Introduction: interleukin-6 signaling

Colorectal cancer represents a significant cause of morbidity and mortality worldwide. The molecular pathogenesis of colorectal cancer is still poorly understood. At least in some subtypes of colon cancer the immune system seems to have tumor-promoting activity. One approach to get deeper insight into the biology of colon cancer is the evaluation of circulating cytokines and cytokine receptors, which have received a great deal of attention as potential diagnostic and prognostic markers in cancer patients. One important cytokine in colon cancer is interleukin-6 (IL-6) with its pleiotropic properties. After IL-6 is secreted from the activated monocyte, macrophage, or tumor cell, it can act on target cells. IL-6 first binds to the membrane-bound IL-6 receptor (IL-6R; mainly expressed on hepatocytes, neutrophils, monocytes/macrophages, and some lymphocytes). This complex of IL-6 and IL-6R associates with the signal-transducing membrane protein gp130, inducing its dimerization and initiation of signaling (reviewed in [1], Fig. 1a). Besides the membrane-bound IL-6R, a naturally occurring soluble form of the IL-6R (sIL-6R) is generated by two independent mechanisms, limited proteolysis of the membrane protein, and translation from an alternatively spliced mRNA [24]. Interestingly, the sIL-6R together with IL-6 stimulates cells that only express gp130 [57], a process named transsignaling (Fig. 1b).

Fig. 1
figure 1

The IL-6R complex and transsignaling process. a Classical IL-6 signaling via the membrane-bound IL-6R. b Transsignalling via generation of sIL-6R. Both signaling processes are dependent on the membrane-bound gp130

Dimerization of gp130 is followed by the rapid activation of tyrosine kinases of the Janus Kinase (Jak) family for initial signal transduction [1]. Jak activation leads to activation of downstream signaling pathways, including phosphatidylinositol 3′-kinase, and mitogen-activated protein kinases. An important target of gp130 signaling is the oncogene STAT3 [810]. STAT3 is a member of the STAT family of latent cytoplasmic transcription factors. After gp130 activation, STAT3 becomes tyrosine phosphorylated, homo- or heterodimerizes, and translocates to the nucleus where it induces gene transcription. STAT3 transcriptionally activates numerous important cellular genes involved in growth regulation, differentiation, and cell cycle, including c-myc and bcl-2 [8].

Interleukin-6 effects on colorectal cancer cells

IL-6 seems to have significant effects on colon cancer cells. IL-6 has been demonstrated to promote the growth of colon cancer cells in vitro [11]. Moreover, IL-6 has been implicated to promote cancer growth, because it has been shown to enhance colony formation of human colon carcinoma cells in vitro in a dose-dependent manner [11]. IL-6 increases invasiveness of colon cancer cells [12] and likely promotes secondary tumor formation through its angiogenic potency. Brozek et al. [13, 14] could show that IL-6 level is low in normal human mucosa and rises only moderately during progression from adenomatous to low grade (G1 and G2) cancer lesions. High levels of IL-6 were observed in rather undifferentiated lesions (G3–G4). These results imply that IL-6 can accelerate tumor progression towards malignancy.

Previous animal studies demonstrated a functional relationship between transforming growth factor (TGF)-ß signaling in tumor-infiltrating cells and IL-6 transsignaling in colon carcinogenesis. It could be shown that TGF-ß production in tumor-infiltrating T lymphocytes suppresses tumor growth in the colon via inhibition of IL-6 production and subsequent IL-6 signal transduction. IL-6 signal transduction was mediated by the soluble rather than the membrane bound IL-6R. Furthermore, it was shown that IL-6 transsignaling is essential for colon carcinogenesis in mice lacking the TGF-ß receptor II and can induce phosphorylation of STAT3 and cell cycle progression in dysplastic epithelial cells [15, 16].

