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Overexpression of AIB1 negatively affects survival of surgically resected non-small-cell lung cancer patients
original article
Annals of Oncology 21: 1675–1681, 2010 doi:10.1093/annonc/mdp592 Published online 11 January 2010
Overexpression of AIB1 negatively affects survival of surgically resected non-small-cell lung cancer patients L.-R. He1,2 , H.-Y. Zhao3 , B.-K. Li4 , L.-J. Zhang4, M.-Z. Liu2, H.-F. Kung1, X.-Y. Guan1, X.-W. Bian5, Y.-X. Zeng1 & D. Xie1* 1 State Key Laboratory of Oncology in South China; Departments of 2Radiotherapy; 3Medical Oncology; 4Surgical Oncology, Cancer Center, Sun Yat-Sen University, Guangzhou and 5Department of Pathology, Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing, China
introduction Lung cancer is responsible for the highest cancer-related morbidity and mortality worldwide [1]. Non-small-cell lung cancer (NSCLC), which accounts for 85%–87% of all lung cancers, is a very heterogenous group of malignancies as far as the pathomorphology, local invasion and metastatic potential are concerned [2]. Clinically, the international lung cancer staging system is still not reliable enough to predict patient prognosis. The most possibly underlying reason might be the heterogeneous biological properties of NSCLC. Therefore, the search for specific genes alterations, which determine biological nature and behavior of NSCLC would be of utmost importance to optimize individual therapy [3]. It is conceivable that amplified DNA may be a component of one or more critical human oncogenes and thus may provide a selective advantage for tumor development. One of the commonly overrepresented chromosomal regions in NSCLC is 20q [4]. Several candidate oncogenes have been isolated from *Correspondence to: Dr D. Xie, State key Laboratory of Oncology in South China, Cancer Center, Sun Yat-Sen University, No 651, Dongfeng Road East, 510060 Guangzhou, China. Tel: +86-20-87343193; Fax: +86-20-87343170; E-mail: [email protected]
These authors contributed equally to this work.
20q, including amplified in breast cancer 1 (AIB1) at 20q12. The AIB1 gene, also known as SRC-3, p/CIP, RAC3, ACTR and TRAM-1, has been found to be involved in a number of biological processes, such as cell proliferation, cell apoptosis, cell migration and others [5]. The oncogenic role of AIB1 was first reported in hormone-sensitive cancers [6], and then, we contiguously reported that AIB1 was also overexpressed and correlated closely with an ascending clinical stage and/or poor patient prognosis in a series of hormone-insensitive malignancies [7–11]. Recently, AIB1 was found to express variably among different types of human NSCLC cells [12]. However, the abnormalities of AIB1 in NSCLC and its clinical/ prognostic significance have not been determined. In this study, immunohistochemistry (IHC) and FISH were used to examine protein expression and amplification of AIB1 in a large cohort of surgically resected NSCLC, and the clinicopathologic/ prognostic significance of AIB1 expression in NSCLC was also assessed.
patients and methods patients In this study, 248 NSCLC patients who received complete resection in Thoracic Department of Cancer Center, Sun Yat-sen University,
ª The Author 2010. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected]
original article
Background: The amplified in breast cancer 1 (AIB1) gene has been considered to play an oncogenic role in human cancers, but its clinical/prognostic significance in non-small-cell lung cancer (NSCLC) is still unclear. Patients and methods: The methods of immunohistochemistry and FISH were utilized to examine protein expression and amplification of AIB1 in 230 informative surgically resected NSCLCs and in 30 samples of normal lung tissues. Results: Overexpression and amplification of AIB1 were found in 48.3% and 8.2% of NSCLCs, respectively. AIB1 overexpression was associated with AIB1 gene amplification and cell proliferation but not related to estrogen receptor (ER)-a, ER-b, progesterone receptor or androgen receptor status. A positive correlation between AIB1 overexpression and an ascending pathologic node stage in lung adenocarcinoma (ADC) was observed (P = 0.043). Univariate survival analysis demonstrated a significant association of AIB1 overexpression with shortened patient survival, especially for those with stage III disease (P < 0.001). Importantly, AIB1 expression was evaluated as the most significant predictor for survival in multivariate analysis (hazards ratio = 2.069, P < 0.001). Conclusion: Overexpression of AIB1 might provide a selective advantage for lymph node metastasis of lung ADC and serve as a useful biomarker for poor prognosis for NSCLC patients. Key words: AIB1, amplification, immunohistochemistry, non-small-cell lung carcinoma, prognosis
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Received 16 September 2009; revised 24 November 2009; accepted 25 November 2009
original article
construction of tissue microarrays The tissue microarray (TMA) was constructed according to a method described previously [13]. Briefly, the individual donor tissue block and the corresponding histological hematoxylin–eosin stained slides were overlaid for tissue TMA sampling. The tissues (248 NSCLC and 30 normal lung tissues taken from regions that were not affected of the same patients) were sampled using a tissue arraying instrument (Beecher Instruments, Silver Spring, MD); a 0.6-mm-diameter cylinder of tissue was removed. Subsequently, the tissue cylinder was re-embedded into a predetermined position in a recipient paraffin block. In our constructed lung tissue TMA, three cores of sample were selected from each primary NSCLC and normal lung tissue. Multiple sections (5 lm thick) were cut from the TMA block and mounted on microscope slides.
immunohistochemistry IHC staining was carried out on 5-lm TMA sections rehydrated through graded alcohols. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 15 min. For antigen retrieval, tissue slides were boiled in 10 mM citrate buffer (pH 6.0) in a pressure cooker for 10 min [AIB1, estrogen receptor (ER) and progesterone receptor (PgR)] or microwave treated for 10 min (androgen receptor (AR) and Ki-67). The slides were incubated with anti-AIB1 [an mAb directed at amino acids 376– 389 of AIB1, Clone 34 (BD Transduction Laboratories, San Jose, CA), diluted 1 : 50 in phosphate-buffered saline (PBS), overnight at 4C], antiER-a [Clone 1D5 (Dako, Glostrup, Denmark), diluted 1 : 100 in PBS, 60 min at room temperature (RT)], anti-ER-b [Clone H-150 (Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1 : 50 in PBS, 80 min at RT], antiPgR [Clone PgR 636 (Dako), diluted 1 : 500 in PBS, 60 min at RT], anti-AR [Clone AR441 (Dako), diluted 1 : 50 in PBS, 60 min at RT] and anti-Ki-67 [Clone MIB-1 (Dako), diluted 1 : 100 in PBS, overnight at 4C]. All incubations were carried out in a moist chamber. Subsequently, the slides were sequentially incubated with biotinylated rabbit anti-mouse immunoglobulin (Ig) at a concentration of 1 : 100 for 30 min at 37C and then reacted with a streptavidin–peroxidase conjugate for 30 min at 37C and 3#,3#-Diaminobenzidine as a chromogen substrate. The nucleus was counterstained using Meyer’s hematoxylin. Known immunostaining positive slides of breast cancer (AIB1, ER-a, ER-b and PgR) and prostate cancer (AR) were used as positive controls. A negative control was obtained by replacing the primary antibody with a normal murine IgG.
