Tumor Biol. DOI 10.1007/s13277-013-1545-x
RESEARCH ARTICLE
CXCR4 as a novel predictive biomarker for metastasis and poor prognosis in colorectal cancer Ying Gao & Chunyu Li & Min Nie & Yao Lu & Shunsen Lin & Peng Yuan & Xun Sun
Received: 16 November 2013 / Accepted: 11 December 2013 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract The clinical significance of CXCR4 expression in colorectal cancer remains unclear. The aim of this study was to investigate the expression and regulatory effects of CXCR4 in colorectal cancer and the association between CXCR4 protein expression and prognosis. The mRNA and protein expression levels of CXCR4 were determined using reverse-transcriptase polymerase chain reaction and immunohistochemistry staining, respectively, and the relationship between the CXCR4 protein level and clinicopathological parameters was analyzed in 720 cases of colorectal cancer. CXCR4 expression was elevated in colorectal cancer tissues compared to pericancerous tissues (P=0.001). Of the 720 enrolled cases, 208 (28.89 %) expressed CXCR4. In univariate analysis, CXCR4 was found to be associated with lymph node metastasis, TNM stage, and liver metastasis (P=0.001, 0.001, and 0.012, respectively). Further multivariate analysis suggested that histological grade, TNM stage, and CXCR4 expression were related to liver metastasis (P=0.020, 0.01, and 0.001, respectively). In the Cox regression test, the histological grade, lymph node metastasis, TNM stage, liver metastasis, and CXCR4 expression were found to be independent prognostic factors (P= 0.02, 0.045, 0.01, 0.001, and 0.001, Y. Gao Department of Pathology, Fourth Affiliated Hospital of China Medical University, Shenyang 110032, China C. Li (*) : Y. Lu : S. Lin : P. Yuan Anorectal Branch, Fourth Affiliated Hospital of China Medical University, Shenyang 110032, China e-mail: [email protected] M. Nie Integrated Surgery, Third Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang 110003, China X. Sun Department of Immunology, China Medical University, Shenyang 110001, China
respectively). CXCR4 protein may be a potential biomarker for liver metastasis and an independent marker for survival in colorectal cancer. Keywords Colorectal cancer . CXCR4 . Metastasis . Prognosis
Introduction Colorectal cancer is one of the most common cancers worldwide and is responsible for thousands of deaths each year. Although the mortality rate of colorectal cancer is decreasing, finding novel targets for its therapy remains an important goal [1–3]. The liver is the most common site of distant metastatic disease, followed by the lung. Indeed, for nearly a third of patients with metastatic colorectal cancer, the liver is the only affected visceral organ [4]. Approximately 15–25 % of colorectal cancer patients have synchronous liver metastasis at the time of initial diagnosis, and 10–25 % of patients develop metachronous liver metastasis sometime after curative resection of the primary lesion [5]. Although some molecular factors have been found to play important roles in the progression and metastasis of colorectal cancer, little is known about the factors that drive metastasis formation [6]. Therefore, identification of predictive markers for postoperative metastasis could improve our understanding of the biological basis for the survival of colorectal cancer patients and may provide important, clinically relevant insights into disease management. The C-X-C chemokine receptor type 4 (CXCR4), also known as fusin or CD184 (cluster of differentiation 184), is a protein encoded by the CXCR4 gene in humans. CXCR4 is an alpha-chemokine receptor specific for stromal-derivedfactor-1 (SDF-1, also called CXCL12), a molecule with potent chemotactic activity for lymphocytes [7, 8]. Some studies
Tumor Biol.
showed that the CXCR4 axis plays an important role in the metastasis of some solid tumors [9, 10]. In a recent study, He et al. examined the clinical and prognostic implications of CXCR4 expression in gastric cancer [9] and found that it was an independent prognostic marker for overall survival. Detection of CXCR4 expression will be helpful for predicting the prognosis for patients with gastric cancer. CXCR4 has also been found to be a prognostic marker in various other types of cancer, including acute myeloid leukemia [11], breast cancer [12], and prostate cancer [13]. Currently, very few studies have investigated the function of CXCR4 in colorectal cancer. Moreover, the relationship between CXCR4 expression and the biological behavior and prognosis of this malignancy remain unclear. In the present study, we investigated the clinical implications of CXCR4 protein expression in colorectal cancer.
