Expression and Significance of GRP78 and HER-2 in Colorectal Cancer

Background and objective GRP78 and HER-2 expression are related to disease behaviors in various cancers. Previous research has shown that GRP78 is overexpressed in colorectal cancer and plays an important role in the development and progression of colorectal cancer. And evidence for HER-2 expression in colorectalcancer is contradictory for the protein expression status and prognostic value. In our study, we aim to investigate GRP78 and HER-2 expression in colorectal cancer and explore their association with clinicopathological features. Methods Immnunohistochemical staining was used to assess the expression of GRP78 and HER-2 in 117 cases of colorectal carcinoma and corresponding adjacent tissues were selected for comparative analysis. Results The positiverate of GRP78 and HER-2 were 68.4% (80/117) and 38.5% (45/117) respectively. The expression of GRP78 and HER-2 in gender, age, tumor size, tumor position, histological grade, depth of invasion and lymph node metastasis had no significant difference (all P > 0.05); No relationship between GRP78 and HER-2 expression was detected ( P = 0.089). Conclusion GRP78 and HER-2 were not predictors for development and progression in CRC patients. The results do not support any relationship between GRP78 and HER-2 expression in this setting. Keyword: GRP78, HER-2, Colorectal cancer, Immunohistochemistry Introduction Colorectal cancer (CRC) is one of the most common gastrointestinal malignancies and still a major cause of cancer death in the world. However, about 30-35% of patients would develop distant metastasis focus, with a median survival period of about 5 years . Although many factors have been reported to be related to the CRC occurrence, the molecular mechanisms which contribute to the pathogenesis and progression of CRC are still unclear. As a chief molecular chaperone in the endoplasmic reticulum (ER), glucose regulated protein (GRP78) is particular in modulating the unfolded protein response (UPR) and then maintaining the cell homeostasis. In addition, it can be translocated to the cell surface and serve as a receptor for one ligand to transmitting some signals for altering cell foundations. However, GRP78 will become a protective protein when the tumor is formed. Recent studies have showed that GRP78 is over-expressed in CRC, and may play an important role in the development and progression of CRC . HER-2 is a proto-oncogene that encodes a transmenbrane tyrosine kinas’ receptor, the current study has found that HER-2 is involved in the development, biological behavior and prognosis in various cancers. Siddiqa et al. 7 demonstrated that HER-2 is overexpressed in 25-30% of patients with breast cancer, and is correlated to the prognosis of breast cancer patients. However, the expression of HER-2 in CRC is contradictory 8, . In the present study, we carried an immunohistochemistry study to identify aberrantly expressed GRP78 and HER-2 in colorectal cancer and analyzed the correlations with the clinicopathological parameters. Materials and Methods All CRC tissues were identified from The Affiliated Hospital of Qingdao University during a 3-year period between October 2010 and October 2013. The patients comprised 73 men and 44 women aged 25-85 years (mean:63.2y). All specimens were fixed in 10% formalin, embedded in paraffin, stained in HE and diagnosed by histology. None of the patients received anticancer treatment prior to surgery. Patients’ clinicopathological parameters including gender, age, tumor differentiation, tumor size, Expression and Significance of GRP78 and HER-2 in Colorectal Cancer http://www.ijSciences.com Volume 5 – April 2016 (04) 60 location, depth of invasion and lymph node metastasis. Immunohistochemistry Immunohistochemical staining was performed following the manufacturer’s instructions. Paraffin sections (4μm thick) were deparaffined in xylene, washed three times for 5 minutes each, then hydrated with an ethanol gradient, and then rinsed in water. After that, with 3% H2O2 for 15 min to block endogenous peroxides activity. The slide were immersed in sodium citrated buffer (PH 6.0) with heating for 3 minutes for antigen retrieval. And then the slides were incubated with the rabbit anti-GRP78 polyclone antibody (1:250, Abcam, USA) and anti-rabbit HER-2 monoclone antibody (1:100, Roche, USA) overnight at 4°C temperature. After that, the slides were incubated with the peroxides-conjugated anti-rabbit antibody (Roche, USA) for 30 min at room temperature, diaminobenzidine (DAB) was used as the chromate agent and counterstained with hematoxylin. Negative controls were performed by replacing the primary antibodies with PBS. The breast cancer specimen was used as positive control according to manufacturer’s instructions. DAB and PBS were purchased from Roche. Immunohistochemistry evaluation Two high qualification pathologist determined the final scores under optics microscope by consensus. The expression of GRP78 