Med Oncol (2014) 31:77 DOI 10.1007/s12032-014-0077-6
ORIGINAL PAPER
Combined detection of Gab1 and Gab2 expression predicts clinical outcome of patients with glioma Hui Liu • Gang Li • Weitao Zeng • Pengxing Zhang Feiyan Fan • Yanyang Tu • Yongsheng Zhang
•
Received: 25 May 2014 / Accepted: 12 June 2014 / Published online: 8 July 2014 Ó Springer Science+Business Media New York 2014
Abstract Grb2-associated binder 1 (Gab1) and Gab2 play important roles in cancer cell signaling. In particular, it has been demonstrated that the upregulation of Gab2 may be correlated with the World Health Organization (WHO) grade of gliomas and that patients with high Gab2 expression levels exhibited shorter survival time. However, the prognostic value of combined expression of Gab1 and Gab2 has not been explored. Gab1 and Gab2 expression in human gliomas and non-neoplastic brain tissues was measured by immunohistochemistry. Both the expression levels of Gab1 and Gab2 proteins in glioma tissues were significantly higher than those in non-neoplastic brain tissues (both P \ 0.001). In addition, the overexpression of Gab1 and Gab2 proteins were both significantly associated with advanced WHO grades (both P \ 0.001) and low KPS (both P = 0.01). Moreover, the overall survival of patients with high Gab1 protein expression or high Gab2 protein expression was obviously lower than those with low expressions (both P \ 0.001). Notably, glioma patients with combined overexpression of Gab1 and Gab2 proteins (Gab1-high/Gab2-
Hui Liu and Gang Li offer the equal contributions to this study. Yanyang Tu and Yongsheng Zhang are co-corresponding authors. H. Liu W. Zeng P. Zhang F. Fan Y. Tu (&) Department of Experimental Surgery, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, China e-mail: [email protected] G. Li Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, China Y. Zhang (&) Administrative Department, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, China e-mail: [email protected]
high) had shortest overall survival (P \ 0.001). Furthermore, multivariate analysis showed that Gab1 expression (P = 0.01), Gab2 expression (P = 0.02), and combined expression of Gab1 and Gab2 (Gab1/Gab2, P = 0.006) were all independent prognostic factors for overall survival in glioma patients. Gab1 and Gab2 proteins are differentially expressed in glioma patients and closely correlated with the biological behavior of this malignancy. Combination of Gab1 and Gab2 expression may represent a promising biomarker for prognostication of human gliomas. Keywords Grb2-associated binder family Glioma Immunohistochemistry Prognosis
Introduction Gliomas account for one of the most common central nervous system neoplasms and divided into four histopathologic grades based on the degree of malignancy, including well-differentiated low-grade astrocytomas [World Health Organization (WHO) grades I–II], anaplastic astrocytomas (WHO grade III), and glioblastoma multiforme (GBM, WHO grade IV) [1]. These tumors may develop at all ages, and the peak incidence is in the fifth and sixth decades of life [2]. Although recent advances in surgery, radiotherapy, and chemotherapy, survival of patient with glioma remains poor. In particular, patients with GBM have a median survival time of roughly 1 year [3]. Recent studies have indicated that age, Karnofsky performance status (KPS) score, WHO tumor grade, and tumor necrosis are important prognostic factors for gliomas [4]. Among them, the WHO tumor grade is the most wellestablished pathological prognostic factor in glioma. However, the prognosis of both high- and low-grade
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tumors remains heterogeneous. For example, Curran et al. [5] demonstrated that the median survival time of patients with high-grade gliomas ranges from 5 to 59 months and some patients with low-grade tumors also present poor outcome. Therefore, it is vital to identify new potential molecular biomarkers for diagnosis and prognosis of human gliomas. Grb2-associated binding (Gab) scaffolding/adapter proteins are a family of three highly conserved members including mammalian Gab1, Gab2, and Gab3, which are homologs of Drosophila Daughter of Sevenless (DOS) and Caenorhabditis elegans suppressor of clear (SOC-1) [6]. Both Gab1 and Gab2 are expressed ubiquitously, but they are most highly expressed in brain, kidney, lung, heart, testis, and ovary. Gab3 also has a widespread expression pattern, although it is most highly expressed in lymphoid tissue [7]. These proteins encompass an N-terminal PH domain, which mediates their interaction with specific membrane lipids and contain a large number of tyrosine residues and proline-rich regions that allow their interaction with signaling proteins encompassing SH2 and SH3 domains and consequently the formation of multimolecular signaling complexes [8]. Since their discovery, an increasing number of work have been done to better understand the functional roles of Gabs in multiple signaling pathways, typically acting as a downstream effectors of receptor tyrosine kinase (RTK)-triggered signal transduction [9]. Gabs have been demonstrated to participate in hematopoiesis, regulation of immune response, and cancer cell signaling [10]. Among them, Gab1 couples to a variety of receptor and non-receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR), c-Met and Src, and, depending on context, is capable of promoting cell growth, differentiation, and migration and is involved in control of cell apoptosis [11]. Accumulating studies have found that Gab1 upregulates proto-oncogene expression via participation in amplification of signaling pathways related to tumor biological behaviors and plays a role in the occurrence and development of tumors [12]. For example, Gab1 is a key binding partner of the Met receptor for induction of cell cycle progression, proliferation, and oncogenic morphological transformation, suggesting Gab1 and its associated signaling partners are potential therapeutic targets to impair proliferation or transformation of cancer cells in human malignancies harboring a deregulated Met receptor [13]; Gab1 mediates tumor progression in Met overexpressing colorectal cancer cells [14]; downregulation of Gab1 could inhibit cell proliferation and migration in hilar cholangiocarcinoma [15]; SHP-2/Gab1 association may be critical for linking EGFR to NF-kappaB transcriptional activity via the PI3 kinase/Akt signaling axis in GBM cells [16]. Regarding to Gab2 that lacks enzymatic activity, it acts downstream of receptor tyrosine
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kinases and non-receptor tyrosine kinases, such as cytokine and G protein-coupled receptors, to transmit and amplify signals to downstream effectors [17]. Gab2 is located on chromosomal band 11q14.1, amplification of which is frequently observed in human malignancies [18]. Gab2 has been identified as a potential oncogene in leukemia, melanoma, breast cancer, and ovarian cancer [19–22]. Although it appears to be insufficient to transform primary mammary epithelial cells, Gab2 has been indicated to cooperate with ErbB2 to potentiate tumorigenic signaling [23]. In addition to four kinds of human malignancies, Gab2 is overexpressed in malignant lung tissues compared with that in normal lung tissues suggesting that Gab2 expression may play a role in lung cancer development [24]. More interestingly, in 2013, Shi et al. [25] reported that the upregulation of Gab2 may be correlated with the WHO grade of gliomas and that patients with high Gab2 expression levels exhibited shorter survival time, suggesting that Gab2 may be a useful prognostic marker for gliomas and a novel therapeutic target for glioma invasion intervention. Both Gab1 and Gab2 have been demonstrated to be closely correlated with the tumorigenesis of human gliomas. However, the prognostic value of combined expression of Gab1 and Gab2 has not been explored. To address this problem, we examined the immunohistochemical expression of Gab1 and Gab2, and their association with clinicopathological characteristics and prognosis in glioma patients.