sections were heated at 70 °C for 1 h, de-waxed in xylene, and dehydrated through a gradient concentration of alcohol. After retrieving and blocking endogenous peroxidase and non-specific staining with 3 % (v/v) H2O2 and normal goat serum, the sections were incubated with anti-CXCR4 antibody (Santa Cruz Biotechnology, Inc.) overnight at 4 °C. The slides were then incubated with horseradish peroxidaseconjugated goat anti-mouse IgG secondary antibody for 10 min at 37 °C. Finally, the sections were visualized using diaminobenzidine solution (DAKO, Carpinteria, CA, USA) and counterstained with hematoxylin (DAKO, Carpinteria, CA, USA). Antibody specificity was confirmed by immunohistochemistry with peptide competition. The intensity of CXCR4 immunohistochemistry staining was scored independently by two gastroenterology pathologists using the semiquantitative immunoreactivity scoring system, as previously described [15, 17]. Negative controls were treated identically, without the primary antibody.
Materials and methods Patients and tissue specimens For the present study, we selected 720 patients with histologically confirmed colorectal cancer who underwent radical operations at the China Medical University between January 2003 and January 2008 and analyzed the immunohistochemistry staining results and patient outcomes. TNM staging was determined based on the American Joint Committee on Cancer Staging Manual [14]. The study protocol was approved by the Ethics Committee of ShengJing Hospital and Liaoning Province Tumor Hospital.
Statistical analysis All data were analyzed using SPSS Statistics software (version 13.0, Chicago, IL, USA). Relationships between tumor marker levels and other parameters were analyzed using the chi-square test, Fisher's extract test, or independent t tests. Disease-specific survival was analyzed using the Kaplan– Meier method. The log-rank test was used to analyze survival differences. Multivariate analysis was performed using the Cox proportional hazards model with forward stepwise selection. A P value of less than 0.05 was considered statistically significant.
Experiment methods Reverse-transcriptase polymerase chain reaction (RT-PCR)
Results
RNA was extracted from cells using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Semi-quantitative RT-PCR was performed using an RT-PCR system (Promega, Madison, WI, USA). Primers with the following sequences were obtained from Invitrogen: for CXCR4, forward 5′-GTA AAA CGA CGG CCA GT-3′ and reverse 5′-CAG GAA ACA GCT ATG AC′ and for the housekeeping gene GAPDH, forward 5′-CCA CCC ATG GCA AAT TCC CAT GGC A-3′ and reverse 5′-TCT AGA CGG CAG GTC AGG TCC ACC-3′. The annealing temperature was 54.5 °C, and 30 cycles were performed. The products were electrophoresed on 2.5 % agarose gel.
CXCR4 expression in colorectal cancer and its relationship with clinical and pathological characteristics The RT-PCR analysis showed that the levels of CXCR4 mRNA were significantly higher in colorectal cancer than in the pericarcinoma tissues (P= 0.001) (Fig. 1).
Immunohistochemistry procedures Briefly, the specimen tissues were cut into 4-μm-thick sections, as were the tissue microarray paraffin blocks. The
Fig. 1 RT-PCR analysis revealed that CXCR4 mRNA expression was significantly higher in colorectal cancer than in pericarcinoma tissues (P= 0.001)
Tumor Biol. Fig. 2 CXCR4 was expressed at a low level in tissue adjacent to the tumor (400×), and at a high level in colorectal cancer tissue (400×)
Immunohistochemical examination showed that CXCR4 was located in the cytoplasm and membrane of colorectal cancer cells and that the level of CXCR4 protein was consistent with the RNA level as determined using RT-PCR analysis (Fig. 2). In univariate analysis, CXCR4 protein expression was significantly related to tumor size, histological grade, lymph node metastasis, TNM stage, and postoperative liver metastasis (P=0.024, 0.001, 0.001, 0.001, and 0.001 respectively), but not to age or gender (P=0.254 and 0.871 respectively) (Table 1). We further performed logistic multiple analysis to correct for confounding factors, after which, histological grade, lymph node metastasis, TNM stage, and postoperative liver metastasis were found to be significantly
Table 1 The correlation between CXCR4 protein expression and clinicopathological features of colorectal cancer (n=720) Characteristic Age (years) 55 Gander Man Female Tumor size
RESEARCH ARTICLE
CXCR4 as a novel predictive biomarker for metastasis and poor prognosis in colorectal cancer Ying Gao & Chunyu Li & Min Nie & Yao Lu & Shunsen Lin & Peng Yuan & Xun Sun
