Tumor Biol. DOI 10.1007/s13277-015-3595-8
RESEARCH ARTICLE
MEK inhibitor diminishes nasopharyngeal carcinoma (NPC) cell growth and NPC-induced osteoclastogenesis via modulating CCL2 and CXCL16 expressions Yu Zhu 1,2 & Chunlin Zou 1,2 & Zhe Zhang 3 & Chao-Nan Qian 4 & Xin Yang 1,2 & Junlin Shi 1,2 & Yudui Xia 1,2 & Jian Zhang 1,2,5 & Yi Lu 1,2
Received: 28 March 2015 / Accepted: 19 May 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Nasopharyngeal carcinoma (NPC) is a common malignancy in southern China and Southeast Asia. NPC frequently metastasizes to the bone in advanced patients resulting in high mortality. The molecular mechanisms for NPC development and cancer-induced bone lesions are unclear. In this study, we firstly determined chemokine receptor CCR2 and CXCR6 expressions in clinical specimens and CNE2, SUNE1, CNE1, and HK1 cell lines. Then, we measured chemokine CCL2 and CXCL16 production in these NPC cell lines by ELISA. Expression levels of these chemokines and their receptors were observed to positively correlate with tumor aggressiveness. Furthermore, U0126 (MEK inhibitor) was used to treat these NPC cell lines. CCL2 and CXCL16 expression levels and cell proliferation were significantly inhibited by U0126 in a dose- and time-dependent manner.
Finally, we collected conditioned medium (CM) from NPC cell cultures in the presence of U0126 treatment. When mouse bone marrow non-adherent cells were treated with the CM, the numbers of multinucleated osteoclast formation were dramatically diminished. These results indicate that MEK inhibitor diminishes NPC cell proliferation and NPC-induced osteoclastogenesis via modulating CCL2 and CXCL16 expressions. This study provides novel therapeutic targets such as CCL2/CCR2 and CXCL16/CXCR6 for advanced NPC patients. Keywords CCL2/CCR2 . CXCL16/CXCR6 . U0126 . Osteoclastogenesis . Nasopharyngeal carcinoma
Introduction * Jian Zhang [email protected] * Yi Lu [email protected] 1
Key Laboratory of Longevity and Aging-related Diseases, Ministry of Education, Nanning, Guangxi, China
2
Center for Translational Medicine, Guangxi Medical University, No.22 Shuangyong Road, 1416 Pharmacology & Biomedical Sciences Building, Nanning, Guangxi 530021, China
3
Department of Otolaryngology Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi 530021, China
4
State Key Laboratory of Oncology in South China and Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong 510060, China
5
Department of Pathology and Internal Medicine, University of Michigan, Ann Arbor, MI, USA
Nasopharyngeal carcinoma (NPC) is one of the most common cancers in southern China and Southeast Asia [1, 2]. Radiation therapy has become one of the main treatments for NPC patients. Addition of chemotherapy is recommended for advanced NPC patients [3]. Locoregional relapse and distant metastasis are key factors that cause failure of NPC treatment [4]. There is about 5 % NPC patients when they were initially diagnosed had already had distance metastasis. About 20– 40 % of patients would appear distant metastases within 5 years after radical radiation therapy. The incidence of distant metastases increases along with higher clinical stages [5, 6]. The bone has been demonstrated as a common site of NPC distant metastasis, followed by the lung and liver. NPC skeletal metastasis mostly refers to the spine, thorax, and pelvis bone. Patients with bone metastasis suffer from severe bone pain, pathologic fractures, and spinal cord compression symptoms [7], and often cause multiple bone lesions including
Tumor Biol.
