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Grb7 is over-expressed in cervical cancer and facilitate invasion and inhibit apoptosis in cervical cancer cells
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Original article
Grb7 is over-expressed in cervical cancer and facilitate invasion and inhibit apoptosis in cervical cancer cells Hong-Bing Zhao a,b,∗ , Xi-Feng Zhang b , Xue-Lin Jia a , Hao-Bin Wang a a b
Department of Oncology, The First Clinical Hospital of Zhengzhou University, Zhengzhou, Henan, 450052, PR China Department of Pathology, Shangqiu Medical College, Shangqiu, Henan, 476100, PR China
a r t i c l e
i n f o
Article history: Received 11 December 2016 Received in revised form 21 April 2017 Accepted 25 May 2017 Keywords: Grb7 Cervical cancer Overall survival Migration MMP-9 Bax Bcl-2
a b s t r a c t Growth factor receptor bound protein-7 (Grb7) is a multi-domain adaptor protein that is co-opted by numerous tyrosine kinases involved in various cellular signaling. The objective of this study was to investigate the expression of Grb7 and its clinicopathological significance in cervical cancer. Utilising immunohistochemical staining, we examined the expression of Grb7 in 120 cases of human cervical cancer tissue and 10 cases of adjacent non-cancerous cervical tissue. The positive rate of Grb7 protein expression was 34.2%, which was significantly higher than that in adjacent non-cancerous cervical tissue (0%, p < 0.05). The expression of Grb7 was found to be correlated with age, tumor size, serosal invasion, differentiated degree, tumor stage, early or advanced stage and lymph node metastasis. Kaplan-Meier survival analysis showed that patients with positive Grb7 protein expression had a lower overall survival rate than patients without Grb7 expression. In addition, Grb7 plays an important role in promoting tumor progression, including invasion and anti-apoptosis, in cervical cancer cell line. Down-regulation of Grb7 repressed the expression of MMP-9 and Bcl-2, and increased the expression of Bax in Grb7 knockdown Hela cells. Cell invasion assay showed decreased number of Grb7 knockdown Hela cells (18.7 ± 2.1) compared to Hela cells (65.3 ± 2.5, P < 0.05). Our results indicated that Grb7 over-expression may facilitate invasion and inhibit apoptosis in cervical cancer and Grb7 is a potentially molecular target of cervical cancer chemotherapy. © 2017 Elsevier GmbH. All rights reserved.
1. Introduction Cervical cancer is the third most common malignancy in women worldwide. 12,990 diagnoses of cervical cancer will be made by the end of 2016 in the United States, over 4000 women die from cervical cancer each year. But 83% of cases occur in developing countries and recurrent or metastatic tumors are the main cause of treatment failures. Growth factor receptor bound protein-7 (Grb7) is a multi-domain adaptor protein that is co-opted by numerous tyrosine kinases involved in various cellular signaling and functions [1], and is found to be over expressed in such metastatic tumors as liver cancer [2], esophageal cancer [3], breast cancer [4–7], pancreatic cancer [8], ovarian cancer [9,10] and chronic lymphocytic leukemia [11]. Grb7 contains a src-homology 2(SH2) domain, which is reported to be mainly involved in Grb7 signal pathways. SH2 domains recruit signaling proteins to phosphotyrosine residues on
aberrantly activated growth factor and cytokine receptors and participate in cancer cell migration, metastasis and angiogenesis. Grb7 is a prime target for the development of novel anti-cancer therapies. Grb7-specific inhibitors are in early development. Recently, Study suggested that G7-18NATE(a nonphosphorylated cyclic peptide 1) inhibits Grb7 via targeting the Grb7-SH2 domain in breast cancer [4]. But the molecular mechanism by which Grb7 involved in invasion and anti-apoptosis in cervical cancer remain unclear yet. In this study, we used immunohistochemical staining, cell invasion assay and shRNA(short hairpin RNA) targeting Grb7 to confirm the correlation between the expression of Grb7 protein and clinicopathological characteristics in cervical cancer and to elucidate the underlying mechanisms by which Grb7 boosted invasion and anti-apoptosis of cervical cancer cells. 2. Materials and methods 2.1. Patients and tissue specimen
∗ Corresponding author at: Department of Oncology, The First Clinical Hospital of Zhengzhou University, Zhengzhou, Henan, 450052, PR China. E-mail address: [email protected] (H.-B. Zhao).
120 cases of cervical cancer and 10 samples of tumor-adjacent tissue were collected from First Clinical Hospital of Zhengzhou Uni-
http://dx.doi.org/10.1016/j.prp.2017.05.013 0344-0338/© 2017 Elsevier GmbH. All rights reserved.
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versity from January 2010 to July 2015. All the patients were aged 33 to71 years old with a median age of 44 years old. No patients were undergoing chemotherapy, radiotherapy, or immunotherapy before radical surgery. All the tissue specimens were confirmed independently by two qualified pathologists without knowing the characteristics of patients.