Materials and methods Patients and tissue samples This study was approved by the Research Ethics Committee of Tangdu Hospital, Fourth Military Medical University, P. R. China. Written informed consent was obtained from all of the patients. All specimens were handled and made anonymous according to the ethical and legal standards. A total of 128 formalin-fixed, paraffin-embedded specimens of gliomas resected between 2000 and 2010 were retrieved from the archives of the Pathology Department of Tangdu Hospital, Fourth Military Medical University, P. R. China. All the slides were re-evaluated according to WHO classifications [1] by two pathologists, with differences resolved by careful discussion. A total of 76 males and 52 females (1.46:1) were enrolled in this study, and the median age was 42 years (range 12–71). Thirty-two of the 128 gliomas were classified as low grade [18 pilocytic astrocytomas (WHO I) and 14 diffuse astrocytomas (WHO II)], and 96 were classified as high-grade gliomas [38 anaplasia
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Table 1 Clinicopathological features of 128 patients with gliomas Features
WHO I
WHO II
WHO III
WHO IV
Case no.
18
14
38
58
Mean age (year)
38.6
45.9
43.1
44.2
Male
12
6
25
33
Female
6
8
13
25
15 3
11 3
9 29
15 43
Gender
KPS [80 \80 Surgery Gross total resection
18
14
28
38
Partial resection
0
0
9
15
Biopsy
0
0
1
5
Adjuvant treatment Radiotherapy
0
0
30
12
Chemotherapy
0
1
0
6
Radiotherapy and chemotherapy combination
0
0
5
28
astrocytomas (WHO III) and 58 primary glioblastomas (WHO IV)]. None of the patients had received chemotherapy or radiotherapy prior to surgery. The clinicopathological features and the treatment strategies of all the patients are indicated in Table 1. Paraffin and snap-frozen sections of non-neoplastic brain tissues from ten patients with intractable epilepsy were also included as controls. Five-year follow-up was performed, and all patients had complete follow-up until death. Overall survival time was calculated from the date of the initial surgical operation to death. Patients, who died of diseases not directly related to their gliomas or due to unexpected events, were excluded from this study. Immunohistochemistry assay Formalin-fixed, paraffin-embedded, sectioned tissues (4 lm thick) were immunostained using the labeled Streptavidin Biotin 2 System (BioGenex; San Ramon, CA, USA). Following peroxidase blocking with 0.3 % H2O2/methanol for 30 min, specimens were blocked with phosphate-buffered saline (PBS) containing 5 % normal horse serum (Vector Laboratories Inc., Burlingame, CA, USA). All incubations with polyclonal rabbit antihuman Gab1 antibody (Santa Cruz Biotechnology, #sc-9049, 1:100 dilution) and polyclonal rabbit antihuman Gab2 antibody (Santa Cruz Biotechnology, #sc-25498, 1:100 dilution), respectively, were carried out overnight at 4 °C. Then, the specimens were briefly washed in PBS and incubated at room temperature with the antirabbit antibody and avidin–biotin peroxidase (Vector Laboratories Inc., Burlingame, CA, USA). The specimens were
then washed in PBS and color developed by diaminobenzidine solution (Dako Corporation, Carpinteria, CA, USA). After washing with water, specimens were counterstained with Meyer’s hematoxylin (Sigma Chemical Co., St Louis, MO, USA). Non-neoplastic brain tissues were used as control tissues, and non-immune IgG was also used as negative control antibody for immunohistochemical staining. Assessment of immunohistochemical staining was evaluated by two independent pathologists. The scores of the two pathologists were compared, and any discrepant scores were trained through re-examining the stainings by both pathologists to achieve a consensus score. The number of positive-staining cells showing immunoreactivity in the cytoplasm for Gab1 and Gab2 in ten representative microscopic fields was counted, and the percentage of positive cells was calculated. The percentage scoring of immunoreactive tumor cells was as follows: 0 (0 %), 1 (1–10 %), 2 (11–50 %) and 3 ([50 %). The staining intensity was visually scored and stratified as follows: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). A final immunoreactivity scores (IRS) was obtained for each case by multiplying the percentage and the intensity score. As there were no absolute criteria for the immunoreactivity of each antibody used in this study, we selected the median of IRS for Gab1 or Gab2 expression levels cutoff points, considering different expression on normal and tumor cells. This method considered the following values as criteria for the high expression group: Gab1 C 4.47 and Gab2 C 4.70. Statistical analysis All computations were carried out using the software of SPSS version 13.0 for Windows (SPSS Inc, IL, USA). Data were expressed as mean ± standard deviation (SD). The analysis of variance (ANOVA) was used to determine the statistical differences among the groups. The correlation between Gab1 and Gab2 protein expression was calculated using Spearman’s test. A life table was calculated according to the Kaplan–Meier method. Hazard ratios for the time-to-event end point were estimated using the multivariate Cox regression analysis in a forward stepwise method to evaluate the effect of multiple independent prognostic factors on survival outcome. Differences were considered statistically significant when P was \0.05.
