Imaging, Diagnosis, Prognosis

Prognostic Significance of Nuclear ING3 Expression in Human Cutaneous Melanoma Ye m in Wa n g , 1Derek L. Dai,1Magdalena Martinka,2 and Gang Li1

Abstract Purpose:The novel tumor-suppressor ING3has been shown to modulate transcription, cell cycle control, and apoptosis. Our previous study showed that ING3promotes UV-induced apoptosis via the Fas/caspase-8^ dependent pathway in melanoma cells.Toinvestigate the putative role of ING3in the development of melanoma, we examined the expression of ING3in melanocytic lesions at different stages and analyzed the correlation between ING3expression and clinico- pathologic variables and patient survival. Experimental Design: Using tissue microarray and immunohistochemistry, we evaluated nuclear and cytoplasmic ING3staining in 58 dysplastic nevi, 114 primary melanomas, and 50 metastatic melanomas. Results: Nuclear ING3expression was remarkably reduced in malignant melanomas compared with dysplastic nevi (P < 0.001), which was significantly correlated with the increased ING3level in cytoplasm (P < 0.05). Furthermore, the reduced nuclear ING3expression was significantly correlated with a poorer disease-specific 5-year survival of patients with primary melanoma, especially for the high-risk melanomas (thickness z2.0 mm) with the survival rate reducing from 93% for patients with strong nuclear ING3 staining in their tumor biopsies to 44% for those with negative-to-moderate nuclear ING3staining ( P = 0.004). Strikingly, our multivariate Cox regres- sion analysis revealed that reduced nuclear ING3expression is an independent prognostic factor to predict patient outcome in primary melanomas (P =0.038). Conclusions: Our data indicate that ING3may be an important marker for human melanoma progression and prognosis as well as a potential therapeutic target.

Human cutaneous malignant melanoma is a type of skin during the tumor initiation and progression will be helpful cancer resulting from uncontrolled proliferation of melanocytes for prevention and treatment of melanoma. in the epidermis. The incidence of melanoma is increasing most The ING3 gene is mapped to 7q31.3, consists of 12 exons, and rapidly in the Caucasian population among all malignancies encodes a 46.8 kDa protein, which shares the conserved except for lung cancer in women (1, 2). Melanoma is a life- carboxyl-terminal plant homeodomain and a nuclear localiza- threatening disease due to its ability to metastasize rapidly tion sequence with other ING family members (8). ING3 is to other organs and high resistance to conventional radiothe- ubiquitously expressed in normal human tissues and can rapy and chemotherapy (3, 4). Patients with metastatic modulate p53-mediated transcription, cell cycle control, and melanoma have a poor prognosis with a median survival of apoptosis in RKO cells (9). As a critical chromatin acetylation only 6 to 10 months and <5% of the patients surviving >5 years regulator, ING3 is predominantly present in the NuA4 histone (5–7). Therefore, understanding of the molecular events acetyltransferase multisubunit complex and required for the histone acetyltransferase activity of Tip60 (10, 11). It is also reported to link the function of p53 to histone acetylation (11). The yeast homologue of ING3, yng2, is also an important component of yeast NuA4 complex and abrogation of yng2 Authors’ Affiliations: Departments of 1Dermatology and Skin Science and function results in a deficient genome-wide nucleosomal histone 2 Pathology, Vancouver Coastal Health Research Institute, University of British H4 acetylation and an increased sensitization to DNA damage in Columbia,Vancouver, British Columbia, Canada Received 2/16/07; revised 5/3/07; accepted 5/10/07. S phase (12). In melanoma cells, we have previously shown that Grant support: Canadian Institutes of Health Research, Canadian Dermatology ING3 promotes UV-induced apoptosis via a Fas/caspase- Foundation, and Canadian Melanoma Foundation; Roman M. Babicki Fellowship 8–dependent pathway, independently of functional p53 (13). from the University of British Columbia (Y.Wang); and Research Scientist Award ING3 is significantly reduced in human head and neck from National Cancer Institute of Canada with funds provided by the Canadian squamous cell carcinomas and higher mortality has been Cancer Society (G. Li). The costs of publication of this article were defrayed in part by the payment of page observed in cases with decreased ING3 expression (14). In charges. This article must therefore be hereby marked advertisement in accordance malignant melanoma, although a frequent copy number gain with 18 U.S.C. Section 1734 solely to indicate this fact. has been reported at the chromosome 7q31-q34 region that Requests for reprints: Gang Li, Jack Bell Research Centre, 2660 Oak Street, contains the ING3 gene with the comparative genomic Vancouver, BC, Canada V6H 3Z6. Phone: 604-875-5826; Fax: 604-875-4497; E-mail: [email protected]. hybridization analysis (15), ING3 expression level and the F 2007 American Association for Cancer Research. cytogenetic characteristics of the ING3 gene in melanoma has doi :10.1158/1078-0432.CCR-07-04 08 never been revealed. To investigate the role of ING3 in the