2.2. Immunohistochemistry assay Grb7 polyclonal antibody and DAB staining kit were purchased from Santa Cruz Biotechnology, Inc. (Texas, U.S.A.). All samples were fixed with 4% formaldehyde, embedded with paraffin, and subjected to serial sectioning for histopathological diagnosis and immunohistochemistry examination. The MaxVision twostep immunohistochemical detection method was used to detect Grb7 expression. Consecutive 3 m sections were deparaffinized, hydrated, and rinsed in distilled H2 O. The samples were boiled in citrate buffer (pH 6.0) in a high pressure cooker for 8 min and cooled to room temperature, and washed 4 times with phosphate-buffered saline (PBS; pH 7.4) for 5 min. All of the samples were incubated with rabbit anti-Grb7 polyclonal antibody (1:50 diluted) overnight at 4 ◦ C. The next day, the samples were washed with PBS 4 times for 5 min, and incubated with anti-rabbit IgG antibody for 1 h. Afterwards, the samples were washed with PBS 3 times for 5 min. DAB chromogenic reagent was dropped on samples and staining was observed under a light microscope. After staining, the samples were rinsed in distilled water to stop the reaction. PBS instead of the primary antibody was used as a negative control and known sample was used as positive controls. We used the following scoring method to evaluate the Grb7 expression. Each sample were graded based on the average percentage of positive cells and the staining intensity in five areas (per × 100 field) at random. The intensity (S) was graded as follows: 0 (no staining); +1 (cells stained yellow); +2 (cells stained orange) and +3 (cells stained brown). According to the mean percentage (P) of positive cells, score of percentage were classified as: 0, <5% staining; 1, 6%–25%; 2, 26%–50%; 3, <51%–75% and 4, >75%. The final H-score were obtained by using the arithmetic: HSCORE = (S × P). The classification was as follow: 0–1 point were considered negative, scores of 2–4 points were considered weakly positive, scores of 5–8 points were considered moderately positive, and scores of 9–12 points were considered strongly positive.
2.5. Quantitative RT-PCR Total RNAs were respectively extracted from cells using Trizol reagent (Toyobo Co., Ltd. Shanghai, China). The ratio of OD260 to OD280 was over 1.7. Subsequently they were reverse transcribed into cDNAs using PrimeScriptTM RT Reagent Kit(Takara Bio Inc. Osaka, Japan). Quantitative RT-PCR was performed with SYBR Green master mix real-time core reagents(Toyobo Co., Ltd. Shanghai, China) on an ABI 7500 (Applied Biosystems) according to the manufacturer’s instructions.All primers and probe combinations were designed using Primer Premier 6.0 (Premier Biosoft, Canada) and purchased from Sangon (Sangon Biotech Co., Ltd. Shanghai, China). Primers for quantitative RT-PCR were as follows: Grb7-F:5 -CTTCGCCAAGTATGAACTATT-3 Grb7-R:5 -GTTGGGCTTGACACAGAAGCC-3 Bcl-2-F: 5 -CATGCGGCCTCTGTTTGATT-3 Bcl-2-R: 5 -TGCATATTTGTTTGGGGCAGG-3 Bax-F: 5 -CGTGTCTGATCAATCCCCGA-3 Bax-R: 5 -GGGCAGAAGGCACTAATCAAG-3 MMP-9-F:5 -GACTCCGTCTTTGAGGAGCC-3 MMP-9-R:5 -GAACTCACGCGCCAGTAGAA-3 -actin-F: 5 -AGAAAATCTGGCACCACACC-3 -actin-R: 5 -TAGCACAGCCTGGATAGCAA-3 All reactions were performed in triplicate, and the expression of mRNAs was normalized to that of the reference gene -actin. Relative quantification of mRNA within the samples was caculated using the 2–Ct method. 2.6. Western blot The concentration of total protein extracted from Hela cells and Grb7 knockdown Hela cells was determined with a BCA Protein Assay Kit (Pierce, USA). Equal amounts of protein were separated by 10% SDS-PAGE and electrophoretically transferred to PVDF membranes (Pierce, USA) using a mini trans-blot (Bio-Rad laboratories, Hercules, CA, USA). Rabbit anti-human Grb7, Bax, Bcl-2, MMP-9 and -actin were purchased from Santa Cruz Biotechnology, Inc. (Texas, U.S.A.). -actin was used as an internal control. Electrochemiluminescence was performed according to the manufacturer’s instructions and read with a Chemi lmager 5500 imaging system (Alpha Innotech Co,San Leandro, CA, USA). 2.7. Cell invasion assay
2.3. Cell culture Hela cell line was preserved in our laboratory. Cells were cultured in RPMI1640 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin(Sigma-Aldrich, St. Louis, MO, USA), 0.5 mg/mL hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA), 100 ng/mL cholera toxin (Sigma-Aldrich, St. Louis, MO, USA), and 10 g/mL insulin (Sigma-Aldrich, St. Louis, MO, USA), and 20 ng/mL recombinant human EGF (Peprotech, 100-15) in a 5% CO2 incubator at 37 ◦ C. Media was changed every 3 days and cells were passaged when 65–80% confluent.
Quantification of cell invasion was performed by Cell Invasion Assay Kits(BD Science, Sparks, MD, USA.) according to the manufacturer’s instructions. 1 × 105 cells were resuspended in serum-free culture medium and seeded on the upper chamber. After incubation at 37 ◦ C in 5% CO2 for 24 h, cells still on the upper surface of the chamber were removed, and the cells that invaded through the membranes were fixed by methanol and stained with 0.1% crystal violet to assess the cell number. Quantification of invaded cells was performed according to published criteria [12]. The experiments were repeated thrice. 2.8. Statistical analysis
2.4. In vitro transfection Grb7 shRNA Plasmid was designed against Grb7 target. Grb7 shRNA Plasmid (h) and Support Reagents were purchased from Santa Cruz Biotechnology, Inc. (Texas, U.S.A.) In vitro transfection was performed according to the manufacture protocol. The transfection rates were measured using fluorescence microscopy.
Data analysis was performed using the Statistical Package for the Social Sciences (SPSS), version 17.0 (USA). Data analysis of quantitative RT-PCR results was performed using a Mann-Whitney U test. A possible association between positive expression of Grb7 and clinicopathological parameters was compared using a 2 test. Differences with a p less than 0.05 were considered to be statistically significant.