Results Gab1 and Gab2 proteins are upregulated in human glioma tissues versus non-neoplastic brain tissues We observed immunoreactivities of Gab1 and Gab2 proteins in the cytoplasm (Fig. 1), and distinctly low levels of
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Fig. 1 Representative sections for Gab1 and Gab2 immunoreactivities in glioma tissues (a, b, respectively) and non-neoplastic brain tissues (c, d, respectively; 9400). The immunoreactivities of Gab1 and Gab2 proteins were shown in the cytoplasm, and distinctly low levels of their expression were observed in non-neoplastic brain tissues compared with glioma tissues. The red arrows refer to the positive area of immunostaining. Statistical analysis showed that both the expression levels of Gab1 and Gab2 proteins in glioma tissues were significantly higher than those in corresponding nonneoplastic brain tissues (both P \ 0.001, e, f, respectively)
proteins in glioma tissues were significantly higher than those in non-neoplastic brain tissues (both P \ 0.001). Of the 128 glioma tissues investigated, 68 cases (53.13 %) were regarded as Gab1-high expression group and 66 cases (51.56 %) as Gab2-high expression group. The Spearman correlation analysis revealed that a corresponding correlation between Gab1 overexpression and Gab2 overexpression was evident in gliomas (r = 0.514; P \ 0.001, Fig. 2). Upregulations of Gab1 and Gab2 proteins associate with tumor progression of human gliomas Fig. 2 Scatter diagram of the Spearman correlation analysis between Gab1 and Gab2 protein expression levels in glioma tissues
their expression were observed in non-neoplastic brain tissues compared with glioma tissues. Statistical analysis showed that both the expression levels of Gab1 and Gab2
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Table 2 summarized the associations between upregulations of Gab1 or Gab2 proteins with clinicopathological characteristics of human gliomas. We found that the upregulations of Gab1 and Gab2 proteins were both significantly associated with advanced WHO grades (both
Med Oncol (2014) 31:77 Table 2 Associations of Gab1 or Gab2 expression in human glioma tissues with different clinicopathological features
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Clinicopathological features
No. of cases
Gab1 expression High (n, %)
P
Low (n, %)
Gab2 expression High (n, %)
P
Low (n, %)
WHO grade I
18
2 (11.11)
16 (88.89)
II III
14
4 (28.57)
38
20 (52.63)
IV
58 52 76
\0.001
\0.001
2 (11.11)
16 (88.89)
10 (71.43)
4 (28.57)
10 (71.43)
18 (47.37)
20 (52.63)
18 (47.37)
42 (72.41)
16 (27.59)
40 (68.97)
18 (31.03)
25 (48.08) 43 (56.58)
27 (51.92) 33 (43.42)
NS
25 (48.08) 41 (53.95)
27 (51.92) 35 (46.05)
NS
NS
40 (52.63)
36 (47.37)
NS
26 (50.00)
26 (50.00)
Age \55 C55 Gender Male
76
42 (55.26)
34 (44.74)
Female
52
26 (50.00)
26 (50.00)
KPS \80
78
50 (64.10)
28 (35.90)
C80
50
18 (36.00)
32 (64.00)
P \ 0.001, Table 2) and low KPS (both P = 0.01, Table 2). No statistically significant association of Gab1 or Gab2 with age at diagnosis and gender of patients was found (both P [ 0.05, Table 2). Association of Gab1 and Gab2 protein expression with overall survival in patients with gliomas During the follow-up period, 100 of the 128 glioma patients (78.1 %) had died after the surgery. The Kaplan– Meier plot showed that patients showing Gab1 overexpression had a poorer overall survival than patients with Gab1-low tumors (log rank, P = 0.01; Fig. 3a). On the other hand, the aberrant expression of Gab2 also was significantly associated with overall survival (P = 0.006; Fig. 3b). With regard to combined expression of Gab1 and Gab2 proteins, we divided the specimens into three groups: group 1, tumors exhibiting high expression of Gab1 and Gab2 (Gab1-high/Gab2-high, 46 specimens); group 2, tumors with different expression of Gab1 and Gab2 (Gab1high/Gab2-low or Gab1-low/Gab2-high, 42 specimens); and group 3, tumors with low expression of both proteins (Gab1-low/Gab2-low, 40 specimens). Notably, there was a trend toward the poorest overall survival in the patient group with high expression of Gab1 and Gab2 (P \ 0.001, Fig. 3c). Interestingly, the overall survival curve for patients in group 2 (Gab1-high/Gab2-low, or Gab1-low/ Gab2-high) was relatively close to those with tumors that were low expression for both Gab1 and Gab2, but were dramatically discrepant from those of the Gab1-high/Gab2high group (Fig. 3c). In multivariate analysis, Cox proportional hazards model involving the expression levels of Gab1 and Gab2
0.01
50 (64.10)
28 (35.90)
16 (32.00)
34 (68.00)
0.01
proteins, and various clinical parameters showed that high Gab1 protein expression (P = 0.01), high Gab2 protein expression (P = 0.02), and combined expression of Gab1 and Gab2 (P = 0.006) were all independent prognostic factors for overall survival in glioma patients. Statistical values of the expression levels of Gab1 and Gab2 proteins and other clinical parameters derived from Cox stepwise proportional hazards model are indicated in Table 3.