Results

IL-6 levels in colorectal cancer patients and healthy controls

Concerning the significance of IL-6 in colorectal cancer patients, published results in PubMed are relatively consistent (Table 1). We found seven different studies comparing serum IL-6 levels in colon cancer patients with levels of healthy subjects, e.g., the investigation of Galizia et al. (2002) [17]. The authors found IL-6 levels to be more elevated in cancer patients (n = 50, 9.3 ± 2.1 pg/ml; median, 8.8) than in the 25 healthy subjects (4.4 ± 0.8 pg/ml; median, 4.2). In addition to the elevated IL-6 level, they found a relationship between IL-6, tumor status, and presence of distant metastases. Furthermore, they compared preoperative IL-6 level of surgically cured patients with level of patients subjected to noncurative resection and found the latter to be significantly higher (10.8 ± 2.2, n = 20) than that of the curative patients (8.3 ± 1.0, n = 30). Similar results were shown by Dymicka-Piekarska et al. (2007) [18]. They measured IL-6 levels in 42 colorectal cancer patients and 38 healthy volunteers and found out that IL-6 levels were significantly increased in patients (stages I and II (n = 24), 2.98 ± 1.49 pg/ml; stage III (n = 18), 4.15 ± 2.45 pg/ml) as compared with the control group (1.31 ± 0.55 pg/ml). Significantly elevated IL-6 levels in patients are also found by studies of Kinoshita et al. (70 patients, 35.7 ± 69.0 pg/ml vs. 34 healthy volunteers, 4.3 ± 1.0 pg/ml) [19], Chung and Chang (164 patients, median value 11.89 pg/ml vs 20 healthy volunteers, median value 3.41 pg/ml) [20] and Ueda et al. (24 patients, 5.96 ± 1.89 pg/ml vs 64 healthy subjects, <3.1 pg/ml) [21]. Nikiteas et al. [22] found elevated median IL-6 levels (n = 74, 8.11 pg/ml) in colorectal cancer patients compared with normal individuals (3.52 pg/ml) in a Greek population. Kaminska et al. [23] analyzed the levels of different cytokines, among them IL-6, in 50 healthy volunteers and 157 patients with previously untreated colorectal cancer. The median IL-6 level of the patients (2.8 pg/ml) was significantly higher than those of the healthy persons (0.8 pg/ml).

Table 1 Association of IL-6 serum concentration and clinical parameters/colon tumor features

Interleukin-6 levels and different tumor stages

The idea that high serum IL-6 is associated with colorectal carcinoma is supported by further studies analyzing IL-6 and different tumor stages. In Table 1, the results are summarized according to the number of analyzed patients in each study. Comparing different stages of colorectal cancer, Esfandi et al. [24] found a significant association between increasing serum IL-6 levels and staging of the tumor (stage I, 2.75 ± 0.2 pg/ml, n = 11; stage II, 4.14 ± 0.51 pg/ml, n = 8; stage III, 5.86 ± 1.68 pg/ml, n = 23; stage IV, 9.56 ± 0.99 pg/ml, n = 8). The authors conclude that serum IL-6 level may reflect the proliferative activity of the tumor in patients with colorectal carcinoma. Similar results were found by Belluco et al. [25]. The study of Kaminska et al. [23] analyzed the levels of different cytokines, among them, IL-6 in 50 healthy volunteers and 157 patients with previously untreated colorectal cancer. IL-6 increased with tumor stage (I, 1.7; II, 3.2; III, 2.8; IV, 4.9 pg/ml; all median values) and also with infiltration of the colon wall. These data support their preliminary studies on pre- and postoperative serum cytokine levels, e.g., IL-6, in colorectal carcinoma patients [26]. Similar data were found by Chung and Chang [20]. The proportion of patients with IL-6 levels ≥12 pg/ml increased in a Dukes’ stage-related manner. Their data confirm those previously reported by Ueda et al. [21] on a group of 24 patients with colorectal carcinoma showing significantly higher concentrations of IL-6 in Dukes’ D patients (n = 8, 12.16 ± 4.99 pg/ml) compared with those in Dukes’ A–C patients (n = 16, 2.86 ± 0.71 pg/ml).