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Fig. 1. Immunohistologic staining of various cervical cancer was carried out as described in Materials and methods. A, Well differentiated cervical cancer; Grb7 expression is seen primarily in cytoplasm. B, Poorly differentiated cervical cancer.
3. Results 3.1. Grb7 expression pattern in cervical cancer To determine the expression of Grb7 in cervical cancer and adjacent normal tissues, expression of Grb7 was detected using immunohistochemistry. The Grb7 protein in cervical cancer tissue were usually seen in cytoplasm(Fig. 1A and B.). 41 out of 120 (34.2%) cases were scored positive for Grb7 staining. In the adjacent normal cervical tissue, none was stained for Grb7 expression. The difference of expression between cervical cancer tissues and adjacent normal tissues was significant (p < 0.05). The result demonstrated that Grb7 is over-expressed in cervical cancer. 3.2. Correlation between the expression of Grb7 protein and clinicopathological characteristics The expression of Grb7 protein was examined in the 120 cervical cancer patients based on patients’ clinnicopatholigical characteristics. There was significant difference between the Grb7 expression and age, tumor size, serosal invasion, differentiated degree, tumor stage, early or advanced stage and lymph node metastasis (p < 0.05) (Table 1).15 (57.7%) of 26 cervical cancer specimens with positive serosal invasion expressed Grb7, 26 (27.7%) of 94 cases with negative serosal invasion expressed Grb7, this was statistically significant(p < 0.05). There was significant difference in Grb7 positivity between early and advanced cervical cancer(p < 0.05). Statistical differences in Grb7 expression reached significance between stage and stages II or III carcinomas(p < 0.05). 3.3. Cumulative survival rates The average follow-up time was 53.4 months, ranging from 2 to 60 months, the follow-up rate was 100%. Twenty patients died of recurrent carcinoma by the end of July 2015. Cumulative survival was analyzed among the patients with cervical cancer by Log rank test. Survival rates of patients who had Grb7 positive tumor was significantly lower than those with Grb7 negative tumors(p = 0.000) (Fig. 2.). 3.4. Cervical cancer cells over-expressed Grb7 and knockdown of Grb7 decreased Grb7 expression These above results promoted us to investigate whether Grb7 expression level has relationship with tumor malignancy. To test this possibility, we tested Grb7 mRNA expression in cervical cancer cells and Grb7 knockdown cervical cancer cells. Compared to
the Grb7 knockdown Hela cells, qRT–PCR analysis showed that the mRNA expression Grb7 was high in the Hela cells, and had significant difference(p < 0.05) (Fig. 3A.). This result suggests that over-expressed Grb7 in cervical cancer cells may be involved in tumorigenesis. 3.5. Cell invasion assay We further used cell invasion assay to examine the effect on Hela cell and Grb7 knockdown Hela cell invasion. Result showed decreased number of Grb7 knockdown Hela cells (18.7 ± 2.1) compared to Hela cells (65.3 ± 2.5, P = 0.000 < 0.05). This result indicated the participation of Grb7 protein in the invasion of Hela cells, by up-regulating Grb7 expression. Next, to study the role of MMP9 expression in cervical cancer, we then investigated the expression of MMP9 in Hela cells and Grb7 knockdown Hela cells. Compared to the Hela cells, qRT–PCR analysis showed that the mRNA expression of MMP9 was lower in the Grb7 knockdown Hela cells, and had significant difference(p < 0.05, Fig. 3D). Western blots analysis confirmed the expression of MMP-9 (Fig. 4.). These data further support the notion that Grb7 up-regulates MMP9 in cervical cancer cells. 3.6. Grb7 inhibits apoptosis through the mitochondria-associated apoptotic pathway As the mitochondria-associated apoptotic pathway participates in cellular response in tumors, we performed an analysis of proapoptotic protein (Bax) and antiapoptotic protein (Bcl-2) in the Hela cells and the Grb7 knockdown Hela cells (Fig. 4). Compared with HeLa cells, qRT–PCR analysis showed that Bcl-2 level decreased, and Bax level markedly increased in the Grb7 knockdown Hela cells, and had significant difference(p < 0.05, p < 0.05, Fig. 3B and C). Western blots analysis confirmed the expression of Bcl-2 and Bax (Fig. 4). Thus, these results suggest that Grb7 inhibits apoptosis through the mitochondria-associated apoptotic pathway in cervical cancer cells. 3.7. Discussions In esophageal squamous cell carcinoma(ESCC), Grb7 was a driver gene by Analysis of survival data and RNAi screening data, and knockdown of Grb7 reduced the proliferation, migration, and invasion capacities of cells in ESCC cell lines [13]. Immunohistochemical analysis showed that Grb7 was frequently increased and associated with high-grade tumors, as well as a high tendency in association with advanced stage ovarian cancer [10]. Grb7 is a ther-
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Table 1 Relationship between Grb7 expression and Clinicopathologic characteristics in 120 patients with cervical cancer. clinicopathologic Characteristics
All patients
Grb7 (N)
p
N
%
Positive
Negative
age
>50 ≤50
64 56
53.3 46.6
24 17
40 39
0.005
Tumor size,cm
≤2 >3
66 54
55 45
13 28
53 26
0.000
Serisal invasion
Negative Positive
94 26
78.3 21.7
26 15
68 11
0.000
differentiated degree
high moderately Poorly
20 54 46
16.7 45 38.3
12 19 10
8 34 36
0.000
Lymph node metastasis
yes no
63 57
52.5 47.5
10 31
53 26
0.000
stage
I II III IV
54 18 45 3
45 15 37.5 2.5
5 10 25 1
49 8 20 2
0.000
Early or advanced
early advanced
31 89
25.8 74.2
2 39
29 50
0.000
Fig. 2. Survival of patients with cervical cancer. Cumulative survival rates of 120 patients with Grb7-positive or negative cervical cancer.