Discussion Treatment of human glioma has been increasingly informed by biomarkers that predict patient prognosis and response to therapy. Although a number of candidate diagnostic and prognostic biomarkers have already been identified for human glioma, previously, clinical trials found that the vast majority of them might be poorly suited to the task of identifying treatments, but effective only in selected subsets of patients. Therefore, it is still a significant challenge to identify novel and efficient biomarkers for gliomas with good clinical application. In the present study, our data showed that the abnormal expressions of Gab1 and Gab2 proteins appeared to be correlated with the WHO grade of glioma, clinicopathological features, as well as patient survivals. These strong associations suggest that Gab1 and Gab2 overexpression promotes tumor progression and that Gab1 and/or Gab2 could possibly be used as a biomarker for a more aggressive phenotype of gliomas. To the best of our knowledge, this is the first study to demonstrate the prognostic value of the co-expression of Gab1 and Gab2 in gliomas. Gab1, the most common Gabs, was originally isolated as a Grb2-binding protein from a human glial tumor
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Fig. 3 Kaplan–Meier survival curves of glioma patients. a The 5-year overall survival of glioma patients with high Gab1 protein expression was significantly lower than that of glioma patients with low Gab1 protein expression (P \ 0.001). b The 5-year overall survival of glioma patients with high Gab2 protein expression was
significantly lower than that of glioma patients with low Gab2 protein expression (P \ 0.001). c The 5-year overall survival of glioma patients with Bmi-high/Gab2-high expression was significantly lowest of all three groups (P \ 0.001)
Table 3 Cox multivariate analysis
the EGFR and Met [13]. It is also a potential player in the nuclear localization of activated MAPK and is capable of binding to MAPK and aiding in nuclear translocation through a chaperone-like function [28]. However, there have been no reports describing the relationship between Gab1 and clinicopathological characteristics of human gliomas to date. In the present study, the upregulation of Gab1 expression was detected in 53.13 % (68/128) of glioma tissues. In contrast, its immunostaining was not or weakly detected in control brain tissues. Of four WHO grades, grade IV exhibited the highest Gab1 level. On the other hand, Gab1 expression was significantly associated with WHO grade and KPS score, and patients with high WHO grade expression had a significantly shorter overall survival than those with low Gab1 expression. According to Cox regression, Gab1 can be suggested as an independent prognostic factor for human glioma. As another Gabs, Gab2 recruits the tyrosine phosphatase Shp2 and the p85 subunit of PI3K and thereby positively regulates the Ras/Erk and PI3K pathways in response to activation of a variety of receptor and receptor-associated tyrosine kinases [17]. An increasing number of evidence has emerged supporting a functional role for Gab2 in the development of several human malignancies. Aberrant expression of Gab2 has been observed in various human cancer tissues, such as glioma, melanoma, breast cancer, lung cancer, gastric cancer, and ovarian cancer [19–22]. Gab2(-/-) breast cancer cells exhibited decreased migration and impaired Erk activation, suggesting its role in promoting mammary tumor metastasis [19]; Gab2 expression in lung tumor samples was significantly higher than that in normal lung samples, and Gab2 may be as a molecular marker for neoplastic progression [20];
Parameter
Risk ratio
95 % confidence interval
P
Age
0.89
0.58–1.65
0.71
Gender
1.02
0.66–1.83
0.33
WHO grade
4.88
1.96–10.16
0.01
KPS
1.99
1.28–2.95
0.06
Extent of resection
1.29
0.89–2.13
0.11
Type of adjuvant treatment
1.37
1.02–2.24
0.11
Gab1 expression
4.23
2.01–9.06
0.01
Gab2 expression
3.78
1.96–7.66
0.02
Combined Gab1/Gab2 expression
6.88
2.39–15.29
0.006
expression library and was also identified independently as a Met-receptor interacting protein in a yeast two-hybrid screen and as the major tyrosine phosphorylated protein in cells transformed by the Tpr-Met oncogene [26]. It is located in the cytoplasm and recruited to the cell membrane after being activated to bind with several activated signal receptors, resulting in its own activation [27]. Gab1, with wide distribution in various body tissues of mammals, is capable of promoting cell growth, proliferation, differentiation, adhesion, migration, and invasion and is implicated in control of cell apoptosis [11]. Accumulating studies have found that Gab1 may upregulate proto-oncogene expression via participation in amplification of signaling pathways related to tumor biological behaviors. It plays a crucial role in the occurrence and development of tumors. For example, Gab1 has been shown to enhance cell growth and transformation of NIH 3T3 cells downstream of
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increased expression of Gab2 in malignant gastric cells compared with normal epithelial cells, implying its possible roles in gastric cancer development [21]; Gab2-mediated signaling has been implicated in chronic myelogenous leukemia and lymphoid leukemia; The Grb2–Gab2 pathway is critical for leukemic transformation by Bcr–Abl [22]. In addition to these cancer types, Gab2 has been found to be high expressed in gliomas and a subset of cancer cell lines. The authors also indicated that the upregulation of Gab2 was correlated with the WHO grade of gliomas and that patients with high Gab2 expression levels exhibited shorter survival time. In an animal experiment and a cell research, they demonstrated that knockdown of Gab2 through siRNA could inhibit the migration and invasion of glioma cells by mediating cytoskeleton rearrangement and MMPs expression. Moreover, IGF-1-induced pAkt and pmTOR phosphorylation was suppressed by the knockdown of Gab2 [23]. In line with these previous findings, our data here validated again the clinical value of Gab2 overexpression in tumorigenesis, tumor progression, and patient prognosis of gliomas using an independently large cohort. Since accumulating studies have indicated that the efficiency of combined biomarkers may be more stronger than single biomarker in the diagnosis and prognosis of various human cancers, we hypothesized that the prognostic value of the combination of Gab1 and Gab2 (Gab1/ Gab2) might be better than Gab1 or Gab2 alone. To validate this hypothesis, we analyzed the correlations of Gab1/ Gab2 combined expression, Gab1 expression, and Gab2 expression with overall survival of patients with gliomas. Our data showed that Gab1/Gab2 combined expression, Gab1 expression, and Gab2 expression were all independent prognostic factors for overall survival of patients with gliomas. More interestingly, Gab1/Gab2 combined expression could more efficiently predict the prognosis in glioma patients, suggesting that the detection of coexpression of Gab1 and Gab2 could be used to design optimal, individualized treatment and to help identify patients who may or may not benefit from close monitoring after surgery. Additional research would be required to confirm our findings. In conclusion, Gab1 and Gab2 proteins are differentially expressed in glioma patients and closely correlated with the biological behavior of this malignancy. Combination of Gab1 and Gab2 expression may represent a promising biomarker for prognostication of human gliomas. Acknowledgments This work was funded by National Natural Science Foundation of China (No. 81272419, 81101736, 81272776) and Natural Science Foundation of Shannxi Province (No. 2014JM4148). Conflict of interest
None.