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was approved by the medical ethical committee of the University of Table 1. Clinicopathologic features of 114 primary British Columbia and was done in accordance with the Declaration melanomas of Helsinki guidelines. The most representative tumor area was carefully selected and marked on the H&E-stained slides. The TMAs were No. patients (%) constructed as previously described with duplicate 0.6-mm-thick tissue Age, y cores taken from each biopsy specimen (16, 17). Three composite high- V56 56 (49) density TMA blocks containing 107, 126, and 111 cases from a total >56 58 (51) of 344 patients were designed. Multiple 4-Am sections were cut with Gender a Leica microtome and transferred to adhesive-coated slides. Male 64 (56) Immunohistochemistry. TMA slides were dewaxed at 55jCfor Female 50 (44) 30 min and washed with xylene. Tissues were rehydrated by a series Tumor thickness, mm of washes in 100%, 95%, and 80% ethanol, and distilled water. Antigen <2.0 76 (67) retrieval was done by heating the samples at 95jC for 30 min in z2.0 38 (33) 10 mmol/L sodium citrate (pH 6.0). Endogenous peroxidase activity Ulceration Absent 98 (86) was blocked with 3% hydrogen peroxide for 20 min. After blocking Present 16 (14) with universal blocking serum for 30 min, slides were incubated with Tumor subtype a polyclonal rabbit anti-ING3 antibody (Protein Tech Group) at 4jC SSM52 (46) overnight. The sections were then incubated with biotin-labeled LMM 17 (15) secondary antibody and streptavidin-peroxidase for 30 min each, Others* 45 (39) ¶ c followed by developing with 3,3 -diaminobenzidine substrate and Site counterstained with hematoxylin. Slides were finally dehydrated Sun exposed 19 (17) and sealed with coverslips. As negative controls, the ING3 antibody Sun protected 95 (83) was omitted during primary antibody incubation. Evaluation of immunostaining. The evaluation of ING3 nuclear and Abbreviations: SSM, superficial spreading melanoma; LMM, len- cytoplasmic expression was made blinded by two independent tigo malignant melanoma. observers (including one dermatopathologist) simultaneously, and a * Includes desmoplastic melanoma, acrolentigous melanoma, and consensus score was reached for each core. The nuclear or cytoplasmic nodular melanoma. ING3 staining was scored into four grades according to the following cSun-protected sites: trunk, arm, leg, and feet. Sun-exposed sites: head and neck. staining intensities: 0, 1+, 2+, and 3+. Percentages of nuclear or cytoplasmic ING3-positive cells were also scored into four categories: 0 (0%), 1 (1-33%), 2 (34-66%), and 3 (67-100%). In the cases with a discrepancy between duplicated cores, the higher score from the development of melanoma, we used tissue microarray (TMA) duplicate tissue cores was taken as the final score. The sum of the technology and immunohistochemistry to evaluate ING3 intensity and percentage scores was used as the final ING3 nuclear or cytoplasmic staining score (17), defined as follows: 0 to 2, negative or expression in different stages of human melanocytic lesions weak; 3 to 4, moderate; and 5 to 6, strong. and analyzed the correlation between ING3 expression and Statistical analysis. Statistical analysis was done with the SPSS 11.5 clinicopathologic variables and patient survival. software. The m2 test was used to compare the quantitative differences of nuclear or cytoplasmic ING3 expression in different stages of melanoma progression. The Spearman test was used to analyze the Materials and Methods correlation between nuclear and cytoplasmic ING3 expression and the correlation between ING3 expression and the clinicopathologic Construction of tissue microarray. Formalin-fixed, paraffin-embed- variables, including age, gender, tumor thickness, ulceration, and ded tissues from 58 dysplastic nevi, 114 primary melanomas, and 50 tumor location. The Kaplan-Meier method and log-rank test were used metastatic melanomas were used for this study. All specimens were to evaluate the correlations between nuclear ING3 staining and patient obtained from the 1990 to 1998 archives of the Department of survival. Cox regression model was used for multivariate analysis. Pathology, Vancouver General Hospital. The use of human skin tissues P < 0.05 is considered significant.