Fig. 3. Grb7, Bcl-2, Bax and MMP-9 mRNA expression in cervical cancer cells and Grb7 knockdown cervical cancer cells.
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First, the level of Grb7 mRNA was significantly higher in Hela cells than in Grb7 knockdown Hela cells. Second, Grb7 may promote transcription of the metastatic gene MMP9, qRT–PCR and western blots analysis confirmed the expression of MMP-9. Cell invasion assay showed decreased number of Grb7 knockdown Hela cells (18.7 ± 2.1) compared to Hela cells (65.3 ± 2.5, P < 0.05). Third, Grb7 inhibits apoptosis through the mitochondria-associated apoptotic pathway. Grb7 represses the level of Bax and increases the level of Bcl-2, qRT–PCR and western blots analysis confirmed the expression of Bax and Bcl-2. So, Grb7 over-expression may facilitate invasion and inhibit apoptosis in cervical cancer and Grb7 is a potentially molecular target of cervical cancer chemotherapy. Declaration of conflict of interest None. References
Fig. 4. Grb7, Bcl-2, Bax and MMP-9 protein expression in cervical cancer cells and Grb7 knockdown cervical cancer cells.
apeutic target in human breast cancer patients. It exacerbated the cellular growth and migratory behaviour of HER2 + ve breast cancer cells [7], and was significantly related to the presence of lymph node metastasis, stage and histological grade of the malignant tumors [14].Co-silencing of STARD3, Grb7, PSMD3 and PERLD1 together with HER2 led to an additive inhibition of cell viability as well as induced apoptosis [15]. Numerous variables have been reported to correlate with the prognosis of patients with cervical cancer. Independent prognostic factors that have been reported include tumor size, depth of invasion, stage, nodal status, differentiated degree and Grb7 expression. These finding showed that Grb7 levels reflected the severity of tumor, and may be used as a prognostic marker and therapeutic target. Our results revealed that Grb7 was over-expressed in cervical cancer, there was significant difference between the Grb7 expression and age, tumor size, serosal invasion, differentiated degree, tumor stage, early or advanced stage and lymph node metastasis. Enhanced Grb7 expression was a statistically significant factor in predicting the survival of patients. Prognosis of patients with cervical cancer may therefore be predicted more accurately by immunohistochemical Grb7 staining. Grb7 plays an important role in promoting tumor progression, including invasion and anti-apoptosis, in cervical cancer cell line.
[1] Y.L. Tai, L.H. Tung, Y.C. Lin, P.J. Lu, P.Y. Chu, M.Y. Wang, W.P. Huang, K.C. Chen, H. Lee, T.L. Shen, Grb7 protein stability modulated by pin1 in association with cell cycle progression, PLoS One 9 (2016). [2] S. Itoh, A. Taketomi, S. Tanaka, N. Harimoto, Y. Yamashita, S. Aishima, T. Maeda, K. Shirabe, M. Shimada, Y. Maehara, Role of growth factor receptor bound protein 7 in hepatocellular carcinoma, Mol. Cancer Res. 7 (2007) 667–673. [3] S. Tanaka, K. Sugimachi, H. Kawaguchi, H. Saeki, S. Ohno, J.R. Wands, Grb7 signal transduction protein mediates metastatic progression of esophageal carcinoma, J. Cell. Physiol. 3 (2000) 411–415. [4] G.M. Watson, M.J. Gunzburg, N.D. Ambaye, W.A. Lucas, D.A. Traore, K. Kulkarni, K.M. Cergol, R.J. Payne, S. Panjikar, S.C. Pero, P. Perlmutter, M.C. Wilce, J.A. Wilce, Cyclic peptides incorporating phosphotyrosine mimetics as potent and specific inhibitors of the Grb7 Breast cancer target, J. Med. Chem. 19 (2015) 7707–7718. [5] P. Morlacchi, F.M. Robertson, J. Klostergaard, J.S. McMurray, Targeting SH2 domains in breast cancer, Future Med. Chem. 17 (2014) 1909–1926. [6] D. Pradip, M. Bouzyk, N. Dey, B. Leyland-Jones, Dissecting GRB7-mediated signals for proliferation and migration in HER2 overexpressing breast tumor cells: GTP-ase rules, Am. J. Cancer. Res. 2 (2013) 173–195. [7] R.C. Lim, J.T. Price, J.A. Wilce, Context-dependent role of Grb7 in HER2 + ve and triple-negative breast cancer cell lines, Breast Cancer Res. Treat. 3 (2014) 593–603. [8] S. Tanaka, S.C. Pero, K. Taguchi, M. Shimada, M. Mori, D.N. Krag, S. Arii, Specific peptide ligand for Grb7 signal transduction protein and pancreatic cancer metastasis, J. Natl. Cancer Inst. 7 (2006) 491–498. [9] M. Zeng, Z. Yang, X. Hu, Y. Liu, X. Yang, H. Ran, Y. Li, X. Li, Q. Yu, Grb7 gene amplification and protein expression by FISH and IHC in ovarian cancer, Int. J. Clin. Exp. Pathol. 