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ORIGINAL PAPER
Combined detection of Gab1 and Gab2 expression predicts clinical outcome of patients with glioma Hui Liu • Gang Li • Weitao Zeng • Pengxing Zhang Feiyan Fan • Yanyang Tu • Yongsheng Zhang
•
Received: 25 May 2014 / Accepted: 12 June 2014 / Published online: 8 July 2014 Ó Springer Science+Business Media New York 2014
Abstract Grb2-associated binder 1 (Gab1) and Gab2 play important roles in cancer cell signaling. In particular, it has been demonstrated that the upregulation of Gab2 may be correlated with the World Health Organization (WHO) grade of gliomas and that patients with high Gab2 expression levels exhibited shorter survival time. However, the prognostic value of combined expression of Gab1 and Gab2 has not been explored. Gab1 and Gab2 expression in human gliomas and non-neoplastic brain tissues was measured by immunohistochemistry. Both the expression levels of Gab1 and Gab2 proteins in glioma tissues were significantly higher than those in non-neoplastic brain tissues (both P \ 0.001). In addition, the overexpression of Gab1 and Gab2 proteins were both significantly associated with advanced WHO grades (both P \ 0.001) and low KPS (both P = 0.01). Moreover, the overall survival of patients with high Gab1 protein expression or high Gab2 protein expression was obviously lower than those with low expressions (both P \ 0.001). Notably, glioma patients with combined overexpression of Gab1 and Gab2 proteins (Gab1-high/Gab2-
Hui Liu and Gang Li offer the equal contributions to this study. Yanyang Tu and Yongsheng Zhang are co-corresponding authors. H. Liu W. Zeng P. Zhang F. Fan Y. Tu (&) Department of Experimental Surgery, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, China e-mail: [email protected] G. Li Department of Neurosurgery, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, China Y. Zhang (&) Administrative Department, Tangdu Hospital, Fourth Military Medical University, Xi’an 710038, China e-mail: [email protected]
high) had shortest overall survival (P \ 0.001). Furthermore, multivariate analysis showed that Gab1 expression (P = 0.01), Gab2 expression (P = 0.02), and combined expression of Gab1 and Gab2 (Gab1/Gab2, P = 0.006) were all independent prognostic factors for overall survival in glioma patients. Gab1 and Gab2 proteins are differentially expressed in glioma patients and closely correlated with the biological behavior of this malignancy. Combination of Gab1 and Gab2 expression may represent a promising biomarker for prognostication of human gliomas. Keywords Grb2-associated binder family Glioma Immunohistochemistry Prognosis
Introduction Gliomas account for one of the most common central nervous system neoplasms and divided into four histopathologic grades based on the degree of malignancy, including well-differentiated low-grade astrocytomas [World Health Organization (WHO) grades I–II], anaplastic astrocytomas (WHO grade III), and glioblastoma multiforme (GBM, WHO grade IV) [1]. These tumors may develop at all ages, and the peak incidence is in the fifth and sixth decades of life [2]. Although recent advances in surgery, radiotherapy, and chemotherapy, survival of patient with glioma remains poor. In particular, patients with GBM have a median survival time of roughly 1 year [3]. Recent studies have indicated that age, Karnofsky performance status (KPS) score, WHO tumor grade, and tumor necrosis are important prognostic factors for gliomas [4]. Among them, the WHO tumor grade is the most wellestablished pathological prognostic factor in glioma. However, the prognosis of both high- and low-grade
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tumors remains heterogeneous. For example, Curran et al. [5] demonstrated that the median survival time of patients with high-grade gliomas ranges from 5 to 59 months and some patients with low-grade tumors also present poor outcome. Therefore, it is vital to identify new potential molecular biomarkers for diagnosis and prognosis of human gliomas. Grb2-associated binding (Gab) scaffolding/adapter proteins are a family of three highly conserved members including mammalian Gab1, Gab2, and Gab3, which are homologs of Drosophila Daughter of Sevenless (DOS) and Caenorhabditis elegans suppressor of clear (SOC-1) [6]. Both Gab1 and Gab2 are expressed ubiquitously, but they are most highly expressed in brain, kidney, lung, heart, testis, and ovary. Gab3 also has a widespread expression pattern, although it is most highly expressed in lymphoid tissue [7]. These proteins encompass an N-terminal PH domain, which mediates their interaction with specific membrane lipids and contain a large number of tyrosine residues and proline-rich regions that allow their interaction with signaling proteins encompassing SH2 and SH3 domains and consequently the formation of multimolecular signaling complexes [8]. Since their discovery, an increasing number of work have been done to better understand the functional roles of Gabs in multiple signaling pathways, typically acting as a downstream effectors of receptor tyrosine kinase (RTK)-triggered signal transduction [9]. Gabs have been demonstrated to participate in hematopoiesis, regulation of immune response, and cancer cell signaling [10]. Among them, Gab1 couples to a variety of receptor and non-receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR), c-Met and Src, and, depending on context, is capable of promoting cell growth, differentiation, and migration and is involved in control of cell apoptosis [11]. Accumulating studies have found that Gab1 upregulates proto-oncogene expression via participation in amplification of signaling pathways related to tumor biological behaviors and plays a role in the occurrence and development of tumors [12]. For example, Gab1 is a key binding partner of the Met receptor for induction of cell cycle progression, proliferation, and oncogenic morphological transformation, suggesting Gab1 and its associated signaling partners are potential therapeutic targets to impair proliferation or transformation of cancer cells in human malignancies harboring a deregulated Met receptor [13]; Gab1 mediates tumor progression in Met overexpressing colorectal cancer cells [14]; downregulation of Gab1 could inhibit cell proliferation and migration in hilar cholangiocarcinoma [15]; SHP-2/Gab1 association may be critical for linking EGFR to NF-kappaB transcriptional activity via the PI3 kinase/Akt signaling axis in GBM cells [16]. Regarding to Gab2 that lacks enzymatic activity, it acts downstream of receptor tyrosine
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kinases and non-receptor tyrosine kinases, such as cytokine and G protein-coupled receptors, to transmit and amplify signals to downstream effectors [17]. Gab2 is located on chromosomal band 11q14.1, amplification of which is frequently observed in human malignancies [18]. Gab2 has been identified as a potential oncogene in leukemia, melanoma, breast cancer, and ovarian cancer [19–22]. Although it appears to be insufficient to transform primary mammary epithelial cells, Gab2 has been indicated to cooperate with ErbB2 to potentiate tumorigenic signaling [23]. In addition to four kinds of human malignancies, Gab2 is overexpressed in malignant lung tissues compared with that in normal lung tissues suggesting that Gab2 expression may play a role in lung cancer development [24]. More interestingly, in 2013, Shi et al. [25] reported that the upregulation of Gab2 may be correlated with the WHO grade of gliomas and that patients with high Gab2 expression levels exhibited shorter survival time, suggesting that Gab2 may be a useful prognostic marker for gliomas and a novel therapeutic target for glioma invasion intervention. Both Gab1 and Gab2 have been demonstrated to be closely correlated with the tumorigenesis of human gliomas. However, the prognostic value of combined expression of Gab1 and Gab2 has not been explored. To address this problem, we examined the immunohistochemical expression of Gab1 and Gab2, and their association with clinicopathological characteristics and prognosis in glioma patients.