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Table 1. AIB1 expression and clinicopathologic variables Characteristics
Age (years) £59b >59 Gender Male Female Tumor grade G1 G2 G3 pT status pT1 pT2 pT3 pT4 pN status pN0 pN1 pN2 Stage I II III Histology SCC ADC ADSCC Othersc SCC subgroup pN0 pN1 pN2 ADC subgroup pN0 pN1 pN2
Total cases
Informative
AIB1 protein Overexpression, n (%)
130 118
121 109
57 (47.1) 54 (49.5)
181 67
168 62
87 (51.8) 24 (38.7)
24 173 51
22 163 45
9 (40.9) 79 (48.5) 23 (51.1)
15 132 81 20
13 128 71 18
5 59 35 12
120 49 79
113 44 73
47 (41.6) 25 (56.8) 39 (53.4)
82 59 107
80 56 94
32 (40.0) 27 (48.2) 52 (55.3)
94 113 32 9
89 105 29 7
49 18 27
46 17 26
20 (43.5) 9 (52.9) 13 (50.0)
53 22 38
50 20 35
16 (32.0) 11 (55.0) 20 (57.1)
Pa 0.712
0.078
0.731
0.360 (38.5) (46.1) (49.3) (66.7) 0.130
0.131
0.169 42 47 16 6
(47.2) (44.8) (55.2) (85.7) 0.755
0.043
a
Chi-square test. Mean age. c Others include anaplastic large-cell carcinoma, sarcoma, adenoid cystic carcinoma, mucoepidermoid carcinoma and carcinoid tumor. AIB1, amplified in breast cancer 1; pT, pathologic tumor; pN, pathologic node; SCC, squamous cell carcinoma; ADC, adenocarcinoma; ADSCC, adenosquamous carcinoma. b
Positive expression of AIB1 in normal and malignant lung tissues was primarily a nuclear pattern (Figure 1). For AIB1, the staining of the nuclei of the normal lung tissues ranged from 0% to 10% (median 2.3%) of epithelium with positive staining (Figure 1A and B); thus, overexpression of AIB1 was scored when >10% of tumor cells were positively stained in the nuclei [8–11]. For evaluation of the AIB1, ER, PgR, AR and Ki-67 IHC staining in the different tissue sections, the cancerous and noncancerous tissues were scored for these proteins by assessing the site of positive staining in the nucleus. The status of nuclear expression of AIB1, ER, PgR,
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Guangzhou, China, from February 1994 to January 1998 were enrolled. The cases were selected consecutively on the basis of availability of resection tissue and follow-up data. Patients who have previous malignant disease, a second primary tumor or those who received preoperative radiotherapy and/or chemotherapy were excluded. In the majority of cases, lobectomy was carried out (223 of 248 patients) and 25 patients underwent pneumonectomy. Because adjuvant chemotherapy become a standard approach only after 2004, systemic chemotherapy using cisplatin-based combinations was administered to patients with stages III NSCLCs, no postoperative therapy was delivered to patients in stage I–II. Patients with stage III disease and pathologic evidence of N2 disease received postoperative mediastinal radiotherapy. The study was approved by the medical ethics committee of Cancer Center of Sun Yat-Sen University. Tumor differentiation grades were defined according to the criteria of the World Health Organization. The pathologic tumor–node–metastasis (pTNM) status of all NSCLCs was assessed according to the criteria of the sixth edition of the TNM classification of the International Union Against Cancer (2002). The clinicopathologic features of the NSCLC cohort are outlined in Table 1. All the patients were followed up regularly and the last follow-up was carried out in December 2008, with the mean observation period of 65.3 months (3.2–172.5 months) and 167 cancer-related deaths.
Annals of Oncology
original article
Annals of Oncology
statistical analysis
Figure 1. Immunohistochemical stainings of the amplified in breast cancer 1 (AIB1) and FISH assay of AIB1 gene in lung tissues. (A) A normal lung tissue (case 7) showed a negative staining of AIB1 in all the epithelium cells (·200). (B) Positive staining of AIB1 was observed in 2% of the epithelium cells of a normal lung tissue (case 28) (·200). (C) A squamous carcinoma of lung (case 215) showed normal expression of AIB1 with a negative staining of AIB1 in all tumor cells (·200). (D) Overexpression of AIB1 was detected in a squamous carcinoma of lung (case 23), in which 90% of tumor cells were observed strong positive staining of AIB1 (·200). (E) Overexpression of AIB1 was examined in an adenocarcinoma (ADC) of lung (case 116), in which 70% of tumor cells showed strong positive staining of AIB1 (·200). (F) Amplification of AIB1 gene was examined by FISH in the same case of ADC of lung (case 116), in which AIB1 gene signals (red) were detected at least three times more than signals on chromosome 20p12 (green) (·1000). AR and Ki-67 was assessed by determining the percentage of positive cells stained in each tissue section [11, 14]. A minimum of 300 epithelial cells was counted for each case.
FISH Two-color FISH was applied to the sections of TMA using spectrum orange-labeled bacterial artificial chromosome (BAC) clone (RP11447G11) containing the AIB1 gene and a spectrum green-labeled BAC clone on 20p12 (reference probe). The FISH reaction was carried out as described previously with slight modification [8]. Briefly, the deparaffinized TMA section was treated with proteinase K (400 lg/ml) at 37C for 45 min, followed by denaturing in 70% formamide, 2· standard saline citrate (SSC) at 75C for 8 min. Fifty nanograms of each probe were mixed in a 20 ll hybridization mixture (containing 55% formamide, 2· SSC, and 2 lg
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Statistical analysis was carried out with the SPSS software (SPSS standard version 13.0; SPSS Inc., Chicago, IL). The association of AIB1 protein expression with NSCLC patient’s clinicopathologic features and the correlations between molecular features detected with each other were assessed by the chi-square test. Disease-specific survival (DSS) was calculated from the date of diagnosis to the date of cancer-related death or last follow-up. Relative risks of cancer-related death associated with AIB1 expression status and other predictor variables were estimated from univariate Cox proportional hazards model. Multivariate survival analysis was carried out on all parameters that were found to be significant on univariate analysis using the Cox regression model. Survival curves were assessed by the Kaplan–Meier method and compared by the log-rank test. Two-sided P values of <0.05 were considered to indicate statistical significance.
results AIB1 expression in NSCLC and normal lung tissues AIB1 expression could be evaluated informatively in 230 of 248 (92.7%) of the NSCLCs and 26 of 30 (86.7%) of normal lung tissues. The noninformative samples included unrepresentative samples, samples with too few tumor cells (<300 cells per case) and lost samples; such were not used in data compilation. Using this criteria described above, overexpression of AIB1 was observed in 111 of 230 (48.3%) of the NSCLCs and correlated with an ascending pathologic node (pN) stage for lung adenocarcinoma (ADC) but not for squamous cell carcinoma (SCC) (Table 1). relationship between clinicopathologic variables, AIB1 expression and NSCLC patient survival In univariate analysis, AIB1 overexpression was evaluated to correlate closely with poor DSS for the whole cohort and could further stratify patient survival in SCC, ADC, pT3/4, pN1/2 and III stages (Figure 2; Table 2). Kaplan–Meier analysis also demonstrated a significant impact of certain clinicopathologic prognostic parameters such as tumor pT status (P = 0.013), pN status (P < 0.001) and clinical stage (P < 0.001) on patient survival. Multivariate analysis showed that AIB1 expression and pN status were independent predictors of DSS for the whole cohort (Table 3).
