Abstract
Aim: To characterize the chemokine pattern in metastatic salivary adenoid cystic carcinoma (SACC). Materials and Methods: Real-time polymerase chain reaction (RT-PCR) was used to compare chemokine and chemokine receptor gene expression in two SACC cell lines: SACC-83 and SACC-LM (lung metastasis). Chemokines and receptor genes were then screened and their expression pattern characterized in human tissue samples of non-recurrent SACC and recurrent SACC with perineural invasion. Results: Expression of chemokine receptors C5AR1, CCR1, CCR3, CCR6, CCR7, CCR9, CCR10, CXCR4, CXCR6, CXCR7, CCRL1 and CCRL2 were higher in SACC-83 compared to SACC-LM. CCRL1, CCBP2, CMKLR1, XCR1 and CXCR2 and 6 chemokine genes (CCL13, CCL27, CXCL14, CMTM1, CMTM2, CKLF) were more highly expressed in tissues of patients without tumor recurrence/perineural invasion compared to those with tumor recurrence. CCRL1 (receptor), CCL27, CMTM1, CMTM2, and CKLF (chemokine) genes were more highly expressed in SACC-83 and human tissues of patients without tumor recurrence/perineural invasion. Conclusion: CCRL1, CCL27, CMTM1, CMTM2 and CKLF may play important roles in the development of tumor metastases in SACC.
- Adenoid cystic carcinoma
- salivary tumors
- distant metastasis
- metastatic salivary adenoid cystic carcinoma
Salivary adenoid cystic carcinoma (SACC) is the second most common malignant tumor of the salivary glands characterized by slow, relentless growth, local recurrence, and latent distant hematogenous dissemination primarily to the lung (1). Involvement of regional lymph nodes is relatively rare but incidence of perineural invasion is high, increasing recurrence risk. Most authors have found that lymph node involvement is a risk factor for subsequent distant metastasis (2-5). According to Ko et al., 75% of patients with initial nodal involvement eventually developed distant metastasis. Patients with lung metastasis have a poor prognosis (6).
The development of distant metastatic disease is the chief cause for mortality (7, 8). Primary treatment is complete surgical resection when feasible with adjuvant radiotherapy. The role of chemotherapy is debatable. Treatment of metastatic ACC has been difficult to date due to lack of specific targets for metastatic cells (1). Though the steps that must occur in the metastatic event are well characterized, it remains unclear why or how ACC cells ultimately “choose” or are ”chosen” to migrate to a specific metastatic site. A mounting body of evidence suggests that cytokine-like molecules called chemokines play a significant role in directing the cellular traffic in metastatic melanoma, lung, breast and ACC cancers (9-15).
In this study, we aimed to characterize the SACC chemokine and receptor pattern that is seen in SACC human cell lines and tissue samples with the hope that a detailed characterization of the chemokine and receptor pattern in lung metastases from SACC could lead to development of novel therapeutic options to prevent the development of disseminated disease or to detect the presence of circulating tumor cells with metastatic potential.
Materials and Methods
Cell lines and human tissues. SACC-83 and -LM (lung metastasis) cells were a gift from Central Laboratory, Peking University School and Hospital of Stomatology, Peking P.R. China. SACC-83 has a low lung metastasis rate, while SACC-LM has a high lung metastatic rate. SACC-83 and SACC-LM cells were cultured in Dulbecco's modified eagle medium (Gibco, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin and 100 U/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Six SACC human tissue samples (3 with and 3 without recurrence and perineural invasion) were received from Wake Forest University Comprehensive Cancer Center Tissue Procurement Core Lab, Wake Forest School of Medicine Tumor Bank, Winston Salem, NC, USA. The study was approved by the Wake Forest University Health Sciences Institutional Review Board.
Gene table: RT2 profiler polymerase chain reaction (PCR) array.
RNA extraction and microarrays. Total RNA was isolated from SACC-LM, SACC-83 cells and human SACC tissues using Trizol reagent (Life Technologies, Carlsbad, CA, USA). Generally, cell lines and tissue samples were homogenized in Trizol reagent and incubated for 5 min at room temperature. Cell debris was removed by centrifuge and supernatant solution was transferred to new tube. Chloroform was added into the Trizol reagent with a ratio (1:5), vortexed for 15 sec, incubated at room temperature for 3 min and centrifuged at 12,000 × g for 15 min at 4°C. The upper aqueous phase was carefully removed into new tube. Then the same volume of isopropyl alcohol was added, mixed up and down for 10 sec, incubated at room temperature for 10 min and centrifuged at 12,000 × g for 15 min at 4°C. RNA pellets were visible on the side and bottom of the tube, then washed once with 75% ethanol and centrifuged at 7,500 × g for 5 min at 4°C. RNA pellets were air-dried for 5-10 min, dissolve with nuclease-free water and quantified with NanoDrop 1000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA).
Reverse transcription for cDNA synthesis and real-time quantitative polymerase chain reaction (qPCR) for chemokine and receptor detection and quantification. RT2 First Strand Kit (QIAGEN, Valencia, CA, USA) was used for cDNA synthesis. RT2 Profiler PCR Array (QIAGEN) was used for real-time qPCR quantification of known human chemokines and receptors (genes tested in this study are shown in Table I). Amplification reactions were performed in triplicate in the Agilent MX 3000P qPCR System (Agilent Technologies, Inc, Folsom, CA, USA). All the procedures were performed strictly following the manufacturer's instructions. Each RNA expression was normalized to the housekeeping gene as internal control provided by the manufacturer.
Data analysis. The empirical Bayes statistics implemented in Bioconductor package Limma was used for the data analysis and detailed method as previously described (16). The modified Student's t-test was used to determine differentially expressed chemokine and receptors between SACC-83 and SACC-LM cell lines as well as the six human samples coded with or without SACC recurrence and perineural invasion.