Materials and methods Patients and tissue samples This study was approved by the Research Ethics Committee of Tangdu Hospital, Fourth Military Medical University, P. R. China. Written informed consent was obtained from all of the patients. All specimens were handled and made anonymous according to the ethical and legal standards. A total of 128 formalin-fixed, paraffin-embedded specimens of gliomas resected between 2000 and 2010 were retrieved from the archives of the Pathology Department of Tangdu Hospital, Fourth Military Medical University, P. R. China. All the slides were re-evaluated according to WHO classifications [1] by two pathologists, with differences resolved by careful discussion. A total of 76 males and 52 females (1.46:1) were enrolled in this study, and the median age was 42 years (range 12–71). Thirty-two of the 128 gliomas were classified as low grade [18 pilocytic astrocytomas (WHO I) and 14 diffuse astrocytomas (WHO II)], and 96 were classified as high-grade gliomas [38 anaplasia
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Table 1 Clinicopathological features of 128 patients with gliomas Features
WHO I
WHO II
WHO III
WHO IV
Case no.
18
14
38
58
Mean age (year)
38.6
45.9
43.1
44.2
Male
12
6
25
33
Female
6
8
13
25
15 3
11 3
9 29
15 43
Gender
KPS [80 \80 Surgery Gross total resection
18
14
28
38
Partial resection
0
0
9
15
Biopsy
0
0
1
5
Adjuvant treatment Radiotherapy
0
0
30
12
Chemotherapy
0
1
0
6
Radiotherapy and chemotherapy combination
0
0
5
28
astrocytomas (WHO III) and 58 primary glioblastomas (WHO IV)]. None of the patients had received chemotherapy or radiotherapy prior to surgery. The clinicopathological features and the treatment strategies of all the patients are indicated in Table 1. Paraffin and snap-frozen sections of non-neoplastic brain tissues from ten patients with intractable epilepsy were also included as controls. Five-year follow-up was performed, and all patients had complete follow-up until death. Overall survival time was calculated from the date of the initial surgical operation to death. Patients, who died of diseases not directly related to their gliomas or due to unexpected events, were excluded from this study. Immunohistochemistry assay Formalin-fixed, paraffin-embedded, sectioned tissues (4 lm thick) were immunostained using the labeled Streptavidin Biotin 2 System (BioGenex; San Ramon, CA, USA). Following peroxidase blocking with 0.3 % H2O2/methanol for 30 min, specimens were blocked with phosphate-buffered saline (PBS) containing 5 % normal horse serum (Vector Laboratories Inc., Burlingame, CA, USA). All incubations with polyclonal rabbit antihuman Gab1 antibody (Santa Cruz Biotechnology, #sc-9049, 1:100 dilution) and polyclonal rabbit antihuman Gab2 antibody (Santa Cruz Biotechnology, #sc-25498, 1:100 dilution), respectively, were carried out overnight at 4 °C. Then, the specimens were briefly washed in PBS and incubated at room temperature with the antirabbit antibody and avidin–biotin peroxidase (Vector Laboratories Inc., Burlingame, CA, USA). The specimens were
then washed in PBS and color developed by diaminobenzidine solution (Dako Corporation, Carpinteria, CA, USA). After washing with water, specimens were counterstained with Meyer’s hematoxylin (Sigma Chemical Co., St Louis, MO, USA). Non-neoplastic brain tissues were used as control tissues, and non-immune IgG was also used as negative control antibody for immunohistochemical staining. Assessment of immunohistochemical staining was evaluated by two independent pathologists. The scores of the two pathologists were compared, and any discrepant scores were trained through re-examining the stainings by both pathologists to achieve a consensus score. The number of positive-staining cells showing immunoreactivity in the cytoplasm for Gab1 and Gab2 in ten representative microscopic fields was counted, and the percentage of positive cells was calculated. The percentage scoring of immunoreactive tumor cells was as follows: 0 (0 %), 1 (1–10 %), 2 (11–50 %) and 3 ([50 %). The staining intensity was visually scored and stratified as follows: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). A final immunoreactivity scores (IRS) was obtained for each case by multiplying the percentage and the intensity score. As there were no absolute criteria for the immunoreactivity of each antibody used in this study, we selected the median of IRS for Gab1 or Gab2 expression levels cutoff points, considering different expression on normal and tumor cells. This method considered the following values as criteria for the high expression group: Gab1 C 4.47 and Gab2 C 4.70. Statistical analysis All computations were carried out using the software of SPSS version 13.0 for Windows (SPSS Inc, IL, USA). Data were expressed as mean ± standard deviation (SD). The analysis of variance (ANOVA) was used to determine the statistical differences among the groups. The correlation between Gab1 and Gab2 protein expression was calculated using Spearman’s test. A life table was calculated according to the Kaplan–Meier method. Hazard ratios for the time-to-event end point were estimated using the multivariate Cox regression analysis in a forward stepwise method to evaluate the effect of multiple independent prognostic factors on survival outcome. Differences were considered statistically significant when P was \0.05.