Fig. 1. Representative images of ING3immunohistochemical staining in human melanocytic lesions. A, strong nuclear ING3staining in dysplastic nevi. B, moderate nuclear ING3staining in primary melanoma. C, weak nuclear ING3staining in metastatic melanoma.

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Fig. 2. Reduced nuclear ING3expression correlates with melanoma progression and correlates with the increased cytoplasmic ING3expression. Nuclear ( A) and cytoplasmic (B) ING3expression in dysplastic nevi (DN), primary melanoma (PM), and metastatic melanoma (MM ). Reduced nuclear ING3expression is correlated with increased cytoplasmic ING3 staining in primary (C) and metastatic melanoma (D).

Results metastases, respectively (Fig. 2B). Significant differences for the cytoplasmic ING3 staining (negative-to-moderate versus strong Clinicopathologic features of TMAs. Due to loss of biopsy staining) were observed between dysplastic nevi and primary cores or insufficient tumor cells present in the cores, 58 cases of melanomas (P < 0.05, m2 test), between primary melanomas dysplastic nevi, 114 cases of primary melanomas, and 50 cases and melanoma metastases (P < 0.05, m2 test) and between of melanoma metastases could be evaluated for ING3 staining. dysplastic nevi and melanoma metastases (P < 0.001, m2 test). As shown in Table 1, for the 114 primary melanoma cases, In addition, the reduced nuclear ING3 expression was there were 64 men and 50 women, with age ranging from 21 to positively associated with the increased cytoplasmic ING3 93 years (median, 56 years). For melanoma staging, we used expression in either primary melanomas or metastatic melano- Breslow thickness as our criteria for evaluating ING3 expres- mas (Fig. 2C and D; P = 0.001 and 0.042, respectively, sion: 76 tumors were <2.0 mm thick (low-risk melanoma), and Spearman test). 38 were thicker than 2.0 mm (high-risk melanoma). Nineteen Reduced nuclear ING3 staining correlates with tumor melanomas were located in sun-exposed sites (head and neck), location. To assess whether reduced ING3 staining correlates and 95 were located in sun-protected sites (other locations). with clinicopathologic variables of the patients, we examined Ulceration was observed in 16 cases. the ING3 nuclear staining in 114 primary melanomas. Because Reduced ING3 nuclear staining correlates with melanoma UV radiation is the main environmental factor for melanoma progression. Various levels of ING3 staining were observed in formation, we first analyzed the ING3 staining in sun-exposed dysplastic nevi and melanoma biopsies (Fig. 1). Strong nuclear or sun-protected sites. Reduced nuclear ING3 staining (nega- ING3 staining was recorded in 60%, 33%, and 10% of the tive-to-moderate versus strong staining) significantly correlated biopsies in dysplastic nevi, primary melanomas, and melanoma with the location of primary melanomas (Fig. 3A; P = 0.021, metastases, respectively (Fig. 2A). Significant differences for Spearman test). Thirty-eight percent (36 of 95) of tumors had nuclear ING3 staining (negative-to-moderate versus strong strong ING3 nuclear staining in sun-protected sites (trunk, arm, staining) were observed between dysplastic nevi and primary leg, feet, etc.), whereas strong ING3 nuclear expression was melanomas (P < 0.001, m2 test), between dysplastic nevi and observed in only 10% (2 of 19) of tumors at sun-exposed sites melanoma metastases (P < 0.001, m2 test), and between (head and neck). However, the increased cytoplasmic ING3 primary melanomas and melanoma metastases (P < 0.001, staining did not significantly correlate with the location of m2 test). To investigate if the reduced nuclear ING3 staining is primary melanomas (Fig. 3B; P > 0.05, Spearman test), due to a subcellular compartment shift to the cytoplasm, we although the strong ING3 cytoplasmic staining increased from evaluated ING3 staining in cytoplasm. Strong cytoplasmic 38% in tumors at sun-protected sites to 55% in tumors at ING3 staining was recorded in 24%, 39%, and 62% of the sun-exposed sites. On the other hand, neither nuclear nor biopsies in dysplastic nevi, primary melanoma, and melanoma cytoplasmic ING3 staining significantly correlated with other

