Chemokines are considered to play a significant part in cancer migration, production and growth [24
]. Previous studies have shown a significant correlation between chemokine receptor status in human cancers, and prognosis and/or metastases in a variety of malignant tumors such as T-cell leukemia (CCR4) [25
], hepatocellular carcinoma (CCR6) [26
], gastric carcinoma (CCR7) [27
], renal cell carcinoma (CXCR3) [28
], ovarian cancer (CXCR4) [29
], osteosarcoma (CXCR4) [30
], colorectal cancer (CCR7 and CXCR4) [31
], and malignant melanoma (CXCR3 and CXCR4) [33
]. In our study, we investigated the expression of CXCL12, CXCR4 and CXCR7, which are the most important chemokines and chemokine receptors, in human mesothelioma. We found that CXCR4 is over-expressed in most of the human mesothelioma samples tested, and that it is expressed at low levels in most normal pleural tissue. CXCL12 is also expressed in mesothelioma, but at somewhat lower levels than CXCR4, though still at higher levels than in normal pleura. Originally characterized as a pre-B-cell stimulatory factor, CXCL12 is a chemotactic factor for T cells, monocytes, pre-B cells, dendritic cells, and hematopoietic progenitor cells.
Because cancer cell migration and invasion share many similarities with leukocyte trafficking [24
], chemokines are thought to be very important in organ-selective cancer metastasis and recurrence. Receptor CXCR4 immunoreactivity is found not only in cancer cells, but also in lymphocytes in the tumor stroma [6
]. CXCL12 and its receptor, CXCR4, were recently found to influence the dissemination, immune rejection, and neoangiogenesis of human gastrointestinal cancer [6
]. Furthermore, a mechanism for CXCR4 activation during tumor-cell evolution has been described in patients with the von Hippel-Lindau (VHL) syndrome. In these patients, inactivation of the VHL tumor suppressor gene in incipient tumor cells results in over-expression of CXCR4, which confers a selective survival advantage and the tendency to home to selected organs [35
]. It is interesting that CXCL12 is normally produced by the stromal cells of lymph nodes, lung, liver, and bone marrow, the organs that are the most frequent sites for metastasis [36
]. Accordingly, the high frequency of mesothelioma recurrence and metastasis is, at least in part, due to the high expression of CXCR4 and CXCL12.
CXCR7 was absent in most normal pleural tissues and expressed in low levels in most mesothelioma samples. The expression and biological importance of CXCR7 are thought to depend on the cell types investigated. CXCR7 was recently shown to be highly expressed in human glioma cells and non-small-cell lung cancer [37
]. In breast cancer cells, CXCR7 activation fails to cause Ca2+ mobilization or cell migration, but provides a growth advantage [19
], whereas in prostate cancer cells, CXCR7 expression is associated with enhanced adhesive and invasive activities in addition to increased survival [21
]. In healthy mice, CXCR7 is expressed in cardiomyocytes, osteocytes, and brain cells [39
]. CXCR7-deficient mice die prenatally with severe heart defects that are also reported for CXCL12 and CXCR4 knockout animals [40
Our finding that CXCR4 and CXCL12 were both highly expressed in five (H2052, ms -1, 211H, H513 and H290) of the six mesothelioma cell lines studied, whereas CXCR7 was expressed in only two (H2052 and H513) and minimally expressed in another three (H28, 211H and H290), suggests that the expression of chemokines and their receptors varies in mesothelioma cells. Interestingly, the two mesothelioma cell lines that express CXCR7 also showed the highest expression level of CXCR4 among all mesothelioma cell lines tested. These data suggest that CXCR7 may be involved in a subset of mesotheliomas that have more aggressive metastatic behavior and CXCR4 may positively alter the expression levels of CXCR7. This suggestion is not in agreement with the possible CXCR4-CXCR7 reciprocal regulation in prostate cancer [19
]. Wang et al. reported that signaling by CXCR7 activates the Akt pathway in prostate cancer [21
]. However, the relationship between CXCR7 and the Akt pathway in mesothelioma is still not clear (). Our data also suggest that a CXCR4 antagonist such as AMD3100, without simultaneous blockage of CXCR7, may be an inefficient strategy for a therapeutic approach in the subset of patients with CXCR7 expression.
Our results suggest that CXCL12 can induce proliferation in most mesothelioma cell lines. At the same time, ADM3100, the CXCR4 antagonist, can inhibit proliferation and viability in most mesothelioma cell lines. These results suggest that the CXCL12/CXCR4 axis is important for most mesothelioma cells and that blocking it impairs their viability. Therefore, inhibiting the chemokines and their receptors may have clinical applicability for mesothelioma treatment. However, CXCL12 did not induce proliferation and AMD3100 did not inhibit viability in mesothelioma cell line H28, probably because CXCR4, CXCL12 and CXCR7 expression was very low in H28. This result is in agreement with our RT-PCR findings. If the inhibitor of chemokines and their receptors is used in the treatment of mesothelioma, the expression level of chemokines and their receptors should be detected first. In short, our data suggest that CXCL12/CXCR4 is a potential therapeutic target for patients with mesothelioma.
Akt was rapidly and strongly phosphorylated by the chemokine CXCL12 (), leading us to hypothesize that the Akt pathway can be activated by the chemokines and their receptors. The PI3K-dependent signals, including Akt, mTOR, S6K and 4E-BP1, can promote the occurrence, proliferation and survival of tumor cells [42
]. Therefore, our western blot results suggest that the chemokines and receptors play an important role in human malignant mesothelioma. AMD3100 is a CXCR4 antagonist. A concentration of 1 µM has been shown to inhibit HIV entry into cells in vitro [47
]. The p70 ribosomal S6 kinase (p70S6K, S6K) and eIF4E-binding protein 1 (4E-BP1) are two major downstream components of Akt and mTOR [48
]. Phosphorylation of p70S6K allows the translation of ribosomal proteins [50
]. Phosphorylation of 4E-BP1 regulates cap-dependent translation by enabling the formation of an active eIF4E complex [51
]. We found that CXCL12 induced the activation of p70S6K and 4E-BP1, which were downstream of Akt-mTOR pathways, and this activation was dose-dependently blocked by CXCR4 inhibitor AMD3100.
In summary, our study shows that CXCL12 and CXCR4 are over-expressed in 5 of 6 human mesothelioma cell lines and in 22/41 and 31/41 mesothelioma tissues, respectively. CXCL12 is also a functional activation factor of Akt downstream pathways and the CXCR4 antagonist AMD3100, which suggests that CXCL12/CXCR4 interaction is a potential therapeutic target for mesothelioma. Our data also indicate that CXCR7 may play a role in a subset of mesotheliomas, and that the CXCR7 factor should be considered when blocking the CXCL12-CXCR4 interaction. The role of CXCR7 in human mesothelioma, and whether the CXCL12-CXCR4 interaction may be a potential therapeutic target, requires further investigation.