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human Cot1 DNA), denatured at 75C for 5 min and then hybridized to the denatured TMA section at 37C for 24 h. After washing, the TMA section was counterstained with 1 lg/ml diamidino-phenyl-indole in an antifade solution and examined with a Zeiss Axiophot microscope equipped with a triple-band-pass filter. FISH signals from 300 cells in each sample were counted. The criteria for AIB1 gene amplification were defined as the presence (in ‡20% of tumor cells) of either six (or more) gene signals or >2.5 times as many gene signals than reference 20p11 signals (Figure 1F) [11]. All samples not meeting the criteria for gain or amplification were considered normal. Control hybridizations to normal fibroblasts and to normal epithelial cells were carried out to confirm that the hybridization efficiency of the test and reference probes was similar. Two independent pathologists (XD and Rao HL) blinded to the clinicopathologic information carried out the scorings. The interobserver disagreements (8% and 3% of the total informative cases for IHC and FISH, respectively) were reviewed a second time, followed by a conclusive judgment by both pathologists.
original article
Annals of Oncology
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Figure 2. Survival analysis of the amplified in breast cancer 1 (AIB1) expression in total cohort and different subsets of non-small-cell lung cancer patients. (A) Total cohort, (B) pT1/2, (C) pT3/4, (D) pN0, (E) pN1/2, (F) stage I, (G) stage II and (H) stage III. pT, pathologic tumor, pN, pathologic node.
expression of ER, PgR and AR in NSCLCs and their correlation with AIB1 expression ER-a, ER-b, PgR and AR expression could be evaluated informatively in 232 of 248 (93.5%), 227 of 248 (91.5%), 229 of 248 (92.3%) and 225 of 248 (90.7%) of the NSCLCs, respectively. The percentage of positive cells with nuclear staining of these hormone receptors was recorded on a semiquantitative scale as previously described [14, 16]; results
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were categorized as either negative (<10% cell labeling) or positive (‡10% cell labeling). Utilizing these criteria, a positive expression of ER-a, ER-b and PgR was shown in 76 of 232 (32.8%), 86 of 227 (37.9%) and 109 of 229 (47.6%) of our NSCLC cohorts, respectively. None of the NSCLC cases showed a positive expression of AR. Statistical analysis showed that there was no significant association between the expression of AIB1 and ER-a, ER-b or PgR (P > 0.05, data not shown).
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original article
Annals of Oncology
Table 2. Prognostic value of AIB1 expression in NSCLC AIB1 expression
Disease-specific survival Relative 95% risk Confidence interval
Pa,b
119 111
1 1.963
1.414–2.723
<0.001
47 42
1 1.904
1.121–3.232
0.015
58 47
1 2.270
1.398–3.685
0.001
77 64
1 1.484
0.970–2.273
0.087
42 47
1 3.169
1.861–5.397
<0.001
66 47
1 1.262
0.761–2.093
0.367
53 64
1 2.677
1.707–4.198
<0.001
48 32
1 1.055
0.575–1.935
0.863
29 27
1 1.565
0.770–3.184
0.216
42 52
1 3.711
2.225–6.190
<0.001
discussion
a
Belongs to overexpression of AIB1. Univariate Cox proportional hazards analysis. AIB1, amplified in breast cancer 1; NSCLC, non-small-cell lung carcinoma; SCC, squamous cell carcinoma; ADC, adenocarcinoma; pT, pathologic tumor; pN, pathologic node.
b
Table 3. Multivariate Cox regression analysis for disease-specific survival Factors
Hazards ratio
95% Confidence interval
P
AIB1 expressiona pT statusb pN statusc Staged
2.069 1.143 1.684 1.300
1.487–2.879 0.839–1.557 1.126–2.519 0.615–2.748
<0.001 0.379 0.011 0.491
a
Normal expression versus overexpression. pT1 versus pT2 versus pT3 versus pT4. c pN0 versus pN1 versus pN2. d Stage I versus stage II versus stage III. AIB1, amplified in breast cancer 1; pT, pathologic tumor; pN, pathologic node. b
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amplification of AIB1 in NSCLC and normal lung tissues The FISH analysis was informative in 53.3% (16 of 30) of the normal lung tissues and 54.0% (134 of 248) of the NCSLCs. Samples without FISH signal and samples with weak target signals or those with a strong signal background were the main reasons for most of the noninformative cases. The amplification of AIB1 was not detected in any of the normal lung tissues but was observed in 8.2% (11 of 134) of the informative NCSLCs. In our study, 121 informative cases were shown to be positive by both IHC and FISH simultaneously, and a significant correlation between AIB1 overexpression and amplification of AIB1 was found (Table 4).
In the present study, we found that the expression of AIB1 by IHC in all the normal lung tissue specimens was absent or at low levels. In contrast, in many of our NSCLC specimens, an overexpression of AIB1 was frequently detected, which indicated a potential important role of AIB1 in carcinogenesis of lung. Similar results were also observed in other human cancers, such as esophageal, gastric, colorectal and nasopharyngeal carcinomas, in which up-regulating of AIB1 was observed and correlated with a late clinical stage and/or poor prognosis [9, 10, 15]. It is well known that the pathogenesis and biologic behaviors of different subtypes of NSCLC are quite distinct. The underline mechanism might contribute, at least partly to the molecular diversity. Although no significant association was found between AIB1 expression and clinicopathologic features for the whole cohort, interestingly, a steady increase of AIB1 expression was observed in ADCs of lung from no lymph node metastasis to peribronchial lymph node metastasis and to mediastinal lymph node metastasis. These findings indicated that up-regulated expression of AIB1 may provide a selective advantage in lymph Table 4. Association of AIB1 expression and amplification in NSCLC AIB1 gene
Informative cases
AIB1 expression Normal Overexpression
Pa
No amplification Amplification
110 11
62 (56.4) 1 (9.1)
0.003
48 (43.6) 10 (90.9)
a
Chi-square test. AIB1, amplified in breast cancer 1; NSCLC, non-small-cell lung carcinoma.