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We then examined whether the nuclear ING3 expression is an independent prognostic marker for melanoma. We did a multivariate Cox regression analysis, including ING3 nuclear staining, age, gender, tumor thickness, ulceration, and loca- tion of the tumors, for 114 primary melanomas. Besides the tumor thickness to be an independent prognostic marker for disease-specific survival, the nuclear ING3 level showed significance in predicting the patient outcome independently of other clinicopathologic variables for disease-specific sur- vival (relative risk, 9.31; 95% confidence interval, 1.13-76.5; P = 0.038; Table 2). In high-risk primary melanoma, the nuclear ING3 expression showed a borderline significance in predicting the patient outcome as an independent factor (relative risk, 8.34; 95% confidence interval, 0.959-72.6; P = 0.055; Table 2), whereas tumor thickness did not show significance in predicting patient outcome independently of other clinicopathologic variables for disease-specific survival (P =0.151;Table2).

Discussion

Acquired resistance to apoptosis is a major hallmark of cancers (18), which allows cancer cells to escape the apop- totic death induced by anticancer agents and enables the establishment of metastasis. The human cutaneous malignant

Fig. 3. Correlation between the nuclear or cytoplasmic ING3expression and the location of primary melanoma. A, reduced nuclear ING3expression is correlated with sun-exposed tumor sites in primary melanoma. B, increased cytoplasmic ING3 expression did not significantly correlate with tumor location in primary melanoma. clinicopathologic variables, including tumor thickness, sub- type, ulceration, or patient’s age or gender (data not shown). Reduced nuclear ING3 staining correlates with disease-specific 5-year patient survival. To evaluate whether the reduced nuclear ING3 staining in human primary melanomas correlates with a worse prognosis, Kaplan-Meier survival curves were constructed using overall or disease-specific 5-year survival to evaluate the biopsies with strong nuclear ING3 staining to those with negative-to-moderate nuclear ING3 staining. The overall 5-year survival rate dropped from 87% in patients with the strong nuclear ING3 staining to 74% in patients showing negative-to-moderate nuclear ING3 staining, but they were not significantly correlated (P = 0.119, log-rank test; data not shown). On the other hand, the disease-specific 5-year survival rate dropped from 97% in patients showing strong nuclear ING3 staining to 82% in those with negative-to-moderate nuclear ING3 staining, and this correlation between the reduced ING3 nuclear staining and a poorer 5-y disease-specific survival is statistically significant (P = 0.0226, log-rank test; Fig. 4A). We noticed that the majority of the death cases occurred in the high-risk group (tumor thickness z2.0 mm), whereas only 1 of 76 patients in the low-risk group (tumor thickness <2.0 mm) died. Therefore, we analyzed the cor- relation between the reduced nuclear ING3 staining with the disease-specific survival in high-risk primary melanomas. We found that the survival rate was 93% in patients with strong nuclear ING3 staining compared with 44% in patients Fig. 4. Correlation between the reduced nuclear ING3expression and 5-y with negative-to-moderate nuclear ING3 staining, and this disease-specific survival in primary melanoma patients. Patients with strong nuclear P ING3staining have a significantly worse 5-y disease-specific survival than those correlation is statistically significant ( = 0.004, log-rank test; with negative-to-moderate ING3staining in 114 primary melanomas ( A)and38 Fig. 4B). high-risk primary melanomas (B).