osteolytic, osteoblastic lesions, or mixture of both. The interaction of tumor cells with bone microenvironment plays important roles in NPC development. Paget proposed the Bseed and soil hypothesis^ in 1889 to account for the predilection of metastasis to certain organs [8–10]. Accumulating evidence has shown that certain cytokines and chemokines, produced by tumor cells and their microenvironment, lead to a modulation of tumor microenvironment that is suitable for tumor cell growth and thus protecting tumor cells from immune cellmediated attacks [11]. Chemokines play a central role in the tumor-bone microenvironment. Certain chemokines and their corresponding receptors have been suggested as important mediators in the regulation of angiogenesis, growth, invasiveness, and metastasis of tumors [12–14]. Chemokine (C-C motif) ligand 2 (CCL2, also known as monocyte chemotactic protein-1, MCP-1) plays a critical role in the recruitment and activation of monocytes during acute inflammation and carcinogenesis. CCL2 can be produced by bone cells, such as human bone marrow endothelial cells, osteoblasts, osteoclasts, as well as tumor cells [15]. Studies have revealed that CCL2 was overexpressed in many cancers including melanoma, ovarian, breast, prostate, esophageal, gastric, renal cell, lung, colon, and papillary thyroid carcinomas [16]. CCL2-induced prostate cancer (PCa) PC3 and VCaP cancer cell proliferation via activation of phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway [17]. CCL2-enhanced RhoA expression and activity through Smad3 and MEK signaling pathway to regulate breast cancer cell motility and survival [18]. Our previous study has shown that CCL2, in addition to interleukin-8 (IL-8), mediated PCa CM-induced osteoclastogenesis and bone resorption. This effect could be partially blocked by CCL2 neutralizing antibody [19]. Recently, a study had reported that higher levels of soluble CCL2 were detected in the serum of NPC patients and proposed that CCL2 might be prognostic marker to predict bone invasion, post-treatment distant metastasis, and poor overall survival [20]. The chemokine receptor CXCR6 and its ligand CXCL16 were found to be overexpressed in breast cancer tissues and cell lines, prostate cancer tissue, and cell lines, as well as pancreatic ductal adenocarcinoma [12, 21, 22]. It has been shown that CXCR6 is expressed in primary and metastatic melanoma [23]. CXCL16/ CXCR6 strengthened the proliferative capability of melanoma cells [24]. Hu et al. showed that CXCR6/ CXCL16 play an important role in prostate cancer bone metastasis [25]. Another study had confirmed that CXCR6/AKT/mTOR pathway plays a central role in the development of prostate cancer [26]. Blocking the CXCR6/AKT/mTOR signaling pathway may be
beneficial for preventing metastasis and providing a more effective therapeutic strategy for prostate cancer [12]. Overall, CCL2/CCR2 and CXCL16/CXCR6 may play crucial roles on growth, invasiveness, and metastasis of certain tumors, particularly tumors with bone metastasis [16]. However, the functions and regulation of CCL2/CCR2 and CXCL16/CXCR6 in NPC progression remain unknown. In addition, the molecular basis of NPC development and cancer-induced bone lesions are still unclear. In current study, we hypothesize that CCL2 and CXCL16, via MEK/ERK signaling pathway, contribute to NPC cell growth and NPC-induced osteoclast differentiation and formation.
Materials and methods Reagents U0126, MAPK/ERK signaling inhibitor, was purchased from Cell Signaling Technology (Boston, MA, USA). Antibodies against phospho-p70S6k, phospho-ERK1/2, c-Myc, p70S6K, ERK1/2, CDK1, and cyclin B1 were purchased from Cell Signaling Technology. Phosphatase inhibitor cocktails 2 and 3 were purchase from Sigma (St. Louis, MO, USA). Antibodies against CCR2 and CXCR6 were purchased from Abcam (Cambridge, Cambs, UK). Alexa Fluor donkey anti-rabbit IgG (H+L) secondary antibody was the product of Invitrogen (Carlsbad, CA, USA). Recombinant human M-CSF and rhRANKL were purchased from R&D systems (Minneapolis, MN, USA). All chemical reagents were purchased from Sigma. Patients Fifty-one cases of NPC clinical tissue paraffin sections were obtained from the First Affiliated Hospital of Guangxi Medical University and Sun Yat-sen University Cancer Center. All NPC clinical specimens, including 15 cases of nonkeratinizing squamous cell carcinoma, and 36 cases of nonkeratinizing carcinoma (undifferentiated), have been confirmed by histopathologic examination. All procedures involving clinical specimens were performed in accordance with protocols approved by Institutional Research Board, Guangxi Medical University. Cell cultures and treatments NPC cell lines, CNE2 and SUNE1, CNE1, and HK1 were cultured in DMEM (Invitrogen) media supplemented with 10 % fetal bovine serum (FBS). For U0126 treatment assay, NPC cell lines were planted at a density of 1×105/well into 12-well plates and incubated for overnight. The medium was
Tumor Biol.
