Different Cytokine and Chemokine Expression Patterns in Malignant Compared to Those in Nonmalignant Renal Cells

Objective Cytokines and chemokines are widely involved in cancer cell progression and thus represent promising candidate factors for new biomarkers. Methods Four renal cell cancer (RCC) cell lines (Caki-1, 786-O, RCC4, and A498) and a nonmalignant renal cell line (RC-124) were examined with respect to their proliferation. The cytokine and chemokine expression pattern was examined by a DNA array (Human Cytokines & Chemokines RT2 Profiler PCR Array; Qiagen, Hilden, Germany), and expression profiles were compared. Results Caki-1 and 786-O cells exhibited significantly increased proliferation rates, whereas RCC4 and A498 cells demonstrated attenuated proliferation, compared to nonmalignant RC-124 cells. Expression analysis revealed 52 cytokines and chemokines primarily involved in proliferation and inflammation and differentially expressed not only in malignant and nonmalignant renal cells but also in the four RCC cell lines. Conclusion This is the first study examining the expression of 84 cytokines and chemokines in four RCC cell lines compared to that in a nonmalignant renal cell line. VEGFA, NODAL, and BMP6 correlated with RCC cell line proliferation and, thus, may represent putative clinical biomarkers for RCC progression as well as for RCC diagnosis and prognosis.


Introduction
Renal cell cancer (RCC) represents the deadliest neoplasm of the urinary tract, due to the fact that a majority of patients are diagnosed at a very advanced stage [1,2]. Tumor progression is based on various molecular mechanisms, and thus, further examinations of RCC tumor biology are necessary in order to understand the progression and, furthermore, to identify novel biomarkers for diagnosis and prognosis of RCC [1,[3][4][5].
Among other mechanisms, cytokines and chemokines are suspected to play a crucial role in proliferation and progression of various malignancies. For instance, CCL11 controls cancer cell growth and invasion in ovarian cancer and CCL21 as well as CCR7 represent pivotal regulators of bladder cancer progression [6,7]. Moreover, IL17, as a representative of interleukins, is associated with lung and colorectal cancer progression and predicts poor prognosis in breast cancer [8][9][10]. Even though some studies suggested an impact in RCC, the role of cytokines and chemokines in RCC progression is poorly understood [1,11,12]. From a clinical point of view, cytokine and chemokine secretion into the blood stream makes them very suitable as noninvasive clinical markers.
In the study presented here, we used an RCC model system consisting of four malignant RCC cell lines compared to a nonmalignant renal cell line. After classification of the RCC cells in cell lines of high and low cell growth rates, a transcriptional profiling specific for 84 cytokines and chemokines (Table 1) was carried out and compared to the  2 Profiler PCR Array (Qiagen). Abbreviation (symbol) and full name/function (description) of the 84 cytokines and chemokines analyzed by the PCR array. The column "detectable" indicates whether the factor was detected (+) or not detected (−) within this study.

Results
In contrast to the elevated proliferation rates of Caki-1 and 786-O cells compared to those of nonmalignant RC-124 cells, both, RCC4 and A498 cells, showed a markedly reduced cell growth (Figure 1). These differences in proliferation prompted us to investigate whether cytokines and chemokines may control the individual growth properties of the RCC cell lines.
Performing the RT 2 Profiler PCR Array Human Cytokines & Chemokines, the expression of 84 target mRNAs  Furthermore, a global normalized PCA was performed and used as quality check. PCA was carried out on the concentrations of all of the detected cytokines and chemokines in all of the five cell lines (Figure 3) dividing cancer and noncancer cells (PC1) and defining individual characteristics of all cell lines (PC2). As expected, the five cell lines used in this study were divided correctly into two major groups corresponding to malignant versus nonmalignant physiology.
Five members of the TNF superfamily were detected (Figure 4(c)); however, only TNFSF13B was strongly overexpressed in all of the four malignant cell lines, dominated by an upto 25-fold overexpression in Caki-1 and RCC4 cells. Considering the family of interleukins (Figure 4(d)), a total of 13 different factors were detected with an upregulated expression of the interleukins IL1A, IL1B, and IL1RN in each of the 4 malignant cell lines.

Discussion
The process of tumorigenesis is driven by several mechanisms including an increased proliferation, angiogenesis, and immunomodulation [13]. Cytokines and chemokines, in general, are primarily regulators of immune defense. CCL5, in particular, supports leukocyte diapedesis by integrin induction [14,15]. Interestingly, CCL5 expression in all four RCC cell lines was highly upregulated. Since tumorigenesis is associated with inflammatory response, elevated CCL5 secretion by RCC cells may be part of cancer-related inflammation. Corresponding to this observation is the finding that the expression levels of proinflammatory interleukins IL1A and IL1B are increased in all of the four RCC cell lines. Interestingly, overexpression of the anti-inflammatory IL1RN [16] could only be observed in the lowly proliferative RCC4 and A498 cells. This could mean that IL1RN may interfere with antiproliferative pathways or that anti-inflammatory processes generally attenuate cell growth.
Within the TNF superfamily, TNFSF13B is significantly overexpressed in all four RCC cell lines. Looking at the large differences in cellular growth of highly proliferative Caki-1 and 786-O and lowly proliferative RCC4 and A498 cell lines, however, classification of TNFSF13B as a proliferative factor in RCC, as has been done in previous studies [17], could not be clearly confirmed.
Vice versa, the expression of BMP6 was significantly increased in the lowly proliferative cell lines RCC4 and A498, whereas an expression in highly proliferative Caki-1 and 786-O cells was not detectable. Since BMP6 is considered to be a tumor suppressor in breast cancer [24], upregulation of BMP6 in RCC cells may contribute to attenuated cell growth of RCC4 and A498 cells compared to that of the nonmalignant cell line RC-124.    IL1B  IL8  IL11  IL1RN  IL2  IL18  IL23A  IL27  IL12A  IL15  IL1A  IL6  Taken together, there are a number of cytokines and chemokines which are differentially expressed not only in malignant and nonmalignant renal cells but also in the four RCC cell lines. These results may reflect heterogeneity among RCC patients as well as among RCC subtypes. In addition, this may suggest a functional substitution of members of the cytokine/chemokine system due to an activation of alternative factors involved in cytokine/chemokine signalling. Notably, two proliferative factors (VEGFA, NODAL) and a tumor suppressive factor (BMP6) have been found which may significantly contribute to the malignant phenotype of RCC and thus may be suitable as novel marker proteins in RCC diagnosis and therapy.

Conflicts of Interest
The authors have no conflicts of interest to disclose.

Authors' Contributions
Nadine Gelbrich and Hannes Ahrend contributed equally to this study.