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was positively correlated with the tumor location at sun- Table 2. Multivariate Cox regression analysis of exposed sites (Fig. 4A), indicating a crucial role of UV radiation ING3 subcellular distribution in 114 cases of in regulating nuclear ING3 level. However, although increased primary melanoma strong cytoplasmic ING3 staining was observed in tumors at sun-exposed sites, the correlation between cytoplasmic ING3 Variable All primary High-risk melanomas melanomas expression and sun exposure was not significant (Fig. 3B). One possible explanation for this discrepancy is that the nuclear-to- Relative risk P Relative risk P (95% CI) (95% CI) cytoplasm translocation of ING3 may be a partial reason for reduced nuclear ING3 expression after UV irradiation because Gender 0.471 (0.151-1.47) 0.194 0.477 (0.145-1.56) 0.222 32% (6 of 19) of tumors at sun-exposed sites with reduced Age 0.648 (0.159-2.64) 0.545 0.430 (0.097-1.92) 0.268 Thickness 54.8 (6.43-470) 0.000 0.391 (0.108-1.41) 0.151 nuclear expression did not show strong cytoplasmic ING3 Site 0.910 (0.304-2.72) 0.865 1.30 (0.397-4.28) 0.662 staining. In addition, we notice that 13% (15 of 114) of Ulceration 0.708 (0.218-2.30) 0.566 0.769 (0.216-2.74) 0.685 primary melanomas and 16% (8 of 50) of metastatic ING3 9.31 (1.13-76.5) 0.038 8.34 (0.959-72.6) 0.055 melanomas with the reduced nuclear ING3 expression did not show a significant increase of the cytoplasmic ING3 level. NOTE: Coding of variables: ING3 nuclear staining was coded Therefore, mechanisms other than the nuclear-to-cytoplasm as 0 (negative to moderate staining) and 1 (strong staining); shift could be involved in the loss of nuclear ING3 expression gender was coded as 0 (male) and 1 (female); age was coded as 0 (56 y) and 1 (<56 y); thickness was coded as 0 (z2.0 mm) and in these tumors. 1 (<2.0 mm) in all primary melanomas or 0 (z4.0 mm) and 1 The defects in the apoptosis pathway, including Apaf-1, (<4.0 mm) in high-risk melanomas (thickness z2.0 mm); PUMA, and XAF1, mostly attribute to the initiation step in ulceration was coded as 0 (absent) and 1 (present); site was melanoma pathogenesis (29), whereas activated survival coded as 0 (head and neck) and 1 (other sites). pathway by increased integrin-linked kinase and phospho-Akt levels or reduced PTEN expression often correlates with tumor progression (16, 17). In this study, the reduced nuclear ING3 melanoma is a particularly aggressive type of cancer in this expression is correlated with the progression from primary regard. Its metastatic form is very resistant to conventional melanoma to metastatic melanoma, indicating that ING3 plays radiotherapy and chemotherapy (19–21). The novel tumor- a role not only in the initiation of melanoma, but also in suppressor ING3 is inducible by UV irradiation or treatment melanoma progression. In agreement with the correlation with genotoxic drugs, and overexpression of ING3 dramatically between reduced ING3 expression and a poorer patient survival enhances UV-induced apoptosis (13). In this study, using the in head and neck squamous cell carcinoma (14), the reduced tissue microarray and immunohistochemistry technology, we nuclear ING3 expression is significantly correlated with a for the first time showed that ING3 nuclear expression is poorer 5-year disease-specific survival of patients with primary remarkably reduced in human melanomas compared with melanoma (Fig. 4A) and is likely an independent factor dysplastic nevi (Fig. 2A). This finding is consistent with the predicting patient outcome (Table 2). report by Gunduz et al. (14) showing that