expression, leading to an increase in angiogenesis and altered p53 expression.28 Therefore, negative DNA-PKcs and Ku70 expression may implicate an increase in tumor cell survival. In conclusion, DNA-PKcs and Ku70 expression may be a malignant sign of tumor in GBC and is associated with the aggressive nature of GBC, such as invasion, metastasis, and poor prognosis. In contrast, DNA-PKcs and Ku70 expression may not be associated with the resistance of GBC to radiotherapy and chemotherapy. REFERENCES 1. Hawkins WG, DeMatteo RP, Jarnagin WR, et al. Jaundice predicts advanced disease and early mortality in patients with gallbladder cancer. Ann Surg Oncol. 2004;11:310–315. 2. Jayaraman S, Jarnagin WR. Management of gallbladder cancer. Gastroenterol Clin North Am. 2010;39:331–342. 3. de Aretxabala X, Roa I, Burgos L, et al. Gallbladder cancer: an analysis of a series of 139 patients with restricted to the subserosal layer. J Gastrointest Surg. 2006;10:186–192. 4. Valle J, Wasan H, Palmer DH, et al. ABC-02 Trial Investigators. Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer. N Engl J Med. 2010;362:1273–1281. 5. Ootani T, Shirai Y, Tsukada K, et al. Relationship between gallbladder carcinoma and the segmental type of adenomyomatosis of the gallbladder. Cancer. 1992;69:2647–2652. 6. Roa JC, Tapia O, Cakir A, et al. Squamous cell and adenosquamous carcinomas of the gallbladder: clinicopathological analysis of 34 cases identified in 606 carcinomas. Mod Pathol. 2011;24:1069–1078. 7. Mahaney BL, Meek K, Lees-Miller SP. Repair of ionizing radiationinduced DNA double-strand breaks by non-homologous endjoining. Biochem J. 2009;417:639–650. 8. Bouchaert P, Guerif S, Debiais C, et al. DNA-PKcs expression predicts response to radiotherapy in prostate cancer. Int J Radiat Oncol Biol Phys. 2012;84:1179–1185.
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9. Lee HS, Yang HK, Kim WH, et al. Loss of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) expression in gastric cancers. Cancer Res Treat. 2005;37:98–102. 10. Hande MP. DNA repair factors and telomere-chromosome integrity in mammalian cells. Cytogenet Genome Res. 2004;104:116–122. 11. Kon T, Zhang X, Huang Q, et al. Oncolytic virus-mediated tumor radiosensitization in mice through DNA-PKcs-specific shRNA. Transl Cancer Res. 2012;1:4–14. 12. Vandersickel V, Mancini M, Marras E, et al. Lentivirus-mediated RNA interference of Ku70 to enhance radiosensitivity of human mammary epithelial cells. Int J Radiat Biol. 2010;86:114–124. 13. Nishihara K, Nagai E, Izumi Y, et al. Adenosquamous carcinoma of the gallbladder: a clinicopathological, immunohistochemical and flow-cytometric study of twenty cases. Jpn J Cancer Res. 1994; 85:389–399. 14. Kondo M, Dono K, Sakon M, et al. Adenosquamous carcinoma of the gallbladder. Hepatogastroenterology. 2002;49:1230–1234. 15. Oohashi Y, Shirai Y, Wakai T, et al. Adenosquamous carcinoma of the gallbladder warrants resection only if curative resection is feasible. Cancer. 2002;94:3000–3005. 16. Beskow C, Skikuniene J, Holgersson A, et al. Radioresistant cervical cancer shows upregulation of the NHEJ proteins DNA-PKcs, Ku70 and Ku86. Br J Cancer. 2009;101:816–821. 17. Wilson CR, Davidson SE, Margison GP, et al. Expression of Ku70 correlates with survival in carcinoma of the cervix. Br J Cancer. 2000;83:1702–1706. 18. Kienker LJ, Shin EK, Meek K. Both V(D)J recombination and radioresistance require DNA-PK kinase activity, though minimal levels suffice for V(D)J recombination. Nucleic Acids Res. 2000;28:2752–2761. 19. Lee HS, Choe G, Park KU, et al. Altered expression of DNAdependent protein kinase catalytic subunit (DNA-PKcs) during gastric carcinogenesis and its clinical implications on gastric cancer. Int J Oncol. 2007;31:859–866. 20. Rigas B, Borgo S, Elhosseining A, et al. Decreased expression of DNA-dependent protein kinase, a DNA repair protein, during human colon carcinogenesis. Cancer Res. 2001;61:8381–8384. 21. Treilleux I, Chapot B, Goddard S, et al. The molecular causes of low ATM protein expression in breast carcinoma; promoter methylation and levels of the catalytic subunit of DNA-dependent protein kinase. Histopathology. 2007;51:63–69. 22. Someva M, Sakata K, Matsumoto Y, et al. Immunohistochemical analysis of Ku70/86 expression of breast cancer tissues. Oncol Rep. 2007;17:1483–1487. 23. Yang MD, Wang HC, Chang WS, et al. Genetic polymorphisms of DNA double strand break gene Ku70 and gastric cancer in Taiwan. BMC Cancer. 2011;11:174. 24. Persson O, Salford LG, Fransson J, et al. stribution, cellular localization, and therapeutic potential of the tumor-associated antigen Ku70/80 in glioblastoma multiforme. J Neuro Oncol. 2010; 97:207–215. 25. Lees-Miller SP, Sakaguchi K, Ullrich SJ, et al. Human DNAactivated protein kinase phosphorylates serines 15 and 37 in the amino-terminal transactivation domain of human p53. Mol Cell Biol. 1992;12:5041–5049. 26. Bernstein C, Bernstein H, Payne CM, et al. DNA repair/proapoptotic dual-role proteins in five major DNA repair pathways: fail-safe protection against carcinogenesis. Mutat Res. 2002;511:145–178. 27. Gullo C, Au M, Feng G, et al. The biology of Ku and its potential oncogenic role in cancer. Biochim Biophys Acta. 2006;1765:223–234. 28. Lara PC, Lloret M, Clavo B, et al. Hypoxia downregulates Ku70/80 expression in cervical carcinoma tumors. Radiother Oncol. 2008; 89:222–226.
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