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Total Normal expression Overexpression Histology SCC Normal expression Overexpression ADC Normal expression Overexpression pT status pT1/2 Normal expression Overexpression pT3/4 Normal expression Overexpression pN status pN0 Normal expression Overexpression pN1/2 Normal expression Overexpression Stage Stage I Normal expression Overexpression Stage II Normal expression Overexpression Stage III Normal expression Overexpression
Cases
correlation of AIB1 expression and cell proliferation in NSCLCs In our NSCLC cohorts, the median value of 25.6% was used as a cut-off value to define low Ki-67 labeling index (LI) (LI <25.6%) and high Ki-67 LI (LI ‡25.6%) [9, 10]. In 212 of the 248 samples, AIB1 and Ki-67 IHC were detected successfully and simultaneously. Correlation analysis demonstrates that the frequency of cases with a high LI of Ki-67 was significantly greater in tumors with an overexpression of AIB1 (54 of 77, 70.1%) than that in cases with normal expression of AIB1 (23 of 77, 29.9%; P < 0.001).
original article
1680 | He et al.
[26, 27], which indicates that AIB1 may play a role in tumorigenesis of specific subsets of hormone-sensitive cancers, perhaps by facilitating hormone-dependent signal transduction pathways. Interestingly, overexpression of AIB1 was detected in breast tumors positive and negative for ER and PgR [28], while AIB1 knockdown impaired the proliferation of both ARpositive and AR-negative prostate cancer cells [27]. In human lung cancer, it has been reported that estrogen-signaling pathway (including AIB1) plays an important role in NSCLC carcinogenesis [12]. Consistent with previous findings [14, 29], ER-a, ER-b and PgR were detected in significant proportions of our NSCLC specimens. However, no significant correlations were observed between the expression of AIB1 and ER or PgR, and none of our NSCLC cases showed IHC-positive staining for AR. These results indicated there might be some hormoneindependent mechanisms exist in the tumorigenesis process of NSCLC. Recently, extensive investigations revealed that AIB1 can interact with a broad spectrum of transcription factors in addition to hormone receptors, and AIB1 also was found to be involved in many non-hormone responsive tumors [5, 6]. In our study, we found a strong positive association of AIB1 overexpression and high cell proliferation in NSCLCs, which might be responsible, at least in part, for NSCLC tumorigenesis and/or progression. Clearly, further work needs to be done to determine the precise signaling pathway of AIB1 that is ultimately involved in the pathogenesis of NSCLC. In summary, our results provide some evidence for the concept that (i) overexpression of AIB1 might provide a selective advantage for lymph node metastasis of lung ADC and (ii) AIB1 plays an adverse role in the prognosis of NSCLC, especially for those with locally advanced disease, which might be helpful to optimize individual therapy management.
funding Major State Basic Research Program (973 project) (2006CB910104, 2010CB529400) and the 863 Project (2006AA02A401, 2007AA021901) of China.
disclosure None of the authors declares a conflict of interest.
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node metastasis of lung ADC. Since the sensitivity and specificity of computed tomography scans for the detection of mediastinal lymph nodes metastasis are only 70% [16], and the existence of lymph node metastasis seriously affects the prognosis of lung cancer, therefore, the expression status of AIB1, as detected by IHC, might be helpful in making accurate staging essential in the selection of a treatment plan for lung ADC patients. Most importantly, AIB1 expression was evaluated as the most significant prognostic factor in multivariate Cox regression analysis, which indicated an important role of AIB1 in the progression process of NSCLC. Furthermore, AIB1 expression could stratify patient survival in pT3/4, pN1/2 and III stages. As we know, surgical resection is the most effective method to control localized NSCLCs, and however, it might be insufficient for locally advanced cases, as inferred from 80% death within 5 years after single modality of surgery [17]; thus, adjuvant or neoadjuvant treatment are actually needed [18]. Although recent studies indicate that multimodality approaches are effective in the treatment of stage III disease, however, such therapies might account for higher mortality in certain subsets of patients [19]. Our results indicated that locally advanced NSCLC patients who have AIB1 overexpression would present with shorter survival time after surgery compared with those who have AIB1 expression, which might provide some information in identifying the patients who actually need and might benefit from the multimodality therapies, thus optimize individual therapeutic management. One of the limitations of this study is that the results of the subgroup analyses were on the basis of relatively small sample size in some subsets of patients. Thus, a larger scale, multicenter study is needed to confirm these results. It is well established that an increase in copy number of an oncogene, due to amplification, in human cancer, often produces an excess of encoded protein. To determine whether the overexpression of AIB1 in our NSCLC series was caused by gene amplification, the copy number of AIB1 was examined by FISH. We observed that overexpression of AIB1 was associated but not always coincided with AIB1 gene amplification. Similar results were also found in breast, colorectal, gastric, esophageal and bladder cancers [8, 9, 11, 15, 20]. These results indicate that the mechanisms of regulation of AIB1 expression are complicated. Recently, AIB1 was found to be self-regulated at transcription levels [21]. Moreover, it has been demonstrated that translation of AIB1 messenger RNA (mRNA) can be regulated by endogenous microRNAs [22], and the AIB1 protein levels are also influenced by proteasomal degradation pathways [23]. In addition, several cytokines, e.g. inhibitor of nuclear factor-kappa B kinase and transforming growth factorb, were also involved in the regulation of AIB1 expression [24, 25]. These results indicated that, in addition to gene amplification, the modification of AIB1 expression could also be regulated by many other mechanisms, such as transcription levels, mRNA stability and post-translational modification. With respect to the function of the AIB1 gene, it has been recognized as a member of the p160 family and a coactivator for nuclear hormone receptors including ER, PgR and AR [6]. Meanwhile, AIB1 was found to frequently amplify and overexpress in a number of hormone-sensitive human tumors
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7. Wang Y, Wu MC, Sham JS et al. Prognostic significance of c-myc and AIB1 amplification in hepatocellular carcinoma. A broad survey using high-throughput tissue microarray. Cancer 2002; 95: 2346–2352. 8. Xie D, Sham JS, Zeng WF et al. Correlation of AIB1 overexpression with advanced clinical stage of human colorectal carcinoma. Hum Pathol 2005; 36: 777–783. 9. Xu FP, Xie D, Wen JM et al. SRC-3/AIB1 protein and gene amplification levels in human esophageal squamous cell carcinomas. Cancer Lett 2007; 245: 69–74. 10. Liu MZ, Xie D, Mai SJ et al. Overexpression of AIB1 in nasopharyngeal carcinomas correlates closely with advanced tumor stage. Am J Clin Pathol 2008; 129: 728–734. 11. Luo JH, Xie D, Liu MZ et al. Protein expression and amplification of AIB1 in human urothelial carcinoma of the bladder and overexpression of AIB1 is a new independent prognostic marker of patient survival. Int J Cancer 2008; 122: 2554–2561. 12. Marquez-Garban DC, Chen HW, Fishbein MC et al. Estrogen receptor signaling pathways in human non-small cell lung cancer. Steroids 2007; 72: 135–143. 13. Kononen J, Bubendorf L, Kallioniemi A et al. Tissue microarrays for highthroughput molecular profiling of tumor specimens. Nat Med 1998; 4: 844–847. 14. Ishibashi H, Suzuki T, Suzuki S et al. Progesterone receptor in non-small cell lung cancer—a potent prognostic factor and possible target for endocrine therapy. Cancer Res 2005; 65: 6450–6458. 15. Sakakura C, Hagiwara A, Yasuoka R et al. Amplification and over-expression of the AIB1 nuclear receptor co-activator gene in primary gastric cancers. Int J Cancer 2000; 89: 217–223. 16. Pieterman RM, van Putten JW, Meuzelaar JJ et al. Preoperative staging of nonsmall-cell lung cancer with positron-emission tomography. N Engl J Med 2000; 343: 254–261. 17. Steger V, Walles T, Kosan B et al. Trimodal therapy for histologically proven N2/3 non-small cell lung cancer: mid-term results and indicators for survival. Ann Thorac Surg 2009; 87: 1676–1683. 18. Gilligan D, Nicolson M, Smith I et al. Preoperative chemotherapy in patients with resectable non-small cell lung cancer: results of the MRC LU22/NVALT 2/EORTC