9 (2015) 11296–11304. [10] D.W. Chan, W.W. Hui, P.C. Cai, M.X. Liu, M.M. Yung, C.S. Mak, T.H. Leung, K.K. Chan, H.Y. Ngan, Targeting GRB7/ERK/FOXM1 signaling pathway impairs aggressiveness of ovarian cancer cells, PLoS One 12 (2012). [11] M. Haran, S. Chebatco, L. Flaishon, F. Lantner, N. Harpaz, L. Valinsky, A. Berrebi, I. Shachar, Grb7 expression and cellular migration in chronic lymphocytic leukemia: a comparative study of early and advanced stage disease, Leukemia 12 (2004) 1948–1950. [12] L. Zheng, T. Qi, D. Yang, M. Qi, D. Li, X. Xiang, K. Huang, Q. Tong, microRNA-9 suppresses the proliferation, invasion and metastasis of gastric cancer cells through targeting cyclin D1 and Ets1, PLoS One 1 (2013). [13] G. Sawada, A. Niida, H. Hirata, H. Komatsu, R. Uchi, T. Shimamura, Y. Takahashi, J. Kurashige, T. Matsumura, H. Ueo, Y. Takano, M. Ueda, S. Sakimura, Y. Shinden, H. Eguchi, T. Sudo, K. Sugimachi, M. Yamasaki, F. Tanaka, Y. Tachimori, Y. Kajiyama, S. Natsugoe, H. Fujita, Y. Tanaka, G. Calin, S. Miyano, Y. Doki, M. Mori, K. Mimori, An integrative analysis to identify driver genes in esophageal squamous cell carcinoma, PLoS One 10 (10) (2015) e0139808. [14] A.S. Darweesh, M.L. Louka, M. Hana, S. Rashad, M. El-Shinawi, A. Sharaf-Eldin, S.K. Kassim, Validation of analytical breast cancer microarray analysis in medical laboratory, Med. Oncol. 31 (10) (2014) 201. [15] K.K. Sahlberg, V. Hongisto, H. Edgren, R. Mäkelä, K. Hellström, E.U. Due, H.K. Moen Vollan, N. Sahlberg, M. Wolf, A.L. Børresen-Dale, M. Perälä, O. Kallioniemi, The HER2 amplicon includes several genes required for the growth and survival of HER2 positive breast cancer cells, Mol. Oncol. 7 (3) (2013) 392–401.
Please cite this article in press as: H.-B. Zhao, et al., Grb7 is over-expressed in cervical cancer and facilitate invasion and inhibit apoptosis in cervical cancer cells, Pathol. – Res. Pract (2017), http://dx.doi.org/10.1016/j.prp.2017.05.013
ARTICLE IN PRESS Pathology – Research and Practice xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Pathology – Research and Practice journal homepage: www.elsevier.com/locate/prp
Original article
Grb7 is over-expressed in cervical cancer and facilitate invasion and inhibit apoptosis in cervical cancer cells Hong-Bing Zhao a,b,∗ , Xi-Feng Zhang b , Xue-Lin Jia a , Hao-Bin Wang a a b
Department of Oncology, The First Clinical Hospital of Zhengzhou University, Zhengzhou, Henan, 450052, PR China Department of Pathology, Shangqiu Medical College, Shangqiu, Henan, 476100, PR China
a r t i c l e
i n f o
Article history: Received 11 December 2016 Received in revised form 21 April 2017 Accepted 25 May 2017 Keywords: Grb7 Cervical cancer Overall survival Migration MMP-9 Bax Bcl-2
a b s t r a c t Growth factor receptor bound protein-7 (Grb7) is a multi-domain adaptor protein that is co-opted by numerous tyrosine kinases involved in various cellular signaling. The objective of this study was to investigate the expression of Grb7 and its clinicopathological significance in cervical cancer. Utilising immunohistochemical staining, we examined the expression of Grb7 in 120 cases of human cervical cancer tissue and 10 cases of adjacent non-cancerous cervical tissue. The positive rate of Grb7 protein expression was 34.2%, which was significantly higher than that in adjacent non-cancerous cervical tissue (0%, p < 0.05). The expression of Grb7 was found to be correlated with age, tumor size, serosal invasion, differentiated degree, tumor stage, early or advanced stage and lymph node metastasis. Kaplan-Meier survival analysis showed that patients with positive Grb7 protein expression had a lower overall survival rate than patients without Grb7 expression. In addition, Grb7 plays an important role in promoting tumor progression, including invasion and anti-apoptosis, in cervical cancer cell line. Down-regulation of Grb7 repressed the expression of MMP-9 and Bcl-2, and increased the expression of Bax in Grb7 knockdown Hela cells. Cell invasion assay showed decreased number of Grb7 knockdown Hela cells (18.7 ± 2.1) compared to Hela cells (65.3 ± 2.5, P < 0.05). Our results indicated that Grb7 over-expression may facilitate invasion and inhibit apoptosis in cervical cancer and Grb7 is a potentially molecular target of cervical cancer chemotherapy. © 2017 Elsevier GmbH. All rights reserved.