Received: 16 November 2013 / Accepted: 11 December 2013 # International Society of Oncology and BioMarkers (ISOBM) 2014
Abstract The clinical significance of CXCR4 expression in colorectal cancer remains unclear. The aim of this study was to investigate the expression and regulatory effects of CXCR4 in colorectal cancer and the association between CXCR4 protein expression and prognosis. The mRNA and protein expression levels of CXCR4 were determined using reverse-transcriptase polymerase chain reaction and immunohistochemistry staining, respectively, and the relationship between the CXCR4 protein level and clinicopathological parameters was analyzed in 720 cases of colorectal cancer. CXCR4 expression was elevated in colorectal cancer tissues compared to pericancerous tissues (P=0.001). Of the 720 enrolled cases, 208 (28.89 %) expressed CXCR4. In univariate analysis, CXCR4 was found to be associated with lymph node metastasis, TNM stage, and liver metastasis (P=0.001, 0.001, and 0.012, respectively). Further multivariate analysis suggested that histological grade, TNM stage, and CXCR4 expression were related to liver metastasis (P=0.020, 0.01, and 0.001, respectively). In the Cox regression test, the histological grade, lymph node metastasis, TNM stage, liver metastasis, and CXCR4 expression were found to be independent prognostic factors (P= 0.02, 0.045, 0.01, 0.001, and 0.001, Y. Gao Department of Pathology, Fourth Affiliated Hospital of China Medical University, Shenyang 110032, China C. Li (*) : Y. Lu : S. Lin : P. Yuan Anorectal Branch, Fourth Affiliated Hospital of China Medical University, Shenyang 110032, China e-mail: [email protected] M. Nie Integrated Surgery, Third Affiliated Hospital of Liaoning University of Traditional Chinese Medicine, Shenyang 110003, China X. Sun Department of Immunology, China Medical University, Shenyang 110001, China
respectively). CXCR4 protein may be a potential biomarker for liver metastasis and an independent marker for survival in colorectal cancer. Keywords Colorectal cancer . CXCR4 . Metastasis . Prognosis
Introduction Colorectal cancer is one of the most common cancers worldwide and is responsible for thousands of deaths each year. Although the mortality rate of colorectal cancer is decreasing, finding novel targets for its therapy remains an important goal [1–3]. The liver is the most common site of distant metastatic disease, followed by the lung. Indeed, for nearly a third of patients with metastatic colorectal cancer, the liver is the only affected visceral organ [4]. Approximately 15–25 % of colorectal cancer patients have synchronous liver metastasis at the time of initial diagnosis, and 10–25 % of patients develop metachronous liver metastasis sometime after curative resection of the primary lesion [5]. Although some molecular factors have been found to play important roles in the progression and metastasis of colorectal cancer, little is known about the factors that drive metastasis formation [6]. Therefore, identification of predictive markers for postoperative metastasis could improve our understanding of the biological basis for the survival of colorectal cancer patients and may provide important, clinically relevant insights into disease management. The C-X-C chemokine receptor type 4 (CXCR4), also known as fusin or CD184 (cluster of differentiation 184), is a protein encoded by the CXCR4 gene in humans. CXCR4 is an alpha-chemokine receptor specific for stromal-derivedfactor-1 (SDF-1, also called CXCL12), a molecule with potent chemotactic activity for lymphocytes [7, 8]. Some studies
Tumor Biol.
showed that the CXCR4 axis plays an important role in the metastasis of some solid tumors [9, 10]. In a recent study, He et al. examined the clinical and prognostic implications of CXCR4 expression in gastric cancer [9] and found that it was an independent prognostic marker for overall survival. Detection of CXCR4 expression will be helpful for predicting the prognosis for patients with gastric cancer. CXCR4 has also been found to be a prognostic marker in various other types of cancer, including acute myeloid leukemia [11], breast cancer [12], and prostate cancer [13]. Currently, very few studies have investigated the function of CXCR4 in colorectal cancer. Moreover, the relationship between CXCR4 expression and the biological behavior and prognosis of this malignancy remain unclear. In the present study, we investigated the clinical implications of CXCR4 protein expression in colorectal cancer.