Annals of Oncology 21: 1675–1681, 2010 doi:10.1093/annonc/mdp592 Published online 11 January 2010
Overexpression of AIB1 negatively affects survival of surgically resected non-small-cell lung cancer patients L.-R. He1,2 , H.-Y. Zhao3 , B.-K. Li4 , L.-J. Zhang4, M.-Z. Liu2, H.-F. Kung1, X.-Y. Guan1, X.-W. Bian5, Y.-X. Zeng1 & D. Xie1* 1 State Key Laboratory of Oncology in South China; Departments of 2Radiotherapy; 3Medical Oncology; 4Surgical Oncology, Cancer Center, Sun Yat-Sen University, Guangzhou and 5Department of Pathology, Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing, China
introduction Lung cancer is responsible for the highest cancer-related morbidity and mortality worldwide [1]. Non-small-cell lung cancer (NSCLC), which accounts for 85%–87% of all lung cancers, is a very heterogenous group of malignancies as far as the pathomorphology, local invasion and metastatic potential are concerned [2]. Clinically, the international lung cancer staging system is still not reliable enough to predict patient prognosis. The most possibly underlying reason might be the heterogeneous biological properties of NSCLC. Therefore, the search for specific genes alterations, which determine biological nature and behavior of NSCLC would be of utmost importance to optimize individual therapy [3]. It is conceivable that amplified DNA may be a component of one or more critical human oncogenes and thus may provide a selective advantage for tumor development. One of the commonly overrepresented chromosomal regions in NSCLC is 20q [4]. Several candidate oncogenes have been isolated from *Correspondence to: Dr D. Xie, State key Laboratory of Oncology in South China, Cancer Center, Sun Yat-Sen University, No 651, Dongfeng Road East, 510060 Guangzhou, China. Tel: +86-20-87343193; Fax: +86-20-87343170; E-mail: [email protected]
These authors contributed equally to this work.
20q, including amplified in breast cancer 1 (AIB1) at 20q12. The AIB1 gene, also known as SRC-3, p/CIP, RAC3, ACTR and TRAM-1, has been found to be involved in a number of biological processes, such as cell proliferation, cell apoptosis, cell migration and others [5]. The oncogenic role of AIB1 was first reported in hormone-sensitive cancers [6], and then, we contiguously reported that AIB1 was also overexpressed and correlated closely with an ascending clinical stage and/or poor patient prognosis in a series of hormone-insensitive malignancies [7–11]. Recently, AIB1 was found to express variably among different types of human NSCLC cells [12]. However, the abnormalities of AIB1 in NSCLC and its clinical/ prognostic significance have not been determined. In this study, immunohistochemistry (IHC) and FISH were used to examine protein expression and amplification of AIB1 in a large cohort of surgically resected NSCLC, and the clinicopathologic/ prognostic significance of AIB1 expression in NSCLC was also assessed.
patients and methods patients In this study, 248 NSCLC patients who received complete resection in Thoracic Department of Cancer Center, Sun Yat-sen University,
ª The Author 2010. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: [email protected]
original article
Background: The amplified in breast cancer 1 (AIB1) gene has been considered to play an oncogenic role in human cancers, but its clinical/prognostic significance in non-small-cell lung cancer (NSCLC) is still unclear. Patients and methods: The methods of immunohistochemistry and FISH were utilized to examine protein expression and amplification of AIB1 in 230 informative surgically resected NSCLCs and in 30 samples of normal lung tissues. Results: Overexpression and amplification of AIB1 were found in 48.3% and 8.2% of NSCLCs, respectively. AIB1 overexpression was associated with AIB1 gene amplification and cell proliferation but not related to estrogen receptor (ER)-a, ER-b, progesterone receptor or androgen receptor status. A positive correlation between AIB1 overexpression and an ascending pathologic node stage in lung adenocarcinoma (ADC) was observed (P = 0.043). Univariate survival analysis demonstrated a significant association of AIB1 overexpression with shortened patient survival, especially for those with stage III disease (P < 0.001). Importantly, AIB1 expression was evaluated as the most significant predictor for survival in multivariate analysis (hazards ratio = 2.069, P < 0.001). Conclusion: Overexpression of AIB1 might provide a selective advantage for lymph node metastasis of lung ADC and serve as a useful biomarker for poor prognosis for NSCLC patients. Key words: AIB1, amplification, immunohistochemistry, non-small-cell lung carcinoma, prognosis
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Received 16 September 2009; revised 24 November 2009; accepted 25 November 2009
original article
construction of tissue microarrays The tissue microarray (TMA) was constructed according to a method described previously [13]. Briefly, the individual donor tissue block and the corresponding histological hematoxylin–eosin stained slides were overlaid for tissue TMA sampling. The tissues (248 NSCLC and 30 normal lung tissues taken from regions that were not affected of the same patients) were sampled using a tissue arraying instrument (Beecher Instruments, Silver Spring, MD); a 0.6-mm-diameter cylinder of tissue was removed. Subsequently, the tissue cylinder was re-embedded into a predetermined position in a recipient paraffin block. In our constructed lung tissue TMA, three cores of sample were selected from each primary NSCLC and normal lung tissue. Multiple sections (5 lm thick) were cut from the TMA block and mounted on microscope slides.
immunohistochemistry IHC staining was carried out on 5-lm TMA sections rehydrated through graded alcohols. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 15 min. For antigen retrieval, tissue slides were boiled in 10 mM citrate buffer (pH 6.0) in a pressure cooker for 10 min [AIB1, estrogen receptor (ER) and progesterone receptor (PgR)] or microwave treated for 10 min (androgen receptor (AR) and Ki-67). The slides were incubated with anti-AIB1 [an mAb directed at amino acids 376– 389 of AIB1, Clone 34 (BD Transduction Laboratories, San Jose, CA), diluted 1 : 50 in phosphate-buffered saline (PBS), overnight at 4C], antiER-a [Clone 1D5 (Dako, Glostrup, Denmark), diluted 1 : 100 in PBS, 60 min at room temperature (RT)], anti-ER-b [Clone H-150 (Santa Cruz Biotechnology, Santa Cruz, CA), diluted 1 : 50 in PBS, 80 min at RT], antiPgR [Clone PgR 636 (Dako), diluted 1 : 500 in PBS, 60 min at RT], anti-AR [Clone AR441 (Dako), diluted 1 : 50 in PBS, 60 min at RT] and anti-Ki-67 [Clone MIB-1 (Dako), diluted 1 : 100 in PBS, overnight at 4C]. All incubations were carried out in a moist chamber. Subsequently, the slides were sequentially incubated with biotinylated rabbit anti-mouse immunoglobulin (Ig) at a concentration of 1 : 100 for 30 min at 37C and then reacted with a streptavidin–peroxidase conjugate for 30 min at 37C and 3#,3#-Diaminobenzidine as a chromogen substrate. The nucleus was counterstained using Meyer’s hematoxylin. Known immunostaining positive slides of breast cancer (AIB1, ER-a, ER-b and PgR) and prostate cancer (AR) were used as positive controls. A negative control was obtained by replacing the primary antibody with a normal murine IgG.