1. Introduction Cervical cancer is the third most common malignancy in women worldwide. 12,990 diagnoses of cervical cancer will be made by the end of 2016 in the United States, over 4000 women die from cervical cancer each year. But 83% of cases occur in developing countries and recurrent or metastatic tumors are the main cause of treatment failures. Growth factor receptor bound protein-7 (Grb7) is a multi-domain adaptor protein that is co-opted by numerous tyrosine kinases involved in various cellular signaling and functions [1], and is found to be over expressed in such metastatic tumors as liver cancer [2], esophageal cancer [3], breast cancer [4–7], pancreatic cancer [8], ovarian cancer [9,10] and chronic lymphocytic leukemia [11]. Grb7 contains a src-homology 2(SH2) domain, which is reported to be mainly involved in Grb7 signal pathways. SH2 domains recruit signaling proteins to phosphotyrosine residues on
aberrantly activated growth factor and cytokine receptors and participate in cancer cell migration, metastasis and angiogenesis. Grb7 is a prime target for the development of novel anti-cancer therapies. Grb7-specific inhibitors are in early development. Recently, Study suggested that G7-18NATE(a nonphosphorylated cyclic peptide 1) inhibits Grb7 via targeting the Grb7-SH2 domain in breast cancer [4]. But the molecular mechanism by which Grb7 involved in invasion and anti-apoptosis in cervical cancer remain unclear yet. In this study, we used immunohistochemical staining, cell invasion assay and shRNA(short hairpin RNA) targeting Grb7 to confirm the correlation between the expression of Grb7 protein and clinicopathological characteristics in cervical cancer and to elucidate the underlying mechanisms by which Grb7 boosted invasion and anti-apoptosis of cervical cancer cells. 2. Materials and methods 2.1. Patients and tissue specimen
∗ Corresponding author at: Department of Oncology, The First Clinical Hospital of Zhengzhou University, Zhengzhou, Henan, 450052, PR China. E-mail address: [email protected] (H.-B. Zhao).
120 cases of cervical cancer and 10 samples of tumor-adjacent tissue were collected from First Clinical Hospital of Zhengzhou Uni-
http://dx.doi.org/10.1016/j.prp.2017.05.013 0344-0338/© 2017 Elsevier GmbH. All rights reserved.
Please cite this article in press as: H.-B. Zhao, et al., Grb7 is over-expressed in cervical cancer and facilitate invasion and inhibit apoptosis in cervical cancer cells, Pathol. – Res. Pract (2017), http://dx.doi.org/10.1016/j.prp.2017.05.013
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versity from January 2010 to July 2015. All the patients were aged 33 to71 years old with a median age of 44 years old. No patients were undergoing chemotherapy, radiotherapy, or immunotherapy before radical surgery. All the tissue specimens were confirmed independently by two qualified pathologists without knowing the characteristics of patients.
2.2. Immunohistochemistry assay Grb7 polyclonal antibody and DAB staining kit were purchased from Santa Cruz Biotechnology, Inc. (Texas, U.S.A.). All samples were fixed with 4% formaldehyde, embedded with paraffin, and subjected to serial sectioning for histopathological diagnosis and immunohistochemistry examination. The MaxVision twostep immunohistochemical detection method was used to detect Grb7 expression. Consecutive 3 m sections were deparaffinized, hydrated, and rinsed in distilled H2 O. The samples were boiled in citrate buffer (pH 6.0) in a high pressure cooker for 8 min and cooled to room temperature, and washed 4 times with phosphate-buffered saline (PBS; pH 7.4) for 5 min. All of the samples were incubated with rabbit anti-Grb7 polyclonal antibody (1:50 diluted) overnight at 4 ◦ C. The next day, the samples were washed with PBS 4 times for 5 min, and incubated with anti-rabbit IgG antibody for 1 h. Afterwards, the samples were washed with PBS 3 times for 5 min. DAB chromogenic reagent was dropped on samples and staining was observed under a light microscope. After staining, the samples were rinsed in distilled water to stop the reaction. PBS instead of the primary antibody was used as a negative control and known sample was used as positive controls. We used the following scoring method to evaluate the Grb7 expression. Each sample were graded based on the average percentage of positive cells and the staining intensity in five areas (per × 100 field) at random. The intensity (S) was graded as follows: 0 (no staining); +1 (cells stained yellow); +2 (cells stained orange) and +3 (cells stained brown). According to the mean percentage (P) of positive cells, score of percentage were classified as: 0, <5% staining; 1, 6%–25%; 2, 26%–50%; 3, <51%–75% and 4, >75%. The final H-score were obtained by using the arithmetic: HSCORE = (S × P). The classification was as follow: 0–1 point were considered negative, scores of 2–4 points were considered weakly positive, scores of 5–8 points were considered moderately positive, and scores of 9–12 points were considered strongly positive.
2.5. Quantitative RT-PCR Total RNAs were respectively extracted from cells using Trizol reagent (Toyobo Co., Ltd. Shanghai, China). The ratio of OD260 to OD280 was over 1.7. Subsequently they were reverse transcribed into cDNAs using PrimeScriptTM RT Reagent Kit(Takara Bio Inc. Osaka, Japan). Quantitative RT-PCR was performed with SYBR Green master mix real-time core reagents(Toyobo Co., Ltd. Shanghai, China) on an ABI 7500 (Applied Biosystems) according to the manufacturer’s instructions.All primers and probe combinations were designed using Primer Premier 6.0 (Premier Biosoft, Canada) and purchased from Sangon (Sangon Biotech Co., Ltd. Shanghai, China). Primers for quantitative RT-PCR were as follows: Grb7-F:5 -CTTCGCCAAGTATGAACTATT-3 Grb7-R:5 -GTTGGGCTTGACACAGAAGCC-3 Bcl-2-F: 5 -CATGCGGCCTCTGTTTGATT-3 Bcl-2-R: 5 -TGCATATTTGTTTGGGGCAGG-3 Bax-F: 5 -CGTGTCTGATCAATCCCCGA-3 Bax-R: 5 -GGGCAGAAGGCACTAATCAAG-3 MMP-9-F:5 -GACTCCGTCTTTGAGGAGCC-3 MMP-9-R:5 -GAACTCACGCGCCAGTAGAA-3 -actin-F: 5 -AGAAAATCTGGCACCACACC-3 -actin-R: 5 -TAGCACAGCCTGGATAGCAA-3 All reactions were performed in triplicate, and the expression of mRNAs was normalized to that of the reference gene -actin. Relative quantification of mRNA within the samples was caculated using the 2–Ct method. 2.6. Western blot The concentration of total protein extracted from Hela cells and Grb7 knockdown Hela cells was determined with a BCA Protein Assay Kit (Pierce, USA). Equal amounts of protein were separated by 10% SDS-PAGE and electrophoretically transferred to PVDF membranes (Pierce, USA) using a mini trans-blot (Bio-Rad laboratories, Hercules, CA, USA). Rabbit anti-human Grb7, Bax, Bcl-2, MMP-9 and -actin were purchased from Santa Cruz Biotechnology, Inc. (Texas, U.S.A.). -actin was used as an internal control. Electrochemiluminescence was performed according to the manufacturer’s instructions and read with a Chemi lmager 5500 imaging system (Alpha Innotech Co,San Leandro, CA, USA). 2.7. Cell invasion assay
2.3. Cell culture Hela cell line was preserved in our laboratory. Cells were cultured in RPMI1640 (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin(Sigma-Aldrich, St. Louis, MO, USA), 0.5 mg/mL hydrocortisone (Sigma-Aldrich, St. Louis, MO, USA), 100 ng/mL cholera toxin (Sigma-Aldrich, St. Louis, MO, USA), and 10 g/mL insulin (Sigma-Aldrich, St. Louis, MO, USA), and 20 ng/mL recombinant human EGF (Peprotech, 100-15) in a 5% CO2 incubator at 37 ◦ C. Media was changed every 3 days and cells were passaged when 65–80% confluent.