Three other studies did not find a close connection between increasing tumor stages and increasing IL-6 concentrations, two medium-sized studies [19, 22] and a small-sized study [18] (Table 1). Although the study of Nikiteas et al. [22] did not find a significant association of IL-6 and tumor stages, the authors found positively correlating results for higher serum IL-6 levels in patients compared with healthy controls, an association of higher IL-6 levels with increasing tumor size and even for higher IL-6 levels and decreased survival. Kinoshita et al. [19] found similar results. They did not find a significant association of high IL-6 and different stages, but IL-6 levels showed a trend to higher levels in stage IV tumors (I, 31.5 ± 76.2; II, 23.2 ± 40.0; III, 22.7 ± 20.2; IV, 62.0 ± 115.2 pg/ml). Furthermore, they found correlating results for higher IL-6 levels in colon cancer patients compared with controls, and an association of higher IL-6 levels and increasing tumor size. Furthermore, patients with liver metastasis had higher IL-6 levels than patients without liver metastasis. No significant association of high IL-6 levels and increasing tumor stages was also found by the relatively small-sized study of Dymicka-Piekarska et al. [18], but also a trend could be seen to higher levels and increasing stages (I/II, 2.98 ± 1.49; III, 4.15 ± 2.45 pg/ml). Furthermore, they found positively correlating results for higher serum IL-6 levels in patients compared with controls.

In summary, Table 1 shows that the three largest studies reveal an association of high serum concentration of IL-6 and increasing tumor staging. Results are supported by two medium-sized and one small-sized study. Three studies showing no association of IL-6 and tumor stages showed at least a trend in IL-6 levels and/or further positive correlations of other tumor features/clinical parameters like survival or tumor size. Therefore, we conclude that high serum concentration of IL-6 is significantly associated with high colon tumor stages.

Interleukin-6 levels and tumor size

We found four studies correlating IL-6 levels with tumor size. Ueda et al. [21] found out that serum IL-6 levels were significantly higher in patients (8.78 ± 3.25 pg/ml, n = 13) whose tumors exceeding 5 cm in diameter compared with IL-6 levels of patients with smaller tumors (2.64 ± 0.96 pg/ml, n = 11). These results are supported by the study of Kinoshita et al. [19]. The authors could show that serum concentration of IL-6 correlated significantly with tumor size. Similar results were shown by Chung and Chang [20]. The maximal size of the tumor in the high (≥12 pg/ml) IL-6 group (5.29 ± 0.27 cm, n = 81) was significantly larger than that in the low IL-6 group (4.43 ± 0.19 cm, n = 83). The fourth study done by Nikiteas et al. [22] used 8 pg/ml as threshold level. Again, the maximum size of the tumor in the high IL-6 group (≥8 pg/ml, 4.8 ± 1.9 cm, n = 35) was significantly larger than that in the low IL-6 group (3.8 ± 1.3 cm, n = 39).

Interleukin-6 levels and metastasis

Three groups found significantly increased IL-6 levels in patients with liver metastasis (76.3 ± 127.3, 34.15 ± 59.51 and 12.24 ± 5.77 pg/ml, respectively) compared with patients without liver metastases (23.2 ± 32.6, 10.77 ± 23.24 and 3.38 ± 0.84 pg/ml, respectively) [1921]. One study compared IL-6 levels of patients with (n = 4) and without (n = 20) lung metastasis and found IL-6 levels of patients with lung involvement to reveal higher IL-6 levels (20.55 ± 7.92 pg/ml) than patients without lung metastasis (3.05 ± 0.73 pg/ml) [21].

Interleukin-6 levels and survival

Chung and Chang [20] analyzed the association of serum IL-6 and survival. They could show that survival of colorectal cancer patients correlated with serum IL-6 levels in a concentration-related manner. Patients with IL-6 ≥ 12 pg/ml were more often found to have advanced disease. Therefore, the authors conclude that IL-6 levels might be used as a tumor marker for monitoring the treatment course of colorectal cancer. We found four further reports analyzing the association of IL-6 and survival (Table 1). The study of Belluco et al. [25] showed that a preoperative IL-6 serum concentration of more than 10 pg/ml was a negative prognostic factor of survival. Patients with an IL-6 serum concentration of more than 10 pg/ml (n = 78) had a significantly shorter 5-year survival than patients with an IL-6 serum concentration of 10 pg/ml or less (n = 120). Similar results were published by Rich et al. [27] analyzing 80 patients with metastatic colorectal cancer. Patients with serum IL-6 level below the overall median IL-6 value had a median survival of 15.7 months, whereas a median survival of 8.8 months was found in those patients with serum IL-6 concentration above median value. Patients with high IL-6 had significantly poorer survival rate at 2 years regardless of their performance status, as estimated by Kaplan-Meier. Nikiteas et al. [22] found that patients with increased serum IL-6 concentrations (≥8 pg/ml) showed a decrease in overall survival. However, the study of Galizia et al. [17], analyzing 30 patients could not show a difference in disease-free survival between high and low IL-6 levels. Although the authors found elevated IL-6 levels in colon cancer patients compared with control and an association of increasing IL-6 and increasing tumor stages and presence of distant metastases, they did not find a difference in disease-free survival between high and low IL-6 levels.