Results Gab1 and Gab2 proteins are upregulated in human glioma tissues versus non-neoplastic brain tissues We observed immunoreactivities of Gab1 and Gab2 proteins in the cytoplasm (Fig. 1), and distinctly low levels of
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Fig. 1 Representative sections for Gab1 and Gab2 immunoreactivities in glioma tissues (a, b, respectively) and non-neoplastic brain tissues (c, d, respectively; 9400). The immunoreactivities of Gab1 and Gab2 proteins were shown in the cytoplasm, and distinctly low levels of their expression were observed in non-neoplastic brain tissues compared with glioma tissues. The red arrows refer to the positive area of immunostaining. Statistical analysis showed that both the expression levels of Gab1 and Gab2 proteins in glioma tissues were significantly higher than those in corresponding nonneoplastic brain tissues (both P \ 0.001, e, f, respectively)
proteins in glioma tissues were significantly higher than those in non-neoplastic brain tissues (both P \ 0.001). Of the 128 glioma tissues investigated, 68 cases (53.13 %) were regarded as Gab1-high expression group and 66 cases (51.56 %) as Gab2-high expression group. The Spearman correlation analysis revealed that a corresponding correlation between Gab1 overexpression and Gab2 overexpression was evident in gliomas (r = 0.514; P \ 0.001, Fig. 2). Upregulations of Gab1 and Gab2 proteins associate with tumor progression of human gliomas Fig. 2 Scatter diagram of the Spearman correlation analysis between Gab1 and Gab2 protein expression levels in glioma tissues
their expression were observed in non-neoplastic brain tissues compared with glioma tissues. Statistical analysis showed that both the expression levels of Gab1 and Gab2
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Table 2 summarized the associations between upregulations of Gab1 or Gab2 proteins with clinicopathological characteristics of human gliomas. We found that the upregulations of Gab1 and Gab2 proteins were both significantly associated with advanced WHO grades (both
Med Oncol (2014) 31:77 Table 2 Associations of Gab1 or Gab2 expression in human glioma tissues with different clinicopathological features
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Clinicopathological features
No. of cases
Gab1 expression High (n, %)
P
Low (n, %)
Gab2 expression High (n, %)
P
Low (n, %)
WHO grade I
18
2 (11.11)
16 (88.89)
II III
14
4 (28.57)
38
20 (52.63)
IV
58 52 76
\0.001
\0.001
2 (11.11)
16 (88.89)
10 (71.43)
4 (28.57)
10 (71.43)
18 (47.37)
20 (52.63)
18 (47.37)
42 (72.41)
16 (27.59)
40 (68.97)
18 (31.03)
25 (48.08) 43 (56.58)
27 (51.92) 33 (43.42)
NS
25 (48.08) 41 (53.95)
27 (51.92) 35 (46.05)
NS
NS
40 (52.63)
36 (47.37)
NS
26 (50.00)
26 (50.00)
Age \55 C55 Gender Male
76
42 (55.26)
34 (44.74)
Female
52
26 (50.00)
26 (50.00)
KPS \80
78
50 (64.10)
28 (35.90)
C80
50
18 (36.00)
32 (64.00)
P \ 0.001, Table 2) and low KPS (both P = 0.01, Table 2). No statistically significant association of Gab1 or Gab2 with age at diagnosis and gender of patients was found (both P [ 0.05, Table 2). Association of Gab1 and Gab2 protein expression with overall survival in patients with gliomas During the follow-up period, 100 of the 128 glioma patients (78.1 %) had died after the surgery. The Kaplan– Meier plot showed that patients showing Gab1 overexpression had a poorer overall survival than patients with Gab1-low tumors (log rank, P = 0.01; Fig. 3a). On the other hand, the aberrant expression of Gab2 also was significantly associated with overall survival (P = 0.006; Fig. 3b). With regard to combined expression of Gab1 and Gab2 proteins, we divided the specimens into three groups: group 1, tumors exhibiting high expression of Gab1 and Gab2 (Gab1-high/Gab2-high, 46 specimens); group 2, tumors with different expression of Gab1 and Gab2 (Gab1high/Gab2-low or Gab1-low/Gab2-high, 42 specimens); and group 3, tumors with low expression of both proteins (Gab1-low/Gab2-low, 40 specimens). Notably, there was a trend toward the poorest overall survival in the patient group with high expression of Gab1 and Gab2 (P \ 0.001, Fig. 3c). Interestingly, the overall survival curve for patients in group 2 (Gab1-high/Gab2-low, or Gab1-low/ Gab2-high) was relatively close to those with tumors that were low expression for both Gab1 and Gab2, but were dramatically discrepant from those of the Gab1-high/Gab2high group (Fig. 3c). In multivariate analysis, Cox proportional hazards model involving the expression levels of Gab1 and Gab2
0.01
50 (64.10)
28 (35.90)
16 (32.00)
34 (68.00)
0.01
proteins, and various clinical parameters showed that high Gab1 protein expression (P = 0.01), high Gab2 protein expression (P = 0.02), and combined expression of Gab1 and Gab2 (P = 0.006) were all independent prognostic factors for overall survival in glioma patients. Statistical values of the expression levels of Gab1 and Gab2 proteins and other clinical parameters derived from Cox stepwise proportional hazards model are indicated in Table 3.