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Table 1. AIB1 expression and clinicopathologic variables Characteristics
Age (years) £59b >59 Gender Male Female Tumor grade G1 G2 G3 pT status pT1 pT2 pT3 pT4 pN status pN0 pN1 pN2 Stage I II III Histology SCC ADC ADSCC Othersc SCC subgroup pN0 pN1 pN2 ADC subgroup pN0 pN1 pN2
Total cases
Informative
AIB1 protein Overexpression, n (%)
130 118
121 109
57 (47.1) 54 (49.5)
181 67
168 62
87 (51.8) 24 (38.7)
24 173 51
22 163 45
9 (40.9) 79 (48.5) 23 (51.1)
15 132 81 20
13 128 71 18
5 59 35 12
120 49 79
113 44 73
47 (41.6) 25 (56.8) 39 (53.4)
82 59 107
80 56 94
32 (40.0) 27 (48.2) 52 (55.3)
94 113 32 9
89 105 29 7
49 18 27
46 17 26
20 (43.5) 9 (52.9) 13 (50.0)
53 22 38
50 20 35
16 (32.0) 11 (55.0) 20 (57.1)
Pa 0.712
0.078
0.731
0.360 (38.5) (46.1) (49.3) (66.7) 0.130
0.131
0.169 42 47 16 6
(47.2) (44.8) (55.2) (85.7) 0.755
0.043
a
Chi-square test. Mean age. c Others include anaplastic large-cell carcinoma, sarcoma, adenoid cystic carcinoma, mucoepidermoid carcinoma and carcinoid tumor. AIB1, amplified in breast cancer 1; pT, pathologic tumor; pN, pathologic node; SCC, squamous cell carcinoma; ADC, adenocarcinoma; ADSCC, adenosquamous carcinoma. b
Positive expression of AIB1 in normal and malignant lung tissues was primarily a nuclear pattern (Figure 1). For AIB1, the staining of the nuclei of the normal lung tissues ranged from 0% to 10% (median 2.3%) of epithelium with positive staining (Figure 1A and B); thus, overexpression of AIB1 was scored when >10% of tumor cells were positively stained in the nuclei [8–11]. For evaluation of the AIB1, ER, PgR, AR and Ki-67 IHC staining in the different tissue sections, the cancerous and noncancerous tissues were scored for these proteins by assessing the site of positive staining in the nucleus. The status of nuclear expression of AIB1, ER, PgR,
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Guangzhou, China, from February 1994 to January 1998 were enrolled. The cases were selected consecutively on the basis of availability of resection tissue and follow-up data. Patients who have previous malignant disease, a second primary tumor or those who received preoperative radiotherapy and/or chemotherapy were excluded. In the majority of cases, lobectomy was carried out (223 of 248 patients) and 25 patients underwent pneumonectomy. Because adjuvant chemotherapy become a standard approach only after 2004, systemic chemotherapy using cisplatin-based combinations was administered to patients with stages III NSCLCs, no postoperative therapy was delivered to patients in stage I–II. Patients with stage III disease and pathologic evidence of N2 disease received postoperative mediastinal radiotherapy. The study was approved by the medical ethics committee of Cancer Center of Sun Yat-Sen University. Tumor differentiation grades were defined according to the criteria of the World Health Organization. The pathologic tumor–node–metastasis (pTNM) status of all NSCLCs was assessed according to the criteria of the sixth edition of the TNM classification of the International Union Against Cancer (2002). The clinicopathologic features of the NSCLC cohort are outlined in Table 1. All the patients were followed up regularly and the last follow-up was carried out in December 2008, with the mean observation period of 65.3 months (3.2–172.5 months) and 167 cancer-related deaths.
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Annals of Oncology
statistical analysis
Figure 1. Immunohistochemical stainings of the amplified in breast cancer 1 (AIB1) and FISH assay of AIB1 gene in lung tissues. (A) A normal lung tissue (case 7) showed a negative staining of AIB1 in all the epithelium cells (·200). (B) Positive staining of AIB1 was observed in 2% of the epithelium cells of a normal lung tissue (case 28) (·200). (C) A squamous carcinoma of lung (case 215) showed normal expression of AIB1 with a negative staining of AIB1 in all tumor cells (·200). (D) Overexpression of AIB1 was detected in a squamous carcinoma of lung (case 23), in which 90% of tumor cells were observed strong positive staining of AIB1 (·200). (E) Overexpression of AIB1 was examined in an adenocarcinoma (ADC) of lung (case 116), in which 70% of tumor cells showed strong positive staining of AIB1 (·200). (F) Amplification of AIB1 gene was examined by FISH in the same case of ADC of lung (case 116), in which AIB1 gene signals (red) were detected at least three times more than signals on chromosome 20p12 (green) (·1000). AR and Ki-67 was assessed by determining the percentage of positive cells stained in each tissue section [11, 14]. A minimum of 300 epithelial cells was counted for each case.
FISH Two-color FISH was applied to the sections of TMA using spectrum orange-labeled bacterial artificial chromosome (BAC) clone (RP11447G11) containing the AIB1 gene and a spectrum green-labeled BAC clone on 20p12 (reference probe). The FISH reaction was carried out as described previously with slight modification [8]. Briefly, the deparaffinized TMA section was treated with proteinase K (400 lg/ml) at 37C for 45 min, followed by denaturing in 70% formamide, 2· standard saline citrate (SSC) at 75C for 8 min. Fifty nanograms of each probe were mixed in a 20 ll hybridization mixture (containing 55% formamide, 2· SSC, and 2 lg
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Statistical analysis was carried out with the SPSS software (SPSS standard version 13.0; SPSS Inc., Chicago, IL). The association of AIB1 protein expression with NSCLC patient’s clinicopathologic features and the correlations between molecular features detected with each other were assessed by the chi-square test. Disease-specific survival (DSS) was calculated from the date of diagnosis to the date of cancer-related death or last follow-up. Relative risks of cancer-related death associated with AIB1 expression status and other predictor variables were estimated from univariate Cox proportional hazards model. Multivariate survival analysis was carried out on all parameters that were found to be significant on univariate analysis using the Cox regression model. Survival curves were assessed by the Kaplan–Meier method and compared by the log-rank test. Two-sided P values of <0.05 were considered to indicate statistical significance.