was located in cytoplasm and membrane. GRP78 staining was scored semi-quantitatively according to the manufacturer′s guidelines. Score0: no staining; score 1+: ≤ 30% of tumor cells expressing; score 2+: between 31% and 50% of tumor cells expressing; score 3+: >51% of tumor cells expressing. Scores of 0 and 1+ were classified as negative and scores of 2+ and 3+ were classified as positive. HER-2 staining was scored semi quantitatively according to the manufacturer’s instructions: 0: no staining or membrane staining < 10% of the tumor cells; 1+: week membrane staining or ≥ 10% of tumor cells expressing; 2+: weak-to-moderately complete membrane staining of ≥10% of tumor cells; 3+: a strongly complete membrane staining of ≥ 10% of tumor cells. Score of 0 indicated negative for HER-2 expression, 1+, 2+ and 3+ were regarded as positive expression of HER-2。 Statistics analyses The results were analyzed using SPSS19.0 (SPSS, USA).Association between expression of the two proteins and patient clinicopathological parameters were analyzed by the Chi-Squared test or Fisher’s exact test. P values < 0.05 were considered statistically significant. Results The expression of GRP78 and HER-2 expression in colorectal cancer GRP78 and HER-2 did not expressed in adjacent tissues. GRP78 expression were positive in 80 of 117 colorectal cancer samples (68.4%), the pattern of GRP78 immunostaining was both membranous and cytoplasm (Fig.1A). Of 117 cases examined, 45 (38.5%) was HER-2 positive (Fig.1B), HER-2 was expressed in membranous. Correlation of GRP78, HER-2expression and clinicopathological features Expression of GRP78 and HER-2 in CRC with clinical features was shown in Table 1. Statistical evaluation of GRP78 and HER-2 expression according to gender, age, tumor location, tumor size, differentiation grade, depth of invasion or lymph node metastasis revealed no significant difference among these variables (all P > 0.05). The association between GRP78 and HER-2 expression There was no significant positive correlation between GRP78 and HER-2 expression in CRC (r = 0.122 ; P > 0.05) (Table 2). Discussion GRP78, a crucial protein that is located in ER and belongs to HSP70 protein family, can be induced by the ER stress response. It is widely accepted that the over-expression of GRP78 plays an important roles in the process of CRC, including: transfer the unfold or misfiled proteins, maintain protein synthesis under the circumstance of ER stress, stabilize intracellular calcium homeostasis. In the development of CRC, ER stress response, cell repair and apoptosis are important process of pathophysiology. In the present study, for 117 CRC samples, 68.4% (80/117) showed positive expression of GRP78. Same result was described in our previous study . In previous study, GRP78 expression was elevated gradually in human normal colorectal tissues, adenoma specimens and colorectal carcinoma specimens. In addition, we found that GRP78 is overexpressed in CRC patients but not associated with clinicopathological parameters. It demonstrated that GRP78 may participate in the development and progression of colorectal neoplasm, although with no significant of prognosis. HER-2 is a member of tyrosine kinas’ receptor super family and homologous to epidermal growth factor receptor (EGFR). It is referred to the growth and progression of malignant cells. The expression of HER-2 in CRC was not consistent with reported 9, 12 as well as the relationship between tumors’ Expression and Significance of GRP78 and HER-2 in Colorectal Cancer http://www.ijSciences.com Volume 5 – April 2016 (04) 61 development and progression. Kapitanovi et.al. 9 demonstrated that normal mucosa was mostly negative, but significant number of benign lesions and CRC overexpressed HER-2. Adenocarcinomas were significantly more positive than benign. The expression level of HER-2 can be used as an independent prognostic factor for CRC patients. Kovaeevi et al demonstrated that this oncoprotein was predictive neither for overall survival nor for invasion and metastases in rectal cancer patients. Our data showed that HER-2 overexpressed in 38.5% of cases while this did not correlate with clinicopathological parameters, similar to the study by Schuel et al.. All of these above indicated that overexpression of HER-2 plays an important role in the progression of CRC and is not considered as an independent indicator for invasion and prognostic of CRC. Previous report has revealed that GRP78-EGFR complex can inhibited the EGFR singling pathway, while EGFR and HER-2 are similar in structure and function. Several studies show that the two proteins co-expression in colorectal cancer, the synergistic effect existed in the tumor genesis and biological behavior , so that there will be a correlation between GRP78 and HER-2. However, our findings suggest that there is no apparent correlation between GRP78 and HER-2 expression in CRC. Therefore, they may no correlation between GRP78 and HER-2 expression, specific regulatory mechanism b