Discussion Treatment of human glioma has been increasingly informed by biomarkers that predict patient prognosis and response to therapy. Although a number of candidate diagnostic and prognostic biomarkers have already been identified for human glioma, previously, clinical trials found that the vast majority of them might be poorly suited to the task of identifying treatments, but effective only in selected subsets of patients. Therefore, it is still a significant challenge to identify novel and efficient biomarkers for gliomas with good clinical application. In the present study, our data showed that the abnormal expressions of Gab1 and Gab2 proteins appeared to be correlated with the WHO grade of glioma, clinicopathological features, as well as patient survivals. These strong associations suggest that Gab1 and Gab2 overexpression promotes tumor progression and that Gab1 and/or Gab2 could possibly be used as a biomarker for a more aggressive phenotype of gliomas. To the best of our knowledge, this is the first study to demonstrate the prognostic value of the co-expression of Gab1 and Gab2 in gliomas. Gab1, the most common Gabs, was originally isolated as a Grb2-binding protein from a human glial tumor
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Fig. 3 Kaplan–Meier survival curves of glioma patients. a The 5-year overall survival of glioma patients with high Gab1 protein expression was significantly lower than that of glioma patients with low Gab1 protein expression (P \ 0.001). b The 5-year overall survival of glioma patients with high Gab2 protein expression was
significantly lower than that of glioma patients with low Gab2 protein expression (P \ 0.001). c The 5-year overall survival of glioma patients with Bmi-high/Gab2-high expression was significantly lowest of all three groups (P \ 0.001)
Table 3 Cox multivariate analysis
the EGFR and Met [13]. It is also a potential player in the nuclear localization of activated MAPK and is capable of binding to MAPK and aiding in nuclear translocation through a chaperone-like function [28]. However, there have been no reports describing the relationship between Gab1 and clinicopathological characteristics of human gliomas to date. In the present study, the upregulation of Gab1 expression was detected in 53.13 % (68/128) of glioma tissues. In contrast, its immunostaining was not or weakly detected in control brain tissues. Of four WHO grades, grade IV exhibited the highest Gab1 level. On the other hand, Gab1 expression was significantly associated with WHO grade and KPS score, and patients with high WHO grade expression had a significantly shorter overall survival than those with low Gab1 expression. According to Cox regression, Gab1 can be suggested as an independent prognostic factor for human glioma. As another Gabs, Gab2 recruits the tyrosine phosphatase Shp2 and the p85 subunit of PI3K and thereby positively regulates the Ras/Erk and PI3K pathways in response to activation of a variety of receptor and receptor-associated tyrosine kinases [17]. An increasing number of evidence has emerged supporting a functional role for Gab2 in the development of several human malignancies. Aberrant expression of Gab2 has been observed in various human cancer tissues, such as glioma, melanoma, breast cancer, lung cancer, gastric cancer, and ovarian cancer [19–22]. Gab2(-/-) breast cancer cells exhibited decreased migration and impaired Erk activation, suggesting its role in promoting mammary tumor metastasis [19]; Gab2 expression in lung tumor samples was significantly higher than that in normal lung samples, and Gab2 may be as a molecular marker for neoplastic progression [20];
Parameter
Risk ratio
95 % confidence interval
P
Age
0.89
0.58–1.65
0.71
Gender
1.02
0.66–1.83
0.33
WHO grade
4.88
1.96–10.16
0.01
KPS
1.99
1.28–2.95
0.06
Extent of resection
1.29
0.89–2.13
0.11
Type of adjuvant treatment
1.37
1.02–2.24
0.11
Gab1 expression
4.23
2.01–9.06
0.01
Gab2 expression
3.78
1.96–7.66
0.02
Combined Gab1/Gab2 expression
6.88
2.39–15.29
0.006
expression library and was also identified independently as a Met-receptor interacting protein in a yeast two-hybrid screen and as the major tyrosine phosphorylated protein in cells transformed by the Tpr-Met oncogene [26]. It is located in the cytoplasm and recruited to the cell membrane after being activated to bind with several activated signal receptors, resulting in its own activation [27]. Gab1, with wide distribution in various body tissues of mammals, is capable of promoting cell growth, proliferation, differentiation, adhesion, migration, and invasion and is implicated in control of cell apoptosis [11]. Accumulating studies have found that Gab1 may upregulate proto-oncogene expression via participation in amplification of signaling pathways related to tumor biological behaviors. It plays a crucial role in the occurrence and development of tumors. For example, Gab1 has been shown to enhance cell growth and transformation of NIH 3T3 cells downstream of
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increased expression of Gab2 in malignant gastric cells compared with normal epithelial cells, implying its possible roles in gastric cancer development [21]; Gab2-mediated signaling has been implicated in chronic myelogenous leukemia and lymphoid leukemia; The Grb2–Gab2 pathway is critical for leukemic transformation by Bcr–Abl [22]. In addition to these cancer types, Gab2 has been found to be high expressed in gliomas and a subset of cancer cell lines. The authors also indicated that the upregulation of Gab2 was correlated with the WHO grade of gliomas and that patients with high Gab2 expression levels exhibited shorter survival time. In an animal experiment and a cell research, they demonstrated that knockdown of Gab2 through siRNA could inhibit the migration and invasion of glioma cells by mediating cytoskeleton rearrangement and MMPs expression. Moreover, IGF-1-induced pAkt and pmTOR phosphorylation was suppressed by the knockdown of Gab2 [23]. In line with these previous findings, our data here validated again the clinical value of Gab2 overexpression in tumorigenesis, tumor progression, and patient prognosis of gliomas using an independently large cohort. Since accumulating studies have indicated that the efficiency of combined biomarkers may be more stronger than single biomarker in the diagnosis and prognosis of various human cancers, we hypothesized that the prognostic value of the combination of Gab1 and Gab2 (Gab1/ Gab2) might be better than Gab1 or Gab2 alone. To validate this hypothesis, we analyzed the correlations of Gab1/ Gab2 combined expression, Gab1 expression, and Gab2 expression with overall survival of patients with gliomas. Our data showed that Gab1/Gab2 combined expression, Gab1 expression, and Gab2 expression were all independent prognostic factors for overall survival of patients with gliomas. More interestingly, Gab1/Gab2 combined expression could more efficiently predict the prognosis in glioma patients, suggesting that the detection of coexpression of Gab1 and Gab2 could be used to design optimal, individualized treatment and to help identify patients who may or may not benefit from close monitoring after surgery. Additional research would be required to confirm our findings. In conclusion, Gab1 and Gab2 proteins are differentially expressed in glioma patients and closely correlated with the biological behavior of this malignancy. Combination of Gab1 and Gab2 expression may represent a promising biomarker for prognostication of human gliomas. Acknowledgments This work was funded by National Natural Science Foundation of China (No. 81272419, 81101736, 81272776) and Natural Science Foundation of Shannxi Province (No. 2014JM4148). Conflict of interest
None.
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