results AIB1 expression in NSCLC and normal lung tissues AIB1 expression could be evaluated informatively in 230 of 248 (92.7%) of the NSCLCs and 26 of 30 (86.7%) of normal lung tissues. The noninformative samples included unrepresentative samples, samples with too few tumor cells (<300 cells per case) and lost samples; such were not used in data compilation. Using this criteria described above, overexpression of AIB1 was observed in 111 of 230 (48.3%) of the NSCLCs and correlated with an ascending pathologic node (pN) stage for lung adenocarcinoma (ADC) but not for squamous cell carcinoma (SCC) (Table 1). relationship between clinicopathologic variables, AIB1 expression and NSCLC patient survival In univariate analysis, AIB1 overexpression was evaluated to correlate closely with poor DSS for the whole cohort and could further stratify patient survival in SCC, ADC, pT3/4, pN1/2 and III stages (Figure 2; Table 2). Kaplan–Meier analysis also demonstrated a significant impact of certain clinicopathologic prognostic parameters such as tumor pT status (P = 0.013), pN status (P < 0.001) and clinical stage (P < 0.001) on patient survival. Multivariate analysis showed that AIB1 expression and pN status were independent predictors of DSS for the whole cohort (Table 3).
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human Cot1 DNA), denatured at 75C for 5 min and then hybridized to the denatured TMA section at 37C for 24 h. After washing, the TMA section was counterstained with 1 lg/ml diamidino-phenyl-indole in an antifade solution and examined with a Zeiss Axiophot microscope equipped with a triple-band-pass filter. FISH signals from 300 cells in each sample were counted. The criteria for AIB1 gene amplification were defined as the presence (in ‡20% of tumor cells) of either six (or more) gene signals or >2.5 times as many gene signals than reference 20p11 signals (Figure 1F) [11]. All samples not meeting the criteria for gain or amplification were considered normal. Control hybridizations to normal fibroblasts and to normal epithelial cells were carried out to confirm that the hybridization efficiency of the test and reference probes was similar. Two independent pathologists (XD and Rao HL) blinded to the clinicopathologic information carried out the scorings. The interobserver disagreements (8% and 3% of the total informative cases for IHC and FISH, respectively) were reviewed a second time, followed by a conclusive judgment by both pathologists.
original article
Annals of Oncology
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Figure 2. Survival analysis of the amplified in breast cancer 1 (AIB1) expression in total cohort and different subsets of non-small-cell lung cancer patients. (A) Total cohort, (B) pT1/2, (C) pT3/4, (D) pN0, (E) pN1/2, (F) stage I, (G) stage II and (H) stage III. pT, pathologic tumor, pN, pathologic node.
expression of ER, PgR and AR in NSCLCs and their correlation with AIB1 expression ER-a, ER-b, PgR and AR expression could be evaluated informatively in 232 of 248 (93.5%), 227 of 248 (91.5%), 229 of 248 (92.3%) and 225 of 248 (90.7%) of the NSCLCs, respectively. The percentage of positive cells with nuclear staining of these hormone receptors was recorded on a semiquantitative scale as previously described [14, 16]; results
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were categorized as either negative (<10% cell labeling) or positive (‡10% cell labeling). Utilizing these criteria, a positive expression of ER-a, ER-b and PgR was shown in 76 of 232 (32.8%), 86 of 227 (37.9%) and 109 of 229 (47.6%) of our NSCLC cohorts, respectively. None of the NSCLC cases showed a positive expression of AR. Statistical analysis showed that there was no significant association between the expression of AIB1 and ER-a, ER-b or PgR (P > 0.05, data not shown).
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original article
Annals of Oncology
Table 2. Prognostic value of AIB1 expression in NSCLC AIB1 expression
Disease-specific survival Relative 95% risk Confidence interval
Pa,b
119 111
1 1.963
1.414–2.723
<0.001
47 42
1 1.904
1.121–3.232
0.015
58 47
1 2.270
1.398–3.685
0.001
77 64
1 1.484
0.970–2.273
0.087
42 47
1 3.169
1.861–5.397
<0.001
66 47
1 1.262
0.761–2.093
0.367
53 64
1 2.677
1.707–4.198
<0.001
48 32
1 1.055
0.575–1.935
0.863
29 27
1 1.565
0.770–3.184
0.216
42 52
1 3.711
2.225–6.190
<0.001
discussion
a
Belongs to overexpression of AIB1. Univariate Cox proportional hazards analysis. AIB1, amplified in breast cancer 1; NSCLC, non-small-cell lung carcinoma; SCC, squamous cell carcinoma; ADC, adenocarcinoma; pT, pathologic tumor; pN, pathologic node.
b
Table 3. Multivariate Cox regression analysis for disease-specific survival Factors
Hazards ratio
95% Confidence interval
P
AIB1 expressiona pT statusb pN statusc Staged
2.069 1.143 1.684 1.300
1.487–2.879 0.839–1.557 1.126–2.519 0.615–2.748
<0.001 0.379 0.011 0.491
a
Normal expression versus overexpression. pT1 versus pT2 versus pT3 versus pT4. c pN0 versus pN1 versus pN2. d Stage I versus stage II versus stage III. AIB1, amplified in breast cancer 1; pT, pathologic tumor; pN, pathologic node. b
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amplification of AIB1 in NSCLC and normal lung tissues The FISH analysis was informative in 53.3% (16 of 30) of the normal lung tissues and 54.0% (134 of 248) of the NCSLCs. Samples without FISH signal and samples with weak target signals or those with a strong signal background were the main reasons for most of the noninformative cases. The amplification of AIB1 was not detected in any of the normal lung tissues but was observed in 8.2% (11 of 134) of the informative NCSLCs. In our study, 121 informative cases were shown to be positive by both IHC and FISH simultaneously, and a significant correlation between AIB1 overexpression and amplification of AIB1 was found (Table 4).
In the present study, we found that the expression of AIB1 by IHC in all the normal lung tissue specimens was absent or at low levels. In contrast, in many of our NSCLC specimens, an overexpression of AIB1 was frequently detected, which indicated a potential important role of AIB1 in carcinogenesis of lung. Similar results were also observed in other human cancers, such as esophageal, gastric, colorectal and nasopharyngeal carcinomas, in which up-regulating of AIB1 was observed and correlated with a late clinical stage and/or poor prognosis [9, 10, 15]. It is well known that the pathogenesis and biologic behaviors of different subtypes of NSCLC are quite distinct. The underline mechanism might contribute, at least partly to the molecular diversity. Although no significant association was found between AIB1 expression and clinicopathologic features for the whole cohort, interestingly, a steady increase of AIB1 expression was observed in ADCs of lung from no lymph node metastasis to peribronchial lymph node metastasis and to mediastinal lymph node metastasis. These findings indicated that up-regulated expression of AIB1 may provide a selective advantage in lymph Table 4. Association of AIB1 expression and amplification in NSCLC AIB1 gene
Informative cases
AIB1 expression Normal Overexpression
Pa
No amplification Amplification
110 11
62 (56.4) 1 (9.1)
0.003
48 (43.6) 10 (90.9)
a
Chi-square test. AIB1, amplified in breast cancer 1; NSCLC, non-small-cell lung carcinoma.