sections were heated at 70 °C for 1 h, de-waxed in xylene, and dehydrated through a gradient concentration of alcohol. After retrieving and blocking endogenous peroxidase and non-specific staining with 3 % (v/v) H2O2 and normal goat serum, the sections were incubated with anti-CXCR4 antibody (Santa Cruz Biotechnology, Inc.) overnight at 4 °C. The slides were then incubated with horseradish peroxidaseconjugated goat anti-mouse IgG secondary antibody for 10 min at 37 °C. Finally, the sections were visualized using diaminobenzidine solution (DAKO, Carpinteria, CA, USA) and counterstained with hematoxylin (DAKO, Carpinteria, CA, USA). Antibody specificity was confirmed by immunohistochemistry with peptide competition. The intensity of CXCR4 immunohistochemistry staining was scored independently by two gastroenterology pathologists using the semiquantitative immunoreactivity scoring system, as previously described [15, 17]. Negative controls were treated identically, without the primary antibody.
Materials and methods Patients and tissue specimens For the present study, we selected 720 patients with histologically confirmed colorectal cancer who underwent radical operations at the China Medical University between January 2003 and January 2008 and analyzed the immunohistochemistry staining results and patient outcomes. TNM staging was determined based on the American Joint Committee on Cancer Staging Manual [14]. The study protocol was approved by the Ethics Committee of ShengJing Hospital and Liaoning Province Tumor Hospital.
Statistical analysis All data were analyzed using SPSS Statistics software (version 13.0, Chicago, IL, USA). Relationships between tumor marker levels and other parameters were analyzed using the chi-square test, Fisher's extract test, or independent t tests. Disease-specific survival was analyzed using the Kaplan– Meier method. The log-rank test was used to analyze survival differences. Multivariate analysis was performed using the Cox proportional hazards model with forward stepwise selection. A P value of less than 0.05 was considered statistically significant.
Experiment methods Reverse-transcriptase polymerase chain reaction (RT-PCR)
Results
RNA was extracted from cells using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Semi-quantitative RT-PCR was performed using an RT-PCR system (Promega, Madison, WI, USA). Primers with the following sequences were obtained from Invitrogen: for CXCR4, forward 5′-GTA AAA CGA CGG CCA GT-3′ and reverse 5′-CAG GAA ACA GCT ATG AC′ and for the housekeeping gene GAPDH, forward 5′-CCA CCC ATG GCA AAT TCC CAT GGC A-3′ and reverse 5′-TCT AGA CGG CAG GTC AGG TCC ACC-3′. The annealing temperature was 54.5 °C, and 30 cycles were performed. The products were electrophoresed on 2.5 % agarose gel.
CXCR4 expression in colorectal cancer and its relationship with clinical and pathological characteristics The RT-PCR analysis showed that the levels of CXCR4 mRNA were significantly higher in colorectal cancer than in the pericarcinoma tissues (P= 0.001) (Fig. 1).
Immunohistochemistry procedures Briefly, the specimen tissues were cut into 4-μm-thick sections, as were the tissue microarray paraffin blocks. The
Fig. 1 RT-PCR analysis revealed that CXCR4 mRNA expression was significantly higher in colorectal cancer than in pericarcinoma tissues (P= 0.001)
Tumor Biol. Fig. 2 CXCR4 was expressed at a low level in tissue adjacent to the tumor (400×), and at a high level in colorectal cancer tissue (400×)
Immunohistochemical examination showed that CXCR4 was located in the cytoplasm and membrane of colorectal cancer cells and that the level of CXCR4 protein was consistent with the RNA level as determined using RT-PCR analysis (Fig. 2). In univariate analysis, CXCR4 protein expression was significantly related to tumor size, histological grade, lymph node metastasis, TNM stage, and postoperative liver metastasis (P=0.024, 0.001, 0.001, 0.001, and 0.001 respectively), but not to age or gender (P=0.254 and 0.871 respectively) (Table 1). We further performed logistic multiple analysis to correct for confounding factors, after which, histological grade, lymph node metastasis, TNM stage, and postoperative liver metastasis were found to be significantly
Table 1 The correlation between CXCR4 protein expression and clinicopathological features of colorectal cancer (n=720) Characteristic Age (years) 55 Gander Man Female Tumor size