Results
Expression of chemokine receptors C5AR1, CCR1, CCR3, CCR6, CCR7, CCR9, CCR10, CXCR4, CXCR6, CXCR7, CCRL1 and CCRL2 was higher in SACC-83 compared to SACC-LM cell lines, p<0.05 (Table II). Table III demonstrates the statistically significant chemokine gene expression between SACC-83 and SACC-LM (p<0.05). Six human SACC were analyzed for expression of chemokines and receptors. Three samples were recurrent tumors and all had perineural invasion (See Table IV for detailed information of patients). CCRL1, CCBP2, CMKLR1, XCR1 and CXCR2 receptors were more highly expressed in tissue samples of patients without tumor recurrence and perineural invasion compared to those with (Table V). CCL13, CCL27, CXCL14, CMTM1, CMTM2 and CKLF chemokine genes were more highly expressed in tissue samples of patients without tumor recurrence and perineural invasion compared to those with tumor recurrence (Table VI).
Discussion
The complexity of the events that incite and control the migration of cells to metastatic sites remains a considerable challenge in oncology research. Recent studies have demonstrated that tumor cells express functionally active chemokine receptors and that expression of these receptors appears to regulate cellular functions associated with the process of metastasis (9, 14, 15). Chemokines are a superfamily of small cytokine-like proteins that selectively attract and activate different cell types (12, 13). The CXC chemokine receptor CXCR4 is expressed in several cancer types, including breast cancer (11), melanoma (13) and lung cancer (13). Studies have suggested that, in these cancer types, CXCR4 may play a role in metastases. Furthermore, CXCR4 receptor expression has been implicated in metastatic spread in vivo, in animal models of breast cancer and melanoma (13, 14). A group of researchers in Germany, working with ACC and squamous cell carcinoma (SCCA) cell lines, discovered that ACC and SCCA expressed a distinct, non-random pattern of chemokine receptors that influenced their metastatic behavior. The chief finding was that ACC cells expressed high levels of CXCR4 both in vitro and in pathologic tumor specimens, whereas CXCR4 expression was undetectable or low in SCC. Chemokine receptor CCR7 was abundantly expressed in primary tumors and lymph node metastases of SCCA but restricted in ACC (17). The authors concluded that significant differences in chemokine expression correlated with known clinical behavior of these two head and neck tumor entities.
Analysis of chemokine receptor mRNA expression between salivary adenoid cystic carcinoma (SACC)-LM and SACC-83 cells using real-time quantitative polymerase chain reaction (PCR), with associated chemokines.
These data taken collectively suggest that chemokine receptor status in primary ACC tumors could predict organ-specific metastases in vivo. In order to gain incite into the process of metastases in ACC, others have attempted to create clinically relevant animal models of metastatic disease. Recently, Liu et al. used 2 ACC cell lines, one with a low and one with a high metastasis rate, and found that autocrine expression of epiregulin-induced epidermal growth factor receptor activation promotes lung metastasis via epithelial-mesenchymal transition (18). We aimed in this study to use the same SACC cell lines and human SACC samples to study the chemokine receptor pattern of these metastatic foci. In our study, we found differential expression of a panel of chemokines and receptors between two SACC metastatic cell lines. These chemokines and receptors may provide direction into further studies investigating chemokine-driven metastasis. Chemokines and receptors were also analyzed in human tissue looking at differential expression between perineural positive and negative samples and recurrent and non-recurrent in order to investigate chemokine-driven local tissue invasion and progression. We found differential expression of a panel of chemokines and receptors between these low and high locally aggressive groups. Interestingly, we found a panel of chemokines and receptors that showed the same pattern of expression between the SACC-LM/SACC-83 group and the recurrent/non-recurrent human tissues. CCRL1, CCL27, CMTM1, CMTM2 and CKLF demonstrated lower expression in both the SACC-LM group, as well as the tumor recurrence/perineural invasion group. As previously stated, the SACC-LM is well established as having high metastatic potential. Similarly those patients with tumor recurrence and/or perineural invasion represent a higher risk group with increased propensity to poor outcomes when compared to non-recurrent. Finding a panel of chemokines and receptors that were associated with lower expression in both high-risk groups (SACC-LM and recurrent tumor/perineural invasion) certainly suggests that these may play a more significant role in chemokine-driven pathways of metastasis and local progression. This may also suggest an activity of gene suppression in those patients with distant metastases and perineural invasion. Further elucidating these chemokines and their expression patterns could lead to the development of therapeutic targets to limit local and distant spread. In addition, based on these profiles, drug development could be directed toward the prevention of disseminated disease with considerable impact on mortality from ACC.
Analysis of chemokine mRNA expression between salivary adenoid cystic carcinoma (SACC)-LM and SACC-83 cells using real-time quantitative polymerase chain reaction (PCR).
Human salivary adenoid cystic carcinoma (SACC) samples.
Analysis of chemokine receptor mRNA expression in human salivary adenoid cystic carcinoma (SACC) tissues.
Analysis of chemokine mRNA expression in human salivary adenoid cystic carcinoma (SACC) tissues.
Conclusion
CCRL1, CCL27, CMTM1, CMTM2 and CKLF may play important roles in the development of tumor metastases in salivary ACC. Further investigation into the mechanisms underlying this chemokine-mediated tumor cell migration and invasion could lead to development of novel prognostic and therapeutic options to predict metastatic potential, prevent the development of metastases or treat disseminated disease.
Acknowledgements
Jeff Chou Ph.D, is acknowledged for his assistance with statistical analysis.
- Received June 16, 2016.
- Revision received July 4, 2016.
- Accepted July 6, 2016.
- Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