Introduction
Colorectal cancer (CRC) is one of the most common gastrointestinal malignancies and still a major cause of cancer death in the world 1 . However, about 30-35% of patients would develop distant metastasis focus, with a median survival period of about 5 years 2 . Although many factors have been reported to be related to the CRC occurrence, the molecular mechanisms which contribute to the pathogenesis and progression of CRC are still unclear. As a chief molecular chaperone in the endoplasmic reticulum (ER), glucose regulated protein (GRP78) is particular in modulating the unfolded protein response (UPR) 3 and then maintaining the cell homeostasis. In addition, it can be translocated to the cell surface and serve as a receptor for one ligand to transmitting some signals for altering cell foundations 4 . However, GRP78 will become a protective protein when the tumor is formed. Recent studies have showed that GRP78 is over-expressed in CRC, and may play an important role in the development and progression of CRC 5, 6 . HER-2 is a proto-oncogene that encodes a transmenbrane tyrosine kinas' receptor, the current study has found that HER-2 is involved in the development, biological behavior and prognosis in various cancers. Siddiqa et al. 7 demonstrated that HER-2 is overexpressed in 25-30% of patients with breast cancer, and is correlated to the prognosis of breast cancer patients. However, the expression of HER-2 in CRC is contradictory 2,8,9 . In the present study, we carried an immunohistochemistry study to identify aberrantly expressed GRP78 and HER-2 in colorectal cancer and analyzed the correlations with the clinicopathological parameters.

Materials and Methods
All CRC tissues were identified from The Affiliated Hospital of Qingdao University during a 3-year period between October 2010 and October 2013. The patients comprised 73 men and 44 women aged 25-85 years (mean:63.2y). All specimens were fixed in 10% formalin, embedded in paraffin, stained in HE and diagnosed by histology. None of the patients received anticancer treatment prior to surgery. Patients' clinicopathological parameters including gender, age, tumor differentiation, tumor size, http://www.ijSciences.com Volume 5 -April 2016 (04) 60 location, depth of invasion and lymph node metastasis.

Immunohistochemistry
Immunohistochemical staining was performed following the manufacturer's instructions. Paraffin sections (4μm thick) were deparaffined in xylene, washed three times for 5 minutes each, then hydrated with an ethanol gradient, and then rinsed in water. After that, with 3% H2O2 for 15 min to block endogenous peroxides activity. The slide were immersed in sodium citrated buffer (PH 6.0) with heating for 3 minutes for antigen retrieval. And then the slides were incubated with the rabbit anti-GRP78 polyclone antibody (1:250, Abcam, USA) and anti-rabbit HER-2 monoclone antibody (1:100, Roche, USA) overnight at 4℃ temperature. After that, the slides were incubated with the peroxides-conjugated anti-rabbit antibody (Roche, USA) for 30 min at room temperature, diaminobenzidine (DAB) was used as the chromate agent and counterstained with hematoxylin. Negative controls were performed by replacing the primary antibodies with PBS. The breast cancer specimen was used as positive control according to manufacturer's instructions. DAB and PBS were purchased from Roche.

Immunohistochemistry evaluation
Two high qualification pathologist determined the final scores under optics microscope by consensus. The expression of GRP78 was located in cytoplasm and membrane. GRP78 staining was scored semi-quantitatively according to the manufacturer′s guidelines. Score0: no staining; score 1+: ≤ 30% of tumor cells expressing; score 2+: between 31% and 50% of tumor cells expressing; score 3+: >51% of tumor cells expressing. Scores of 0 and 1+ were classified as negative and scores of 2+ and 3+ were classified as positive. HER-2 staining was scored semi quantitatively according to the manufacturer's instructions: 0: no staining or membrane staining < 10% of the tumor cells; 1+: week membrane staining or ≥ 10% of tumor cells expressing; 2+: weak-to-moderately complete membrane staining of ≥10% of tumor cells; 3+: a strongly complete membrane staining of ≥ 10% of tumor cells. Score of 0 indicated negative for HER-2 expression, 1+, 2+ and 3+ were regarded as positive expression of HER-2。

Statistics analyses
The results were analyzed using SPSS19.0 (SPSS, USA).Association between expression of the two proteins and patient clinicopathological parameters were analyzed by the Chi-Squared test or Fisher's exact test. P values < 0.05 were considered statistically significant.