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Total Normal expression Overexpression Histology SCC Normal expression Overexpression ADC Normal expression Overexpression pT status pT1/2 Normal expression Overexpression pT3/4 Normal expression Overexpression pN status pN0 Normal expression Overexpression pN1/2 Normal expression Overexpression Stage Stage I Normal expression Overexpression Stage II Normal expression Overexpression Stage III Normal expression Overexpression
Cases
correlation of AIB1 expression and cell proliferation in NSCLCs In our NSCLC cohorts, the median value of 25.6% was used as a cut-off value to define low Ki-67 labeling index (LI) (LI <25.6%) and high Ki-67 LI (LI ‡25.6%) [9, 10]. In 212 of the 248 samples, AIB1 and Ki-67 IHC were detected successfully and simultaneously. Correlation analysis demonstrates that the frequency of cases with a high LI of Ki-67 was significantly greater in tumors with an overexpression of AIB1 (54 of 77, 70.1%) than that in cases with normal expression of AIB1 (23 of 77, 29.9%; P < 0.001).
original article
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[26, 27], which indicates that AIB1 may play a role in tumorigenesis of specific subsets of hormone-sensitive cancers, perhaps by facilitating hormone-dependent signal transduction pathways. Interestingly, overexpression of AIB1 was detected in breast tumors positive and negative for ER and PgR [28], while AIB1 knockdown impaired the proliferation of both ARpositive and AR-negative prostate cancer cells [27]. In human lung cancer, it has been reported that estrogen-signaling pathway (including AIB1) plays an important role in NSCLC carcinogenesis [12]. Consistent with previous findings [14, 29], ER-a, ER-b and PgR were detected in significant proportions of our NSCLC specimens. However, no significant correlations were observed between the expression of AIB1 and ER or PgR, and none of our NSCLC cases showed IHC-positive staining for AR. These results indicated there might be some hormoneindependent mechanisms exist in the tumorigenesis process of NSCLC. Recently, extensive investigations revealed that AIB1 can interact with a broad spectrum of transcription factors in addition to hormone receptors, and AIB1 also was found to be involved in many non-hormone responsive tumors [5, 6]. In our study, we found a strong positive association of AIB1 overexpression and high cell proliferation in NSCLCs, which might be responsible, at least in part, for NSCLC tumorigenesis and/or progression. Clearly, further work needs to be done to determine the precise signaling pathway of AIB1 that is ultimately involved in the pathogenesis of NSCLC. In summary, our results provide some evidence for the concept that (i) overexpression of AIB1 might provide a selective advantage for lymph node metastasis of lung ADC and (ii) AIB1 plays an adverse role in the prognosis of NSCLC, especially for those with locally advanced disease, which might be helpful to optimize individual therapy management.
funding Major State Basic Research Program (973 project) (2006CB910104, 2010CB529400) and the 863 Project (2006AA02A401, 2007AA021901) of China.
disclosure None of the authors declares a conflict of interest.
references 1. Jemal A, Siegel R, Ward E et al. Cancer statistics, 2008. CA Cancer J Clin 2008; 58: 71–96. 2. Grondin SC, Liptay MJ. Current concepts in the staging of non-small cell lung cancer. Surg Oncol 2002; 11: 181–190. 3. Tomida S, Koshikawa K, Yatabe Y et al. Gene expression-based, individualized outcome prediction for surgically treated lung cancer patients. Oncogene 2004; 23: 5360–5370. 4. Shen H, Zhu Y, Wu YJ et al. Genomic alterations in lung adenocarcinomas detected by multicolor fluorescence in situ hybridization and comparative genomic hybridization. Cancer Genet Cytogenet 2008; 181: 100–107. 5. Liao L, Kuang SQ, Yuan Y et al. Molecular structure and biological function of the cancer-amplified nuclear receptor coactivator SRC-3/AIB1. J Steroid Biochem Mol Biol 2002; 83: 3–14. 6. Yan J, Tsai SY, Tsai MJ. SRC-3/AIB1: transcriptional coactivator in oncogenesis. Acta Pharmacol Sin 2006; 27: 387–394.
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node metastasis of lung ADC. Since the sensitivity and specificity of computed tomography scans for the detection of mediastinal lymph nodes metastasis are only 70% [16], and the existence of lymph node metastasis seriously affects the prognosis of lung cancer, therefore, the expression status of AIB1, as detected by IHC, might be helpful in making accurate staging essential in the selection of a treatment plan for lung ADC patients. Most importantly, AIB1 expression was evaluated as the most significant prognostic factor in multivariate Cox regression analysis, which indicated an important role of AIB1 in the progression process of NSCLC. Furthermore, AIB1 expression could stratify patient survival in pT3/4, pN1/2 and III stages. As we know, surgical resection is the most effective method to control localized NSCLCs, and however, it might be insufficient for locally advanced cases, as inferred from 80% death within 5 years after single modality of surgery [17]; thus, adjuvant or neoadjuvant treatment are actually needed [18]. Although recent studies indicate that multimodality approaches are effective in the treatment of stage III disease, however, such therapies might account for higher mortality in certain subsets of patients [19]. Our results indicated that locally advanced NSCLC patients who have AIB1 overexpression would present with shorter survival time after surgery compared with those who have AIB1 expression, which might provide some information in identifying the patients who actually need and might benefit from the multimodality therapies, thus optimize individual therapeutic management. One of the limitations of this study is that the results of the subgroup analyses were on the basis of relatively small sample size in some subsets of patients. Thus, a larger scale, multicenter study is needed to confirm these results. It is well established that an increase in copy number of an oncogene, due to amplification, in human cancer, often produces an excess of encoded protein. To determine whether the overexpression of AIB1 in our NSCLC series was caused by gene amplification, the copy number of AIB1 was examined by FISH. We observed that overexpression of AIB1 was associated but not always coincided with AIB1 gene amplification. Similar results were also found in breast, colorectal, gastric, esophageal and bladder cancers [8, 9, 11, 15, 20]. These results indicate that the mechanisms of regulation of AIB1 expression are complicated. Recently, AIB1 was found to be self-regulated at transcription levels [21]. Moreover, it has been demonstrated that translation of AIB1 messenger RNA (mRNA) can be regulated by endogenous microRNAs [22], and the AIB1 protein levels are also influenced by proteasomal degradation pathways [23]. In addition, several cytokines, e.g. inhibitor of nuclear factor-kappa B kinase and transforming growth factorb, were also involved in the regulation of AIB1 expression [24, 25]. These results indicated that, in addition to gene amplification, the modification of AIB1 expression could also be regulated by many other mechanisms, such as transcription levels, mRNA stability and post-translational modification. With respect to the function of the AIB1 gene, it has been recognized as a member of the p160 family and a coactivator for nuclear hormone receptors including ER, PgR and AR [6]. Meanwhile, AIB1 was found to frequently amplify and overexpress in a number of hormone-sensitive human tumors
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19.
20. 21.
22. 23.
24.
25.
26.
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