changed to serum-free DMEM with indicated doses of U0126. Then the cells were continued to culture for another 24 h. The cell lysate and CM in each well was collected, and total protein was extracted. Reverse transcription-PCR Total RNA was extracted from NPC cell lines, using TRIzol reagent (Invitrogen), and then subjected to reverse transcription (RT)-PCR for detection of CCL2, CCR2, CXCL16 and CXCR6 mRNA expression levels. PCR primers for CCL2 consisted of sense 5′-TAGCAGCCACCTTCATTC-3′ and antisense 5′-CTTGGGGTCAGCACAGAT-3′. PCR primers for CCR2 consisted of sense 5′-CGTTAGAAACAGGAGCAG3′ and antisense 5′-ACAGGGCAACTAATGGTA-3′. PCR primers for CXCL16 consisted of sense 5′-CTGTACCCGC AACGAAGG-3′ and antisense 5′-AGGCAAGGGACTGA GGAGG-3′. PCR primers for CXCR6 consisted of sense 5′GGCGACTAAGTCATTCCG-3′ and antisense 5′-GCTGCC TTGGGTGTTGTA-3′. The expression of glyceraldehyde-3phosphate dehydrogenase (GAPDH) was used as internal control. RT-PCR was performed as described previously [17, 26].
cells (1×105/well) in 96-well plates for 10 days. The cells were incubated with rhM-CSF (10 ng/ml) and rhRANKL (25 ng/ml) (R&D system), absent or present 25 % CM from NPC cell lines as described above. After 7–10 days of culture, the cells were fixed with 3.7 % formaldehyde and TRAP staining was carried out. Positive staining cells that contained three or more nuclei were counted as osteoclast-like cells. All osteoclast formation was scored from three independent experiments. Immunofluorescence staining Clinical specimen slides were heated at 57 °C for 2 h, and dewaxed, hydrated, and washed with PBS. Then, the slides were blocked with goat serum (ZSGB-Bio, Beijing, China) at room temperature for 30 min. After washing, the slides were incubated with primary antibodies to CCR2 or CXCR6 (Abcam) at 37 °C for 1 h, and then washed with PBS, and finally were incubated with Alexa Fluor donkey anti-rabbit IgG secondary antibodies at 37 °C for 1 h, followed by counterstaining of the cell nucleus by DAPI and observation under a fluorescence microscope.
Western blot
Statistical analysis
Cell lysates from the NPC cells were prepared using standard procedures [15]. The blots were incubated with primary antibody of phospho-p70S6k, phospho-ERK1/2, c-Myc, p70S6K, ERK1/2, and cyclin B1. As control for equal loading of the proteins, immunoblots for β-actin (Sigma) were performed.
All statistical analyses were done by SPSS version 16.0 for Windows. ANOVA was used for comparisons among three groups or more. Student’s t test and Fisher’s exact test were used for comparisons between two groups. Differences with a P
RESEARCH ARTICLE
MEK inhibitor diminishes nasopharyngeal carcinoma (NPC) cell growth and NPC-induced osteoclastogenesis via modulating CCL2 and CXCL16 expressions Yu Zhu 1,2 & Chunlin Zou 1,2 & Zhe Zhang 3 & Chao-Nan Qian 4 & Xin Yang 1,2 & Junlin Shi 1,2 & Yudui Xia 1,2 & Jian Zhang 1,2,5 & Yi Lu 1,2
Received: 28 March 2015 / Accepted: 19 May 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Nasopharyngeal carcinoma (NPC) is a common malignancy in southern China and Southeast Asia. NPC frequently metastasizes to the bone in advanced patients resulting in high mortality. The molecular mechanisms for NPC development and cancer-induced bone lesions are unclear. In this study, we firstly determined chemokine receptor CCR2 and CXCR6 expressions in clinical specimens and CNE2, SUNE1, CNE1, and HK1 cell lines. Then, we measured chemokine CCL2 and CXCL16 production in these NPC cell lines by ELISA. Expression levels of these chemokines and their receptors were observed to positively correlate with tumor aggressiveness. Furthermore, U0126 (MEK inhibitor) was used to treat these NPC cell lines. CCL2 and CXCL16 expression levels and cell proliferation were significantly inhibited by U0126 in a dose- and time-dependent manner.