ING3 is reduced in We have recently found that nuclear ING2 expression was head and neck squamous cell carcinoma. However, different significantly reduced in human melanomas (30). In an earlier mechanisms may be involved for the reduction of ING3 report, Nouman et al. (27) also showed that nuclear ING1b expression in head and neck squamous cell carcinoma and expression was significantly decreased and shifted to cytoplasm melanoma. Fifty percent of the head and neck squamous cell in melanocytic lesions. These results together with our current carcinoma cases showed reduced or absent ING3 mRNA and data suggest that the ING family proteins play a crucial role 48% of cases had allelic deletion in 7q31 region (14), whereas in the development of melanoma. This is also supported by in melanoma the reduced nuclear ING3 expression is positively our in vitro studies showing that both ING1b and ING2 signi- correlated with the increased cytoplasmic ING3 level, indicat- ficantly enhance the repair of UV-damaged DNA and promote ing an obvious translocation of ING3 from nucleus to apoptosis in melanoma cells in a p53-dependent pathway cytoplasm compartment (Fig. 2C). Similarly, the mRNA level (31–34), whereas ING3 promote UV-induced apoptosis by of ING1b, an ING3 homologue, is decreased in 61% of human activating the p53-independent Fas/caspase-8 pathway (13). exocrine pancreatic carcinoma (22), 42% of non–small cell Because the p53 gene is rarely mutated in melanoma (35–37), lung cancer (23), and 44% of primary breast cancers (24), the aberrant expression of ING family tumor suppressors whereas the translocation of ING1b from nucleus to cytoplasm may be an important step during melanoma development. was also observed in the invasive breast cancer (25), childhood Further analysis on the correlation between ING protein acute lymphoblastic leukemia (26), and melanoma (27), which expression and their mutational status will provide new insight may be caused by the sequestration of ING1b by 14-3-3 on the mechanisms of aberrant expression of ING proteins in proteins in cytoplasm (28). The reason for ING3 nuclear-to- melanoma. cytoplasm translocation remains to be determined. In conclusion, the data presented in this report showed that Although the nuclear ING3 expression was significantly nuclear ING3 is significantly reduced with human melanoma reduced in melanomas compared with dysplastic nevi, only progression. The reduced nuclear ING3 level was correlated 60% (35 of 58) of the dysplastic nevi showed strong nuclear with the increased cytoplasmic ING3 level. Strikingly, the ING3 expression and 24% (14 of 58) of them had strong ING3 reduced nuclear ING3 expression correlates with a worse 5-year staining in cytoplasm. This suggests that the ING3 nuclear-to- disease-specific survival of primary melanoma patients and is cytoplasm translocation may be an early event in melanocytic an independent prognostic factor for primary melanomas. neoplasia. Supportively, the reduced nuclear ING3 expression These data, together with the regulation of ING3 in cell growth

www.aacrjournals.org 4115 Clin Cancer Res 2007;13(14) July 15, 2007 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2007 American Association for Cancer Research. Imaging, Diagnosis, Prognosis and apoptosis, indicate that ING3 may play an important role Acknowledgments in both melanoma initiation and progression and serve as a promising prognostic marker and therapeutic target for We thank Drs. D. Huntsman, N. Makretsov, and H. Masoudi for the technical malignant melanoma. assistance inTMA construction.

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