Results
The expression of GRP78 and HER-2 expression in colorectal cancer GRP78 and HER-2 did not expressed in adjacent tissues. GRP78 expression were positive in 80 of 117 colorectal cancer samples (68.4%), the pattern of GRP78 immunostaining was both membranous and cytoplasm (Fig.1A). Of 117 cases examined, 45 (38.5%) was HER-2 positive (Fig.1B), HER-2 was expressed in membranous.

Correlation of GRP78, HER-2expression and clinicopathological features
Expression of GRP78 and HER-2 in CRC with clinical features was shown in Table 1. Statistical evaluation of GRP78 and HER-2 expression according to gender, age, tumor location, tumor size, differentiation grade, depth of invasion or lymph node metastasis revealed no significant difference among these variables (all P > 0.05).

The association between GRP78 and HER-2 expression
There was no significant positive correlation between GRP78 and HER-2 expression in CRC (r = 0.122 ; P > 0.05) ( Table 2).

Discussion
GRP78, a crucial protein that is located in ER and belongs to HSP70 protein family, can be induced by the ER stress response 10 . It is widely accepted that the over-expression of GRP78 plays an important roles in the process of CRC, including: transfer the unfold or misfiled proteins, maintain protein synthesis under the circumstance of ER stress, stabilize intracellular calcium homeostasis 11 . In the development of CRC, ER stress response, cell repair and apoptosis are important process of pathophysiology.
In the present study, for 117 CRC samples, 68.4% (80/117) showed positive expression of GRP78. Same result was described in our previous study 5, 6 . In previous study, GRP78 expression was elevated gradually in human normal colorectal tissues, adenoma specimens and colorectal carcinoma specimens. In addition, we found that GRP78 is overexpressed in CRC patients but not associated with clinicopathological parameters. It demonstrated that GRP78 may participate in the development and progression of colorectal neoplasm, although with no significant of prognosis. HER-2 is a member of tyrosine kinas' receptor super family and homologous to epidermal growth factor receptor (EGFR). It is referred to the growth and progression of malignant cells. The expression of HER-2 in CRC was not consistent with reported 8,9,12 as well as the relationship between tumors' development and progression. Kapitanovi et.al. 9 demonstrated that normal mucosa was mostly negative, but significant number of benign lesions and CRC overexpressed HER-2. Adenocarcinomas were significantly more positive than benign. The expression level of HER-2 can be used as an independent prognostic factor for CRC patients. Kovaeevi et al 13 demonstrated that this oncoprotein was predictive neither for overall survival nor for invasion and metastases in rectal cancer patients. Our data showed that HER-2 overexpressed in 38.5% of cases while this did not correlate with clinicopathological parameters, similar to the study by Schuel et al. 14 . All of these above indicated that overexpression of HER-2 plays an important role in the progression of CRC and is not considered as an independent indicator for invasion and prognostic of CRC.
Previous report 15 has revealed that GRP78-EGFR complex can inhibited the EGFR singling pathway, while EGFR and HER-2 are similar in structure and function. Several studies show that the two proteins co-expression in colorectal cancer, the synergistic effect existed in the tumor genesis and biological behavior 16, 17 , so that there will be a correlation between GRP78 and HER-2. However, our findings suggest that there is no apparent correlation between GRP78 and HER-2 expression in CRC. Therefore, they may no correlation between GRP78 and HER-2 expression, specific regulatory mechanism between the two has no relevant research reports.
Our study aimed to evaluate the expression of GRP78 and HER-2 in CRC by IHC methods. We indicated that both GRP78 and HER-2 were not predictors for development and progression in CRC patients, and our results do not support an association between GRP78 and HER2 expression.