Finally, we collected conditioned medium (CM) from NPC cell cultures in the presence of U0126 treatment. When mouse bone marrow non-adherent cells were treated with the CM, the numbers of multinucleated osteoclast formation were dramatically diminished. These results indicate that MEK inhibitor diminishes NPC cell proliferation and NPC-induced osteoclastogenesis via modulating CCL2 and CXCL16 expressions. This study provides novel therapeutic targets such as CCL2/CCR2 and CXCL16/CXCR6 for advanced NPC patients. Keywords CCL2/CCR2 . CXCL16/CXCR6 . U0126 . Osteoclastogenesis . Nasopharyngeal carcinoma
Introduction * Jian Zhang [email protected] * Yi Lu [email protected] 1
Key Laboratory of Longevity and Aging-related Diseases, Ministry of Education, Nanning, Guangxi, China
2
Center for Translational Medicine, Guangxi Medical University, No.22 Shuangyong Road, 1416 Pharmacology & Biomedical Sciences Building, Nanning, Guangxi 530021, China
3
Department of Otolaryngology Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi 530021, China
4
State Key Laboratory of Oncology in South China and Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, Guangdong 510060, China
5
Department of Pathology and Internal Medicine, University of Michigan, Ann Arbor, MI, USA
Nasopharyngeal carcinoma (NPC) is one of the most common cancers in southern China and Southeast Asia [1, 2]. Radiation therapy has become one of the main treatments for NPC patients. Addition of chemotherapy is recommended for advanced NPC patients [3]. Locoregional relapse and distant metastasis are key factors that cause failure of NPC treatment [4]. There is about 5 % NPC patients when they were initially diagnosed had already had distance metastasis. About 20– 40 % of patients would appear distant metastases within 5 years after radical radiation therapy. The incidence of distant metastases increases along with higher clinical stages [5, 6]. The bone has been demonstrated as a common site of NPC distant metastasis, followed by the lung and liver. NPC skeletal metastasis mostly refers to the spine, thorax, and pelvis bone. Patients with bone metastasis suffer from severe bone pain, pathologic fractures, and spinal cord compression symptoms [7], and often cause multiple bone lesions including
Tumor Biol.
osteolytic, osteoblastic lesions, or mixture of both. The interaction of tumor cells with bone microenvironment plays important roles in NPC development. Paget proposed the Bseed and soil hypothesis^ in 1889 to account for the predilection of metastasis to certain organs [8–10]. Accumulating evidence has shown that certain cytokines and chemokines, produced by tumor cells and their microenvironment, lead to a modulation of tumor microenvironment that is suitable for tumor cell growth and thus protecting tumor cells from immune cellmediated attacks [11]. Chemokines play a central role in the tumor-bone microenvironment. Certain chemokines and their corresponding receptors have been suggested as important mediators in the regulation of angiogenesis, growth, invasiveness, and metastasis of tumors [12–14]. Chemokine (C-C motif) ligand 2 (CCL2, also known as monocyte chemotactic protein-1, MCP-1) plays a critical role in the recruitment and activation of monocytes during acute inflammation and carcinogenesis. CCL2 can be produced by bone cells, such as human bone marrow endothelial cells, osteoblasts, osteoclasts, as well as tumor cells [15]. Studies have revealed that CCL2 was overexpressed in many cancers including melanoma, ovarian, breast, prostate, esophageal, gastric, renal cell, lung, colon, and papillary thyroid carcinomas [16]. CCL2-induced prostate cancer (PCa) PC3 and VCaP cancer cell proliferation via activation of phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway [17]. CCL2-enhanced RhoA expression and activity through Smad3 and MEK signaling pathway to regulate breast cancer cell motility and survival [18]. Our previous study has shown that CCL2, in addition to interleukin-8 (IL-8), mediated PCa CM-induced osteoclastogenesis and bone resorption. This effect could be partially blocked by CCL2 neutralizing antibody [19]. Recently, a study had reported that higher levels of soluble CCL2 were detected in the serum of NPC patients and proposed that CCL2 might be prognostic marker to predict bone invasion, post-treatment distant metastasis, and poor overall survival [20]. The chemokine receptor CXCR6 and its ligand CXCL16 were found to be overexpressed in breast cancer tissues and cell lines, prostate cancer tissue, and cell lines, as well as pancreatic ductal adenocarcinoma [12, 21, 22]. It has been shown that CXCR6 is expressed in primary and metastatic melanoma [23]. CXCL16/ CXCR6 strengthened the proliferative capability of melanoma cells [24]. Hu et al. showed that CXCR6/ CXCL16 play an important role in prostate cancer bone metastasis [25]. Another study had confirmed that CXCR6/AKT/mTOR pathway plays a central role in the development of prostate cancer [26]. Blocking the CXCR6/AKT/mTOR signaling pathway may be
beneficial for preventing metastasis and providing a more effective therapeutic strategy for prostate cancer [12]. Overall, CCL2/CCR2 and CXCL16/CXCR6 may play crucial roles on growth, invasiveness, and metastasis of certain tumors, particularly tumors with bone metastasis [16]. However, the functions and regulation of CCL2/CCR2 and CXCL16/CXCR6 in NPC progression remain unknown. In addition, the molecular basis of NPC development and cancer-induced bone lesions are still unclear. In current study, we hypothesize that CCL2 and CXCL16, via MEK/ERK signaling pathway, contribute to NPC cell growth and NPC-induced osteoclast differentiation and formation.