Quantification of cell invasion was performed by Cell Invasion Assay Kits(BD Science, Sparks, MD, USA.) according to the manufacturer’s instructions. 1 × 105 cells were resuspended in serum-free culture medium and seeded on the upper chamber. After incubation at 37 ◦ C in 5% CO2 for 24 h, cells still on the upper surface of the chamber were removed, and the cells that invaded through the membranes were fixed by methanol and stained with 0.1% crystal violet to assess the cell number. Quantification of invaded cells was performed according to published criteria [12]. The experiments were repeated thrice. 2.8. Statistical analysis
2.4. In vitro transfection Grb7 shRNA Plasmid was designed against Grb7 target. Grb7 shRNA Plasmid (h) and Support Reagents were purchased from Santa Cruz Biotechnology, Inc. (Texas, U.S.A.) In vitro transfection was performed according to the manufacture protocol. The transfection rates were measured using fluorescence microscopy.
Data analysis was performed using the Statistical Package for the Social Sciences (SPSS), version 17.0 (USA). Data analysis of quantitative RT-PCR results was performed using a Mann-Whitney U test. A possible association between positive expression of Grb7 and clinicopathological parameters was compared using a 2 test. Differences with a p less than 0.05 were considered to be statistically significant.
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Fig. 1. Immunohistologic staining of various cervical cancer was carried out as described in Materials and methods. A, Well differentiated cervical cancer; Grb7 expression is seen primarily in cytoplasm. B, Poorly differentiated cervical cancer.
3. Results 3.1. Grb7 expression pattern in cervical cancer To determine the expression of Grb7 in cervical cancer and adjacent normal tissues, expression of Grb7 was detected using immunohistochemistry. The Grb7 protein in cervical cancer tissue were usually seen in cytoplasm(Fig. 1A and B.). 41 out of 120 (34.2%) cases were scored positive for Grb7 staining. In the adjacent normal cervical tissue, none was stained for Grb7 expression. The difference of expression between cervical cancer tissues and adjacent normal tissues was significant (p < 0.05). The result demonstrated that Grb7 is over-expressed in cervical cancer. 3.2. Correlation between the expression of Grb7 protein and clinicopathological characteristics The expression of Grb7 protein was examined in the 120 cervical cancer patients based on patients’ clinnicopatholigical characteristics. There was significant difference between the Grb7 expression and age, tumor size, serosal invasion, differentiated degree, tumor stage, early or advanced stage and lymph node metastasis (p < 0.05) (Table 1).15 (57.7%) of 26 cervical cancer specimens with positive serosal invasion expressed Grb7, 26 (27.7%) of 94 cases with negative serosal invasion expressed Grb7, this was statistically significant(p < 0.05). There was significant difference in Grb7 positivity between early and advanced cervical cancer(p < 0.05). Statistical differences in Grb7 expression reached significance between stage and stages II or III carcinomas(p < 0.05). 3.3. Cumulative survival rates The average follow-up time was 53.4 months, ranging from 2 to 60 months, the follow-up rate was 100%. Twenty patients died of recurrent carcinoma by the end of July 2015. Cumulative survival was analyzed among the patients with cervical cancer by Log rank test. Survival rates of patients who had Grb7 positive tumor was significantly lower than those with Grb7 negative tumors(p = 0.000) (Fig. 2.). 3.4. Cervical cancer cells over-expressed Grb7 and knockdown of Grb7 decreased Grb7 expression These above results promoted us to investigate whether Grb7 expression level has relationship with tumor malignancy. To test this possibility, we tested Grb7 mRNA expression in cervical cancer cells and Grb7 knockdown cervical cancer cells. Compared to
the Grb7 knockdown Hela cells, qRT–PCR analysis showed that the mRNA expression Grb7 was high in the Hela cells, and had significant difference(p < 0.05) (Fig. 3A.). This result suggests that over-expressed Grb7 in cervical cancer cells may be involved in tumorigenesis. 3.5. Cell invasion assay We further used cell invasion assay to examine the effect on Hela cell and Grb7 knockdown Hela cell invasion. Result showed decreased number of Grb7 knockdown Hela cells (18.7 ± 2.1) compared to Hela cells (65.3 ± 2.5, P = 0.000 < 0.05). This result indicated the participation of Grb7 protein in the invasion of Hela cells, by up-regulating Grb7 expression. Next, to study the role of MMP9 expression in cervical cancer, we then investigated the expression of MMP9 in Hela cells and Grb7 knockdown Hela cells. Compared to the Hela cells, qRT–PCR analysis showed that the mRNA expression of MMP9 was lower in the Grb7 knockdown Hela cells, and had significant difference(p < 0.05, Fig. 3D). Western blots analysis confirmed the expression of MMP-9 (Fig. 4.). These data further support the notion that Grb7 up-regulates MMP9 in cervical cancer cells. 