In Table 1, results are summarized according to patients’ numbers. Two large studies with more than 100 patients found a significant association of IL-6 and survival [20, 25]. Also, two medium-sized studies showed the same results [22, 27]. The study of Galizia et al. [17] showed no correlation of IL-6 and survival. This study is a comparatively small study. One should take into consideration that only 30 patients were evaluated for IL-6 and survival. Results might be different when analyzing a larger number of patients. Therefore, we conclude that a high IL-6 level is a negative prognosticator in colon cancer in regard to survival.

Interleukin-6 levels and lymph node involvement

Concerning different aspects of the colon tumor biology like tumor stages, tumor size, involvement of liver and/or lung metastases, and patients’ survival, the overwhelming majority of studies shows a significant correlation of high IL-6 levels and colon cancer. Concerning lymph node involvement situation seems to be contradictory. The study of Chung and Chang [20] found higher IL-6 levels in patients with lymph node metastasis (n = 85, 21.44 ± 45.51 pg/ml) compared with patients without lymph node involvement (n = 79, 7.80 ± 19.09 pg/ml). Contrary to this study are the results of Nikiteas et al. [22] and Kinoshita et al. [19]. IL-6 levels of patients with and without lymph node involvement were not significantly different. Therefore, the correlation of IL-6 levels and lymph node metastasis is not clear and needs further investigation.

Discussion

In analogy to breast tumors [28], serum levels of IL-6 are increased in colon cancer patients and correlate with prognosis. The mechanism by which serum IL-6 raises is not known. One explanation might be the G/C polymorphism at position −174 in the IL-6 gene promoter region. Previously, it could be shown that the −174C allele is less efficiently translated into protein than its −174G counterpart [29]. Hefler et al. [30], analyzing ovarian cancers, found that at an early tumor stage ovarian cancer cells expressed predominantly −174C alleles, whereas at later stages the −174G polymorphism prevailed. In accordance with these results and in regard to colon cancer, Belluco et al. [31] found that colon cancer patients carrying the −174G polymorphic IL-6 gene had significantly higher IL-6 serum levels than patients with the −174C genotype. The authors conclude that this genetic variant might be a prognostic molecular marker, and it should be investigated additionally in prospective studies.

Another possible explanation for the cancer-associated IL-6 elevation might be gene amplification. In a recent publication, Tchirkov et al. [32] evaluated IL-6 gene amplification in tumors from 53 glioma patients using fluorescence in situ hybridization (FISH approach). No IL-6 gene amplification was detected in low-grade or anaplastic tumors, whereas high-level amplification was found in 15 out of 36 glioblastomas (GBM). Furthermore, GBM patients with amplified IL-6 gene had significantly shorter survival than patients without amplification. Multiple Cox analysis for overall survival, including IL-6 amplification, extent of tumor resection, and age as variables, demonstrated that IL-6 amplification was an independent factor of poor prognosis. The authors conclude that amplification of the IL-6 gene leading to its overexpression is likely one of the major factors contributing to the aggressiveness and poor response to therapies of GBM.

Summary and conclusion

The summary of published results in regard to IL-6 levels in colorectal carcinoma patients and different tumor features/clinical parameters shows that high serum concentrations of IL-6 are associated with tumor progression, metastasis and poor survival. Based on the present data of the literature search, we conclude that circulating IL-6 might be a prognostic indicator in colorectal cancer. The cytokine IL-6 emerges as at least a companion of colon cancer pathogenesis. Furthermore, these results let us speculate that IL-6 might act as a tumor-promoting cytokine in colon cancer or it might be (among others) a key player. IL-6 might even represent a therapeutic target, at least in some subtypes of colon cancer which are connected to inflammation-related causes.