Materials and methods Reagents U0126, MAPK/ERK signaling inhibitor, was purchased from Cell Signaling Technology (Boston, MA, USA). Antibodies against phospho-p70S6k, phospho-ERK1/2, c-Myc, p70S6K, ERK1/2, CDK1, and cyclin B1 were purchased from Cell Signaling Technology. Phosphatase inhibitor cocktails 2 and 3 were purchase from Sigma (St. Louis, MO, USA). Antibodies against CCR2 and CXCR6 were purchased from Abcam (Cambridge, Cambs, UK). Alexa Fluor donkey anti-rabbit IgG (H+L) secondary antibody was the product of Invitrogen (Carlsbad, CA, USA). Recombinant human M-CSF and rhRANKL were purchased from R&D systems (Minneapolis, MN, USA). All chemical reagents were purchased from Sigma. Patients Fifty-one cases of NPC clinical tissue paraffin sections were obtained from the First Affiliated Hospital of Guangxi Medical University and Sun Yat-sen University Cancer Center. All NPC clinical specimens, including 15 cases of nonkeratinizing squamous cell carcinoma, and 36 cases of nonkeratinizing carcinoma (undifferentiated), have been confirmed by histopathologic examination. All procedures involving clinical specimens were performed in accordance with protocols approved by Institutional Research Board, Guangxi Medical University. Cell cultures and treatments NPC cell lines, CNE2 and SUNE1, CNE1, and HK1 were cultured in DMEM (Invitrogen) media supplemented with 10 % fetal bovine serum (FBS). For U0126 treatment assay, NPC cell lines were planted at a density of 1×105/well into 12-well plates and incubated for overnight. The medium was
Tumor Biol.
changed to serum-free DMEM with indicated doses of U0126. Then the cells were continued to culture for another 24 h. The cell lysate and CM in each well was collected, and total protein was extracted. Reverse transcription-PCR Total RNA was extracted from NPC cell lines, using TRIzol reagent (Invitrogen), and then subjected to reverse transcription (RT)-PCR for detection of CCL2, CCR2, CXCL16 and CXCR6 mRNA expression levels. PCR primers for CCL2 consisted of sense 5′-TAGCAGCCACCTTCATTC-3′ and antisense 5′-CTTGGGGTCAGCACAGAT-3′. PCR primers for CCR2 consisted of sense 5′-CGTTAGAAACAGGAGCAG3′ and antisense 5′-ACAGGGCAACTAATGGTA-3′. PCR primers for CXCL16 consisted of sense 5′-CTGTACCCGC AACGAAGG-3′ and antisense 5′-AGGCAAGGGACTGA GGAGG-3′. PCR primers for CXCR6 consisted of sense 5′GGCGACTAAGTCATTCCG-3′ and antisense 5′-GCTGCC TTGGGTGTTGTA-3′. The expression of glyceraldehyde-3phosphate dehydrogenase (GAPDH) was used as internal control. RT-PCR was performed as described previously [17, 26].
cells (1×105/well) in 96-well plates for 10 days. The cells were incubated with rhM-CSF (10 ng/ml) and rhRANKL (25 ng/ml) (R&D system), absent or present 25 % CM from NPC cell lines as described above. After 7–10 days of culture, the cells were fixed with 3.7 % formaldehyde and TRAP staining was carried out. Positive staining cells that contained three or more nuclei were counted as osteoclast-like cells. All osteoclast formation was scored from three independent experiments. Immunofluorescence staining Clinical specimen slides were heated at 57 °C for 2 h, and dewaxed, hydrated, and washed with PBS. Then, the slides were blocked with goat serum (ZSGB-Bio, Beijing, China) at room temperature for 30 min. After washing, the slides were incubated with primary antibodies to CCR2 or CXCR6 (Abcam) at 37 °C for 1 h, and then washed with PBS, and finally were incubated with Alexa Fluor donkey anti-rabbit IgG secondary antibodies at 37 °C for 1 h, followed by counterstaining of the cell nucleus by DAPI and observation under a fluorescence microscope.
Western blot
Statistical analysis
Cell lysates from the NPC cells were prepared using standard procedures [15]. The blots were incubated with primary antibody of phospho-p70S6k, phospho-ERK1/2, c-Myc, p70S6K, ERK1/2, and cyclin B1. As control for equal loading of the proteins, immunoblots for β-actin (Sigma) were performed.
All statistical analyses were done by SPSS version 16.0 for Windows. ANOVA was used for comparisons among three groups or more. Student’s t test and Fisher’s exact test were used for comparisons between two groups. Differences with a P