3.6. Grb7 inhibits apoptosis through the mitochondria-associated apoptotic pathway As the mitochondria-associated apoptotic pathway participates in cellular response in tumors, we performed an analysis of proapoptotic protein (Bax) and antiapoptotic protein (Bcl-2) in the Hela cells and the Grb7 knockdown Hela cells (Fig. 4). Compared with HeLa cells, qRT–PCR analysis showed that Bcl-2 level decreased, and Bax level markedly increased in the Grb7 knockdown Hela cells, and had significant difference(p < 0.05, p < 0.05, Fig. 3B and C). Western blots analysis confirmed the expression of Bcl-2 and Bax (Fig. 4). Thus, these results suggest that Grb7 inhibits apoptosis through the mitochondria-associated apoptotic pathway in cervical cancer cells. 3.7. Discussions In esophageal squamous cell carcinoma(ESCC), Grb7 was a driver gene by Analysis of survival data and RNAi screening data, and knockdown of Grb7 reduced the proliferation, migration, and invasion capacities of cells in ESCC cell lines [13]. Immunohistochemical analysis showed that Grb7 was frequently increased and associated with high-grade tumors, as well as a high tendency in association with advanced stage ovarian cancer [10]. Grb7 is a ther-
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Table 1 Relationship between Grb7 expression and Clinicopathologic characteristics in 120 patients with cervical cancer. clinicopathologic Characteristics
All patients
Grb7 (N)
p
N
%
Positive
Negative
age
>50 ≤50
64 56
53.3 46.6
24 17
40 39
0.005
Tumor size,cm
≤2 >3
66 54
55 45
13 28
53 26
0.000
Serisal invasion
Negative Positive
94 26
78.3 21.7
26 15
68 11
0.000
differentiated degree
high moderately Poorly
20 54 46
16.7 45 38.3
12 19 10
8 34 36
0.000
Lymph node metastasis
yes no
63 57
52.5 47.5
10 31
53 26
0.000
stage
I II III IV
54 18 45 3
45 15 37.5 2.5
5 10 25 1
49 8 20 2
0.000
Early or advanced
early advanced
31 89
25.8 74.2
2 39
29 50
0.000
Fig. 2. Survival of patients with cervical cancer. Cumulative survival rates of 120 patients with Grb7-positive or negative cervical cancer.
Fig. 3. Grb7, Bcl-2, Bax and MMP-9 mRNA expression in cervical cancer cells and Grb7 knockdown cervical cancer cells.
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First, the level of Grb7 mRNA was significantly higher in Hela cells than in Grb7 knockdown Hela cells. Second, Grb7 may promote transcription of the metastatic gene MMP9, qRT–PCR and western blots analysis confirmed the expression of MMP-9. Cell invasion assay showed decreased number of Grb7 knockdown Hela cells (18.7 ± 2.1) compared to Hela cells (65.3 ± 2.5, P < 0.05). Third, Grb7 inhibits apoptosis through the mitochondria-associated apoptotic pathway. Grb7 represses the level of Bax and increases the level of Bcl-2, qRT–PCR and western blots analysis confirmed the expression of Bax and Bcl-2. So, Grb7 over-expression may facilitate invasion and inhibit apoptosis in cervical cancer and Grb7 is a potentially molecular target of cervical cancer chemotherapy. Declaration of conflict of interest None. References
Fig. 4. Grb7, Bcl-2, Bax and MMP-9 protein expression in cervical cancer cells and Grb7 knockdown cervical cancer cells.
apeutic target in human breast cancer patients. It exacerbated the cellular growth and migratory behaviour of HER2 + ve breast cancer cells [7], and was significantly related to the presence of lymph node metastasis, stage and histological grade of the malignant tumors [14].Co-silencing of STARD3, Grb7, PSMD3 and PERLD1 together with HER2 led to an additive inhibition of cell viability as well as induced apoptosis [15]. Numerous variables have been reported to correlate with the prognosis of patients with cervical cancer. Independent prognostic factors that have been reported include tumor size, depth of invasion, stage, nodal status, differentiated degree and Grb7 expression. These finding showed that Grb7 levels reflected the severity of tumor, and may be used as a prognostic marker and therapeutic target. Our results revealed that Grb7 was over-expressed in cervical cancer, there was significant difference between the Grb7 expression and age, tumor size, serosal invasion, differentiated degree, tumor stage, early or advanced stage and lymph node metastasis. Enhanced Grb7 expression was a statistically significant factor in predicting the survival of patients. Prognosis of patients with cervical cancer may therefore be predicted more accurately by immunohistochemical Grb7 staining. Grb7 plays an important role in promoting tumor progression, including invasion and anti-apoptosis, in cervical cancer cell line.
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Please cite this article in press as: H.-B. Zhao, et al., Grb7 is over-expressed in cervical cancer and facilitate invasion and inhibit apoptosis in cervical cancer cells, Pathol. – Res. Pract (2017), http://dx.doi.org/10.1016/j.prp.2017.05.013