|Abstract: ||Abstract:Pro-inflammatory cytokines and chemokines are involved in promoting tumorigenesis by facilitating tumor proliferation and metastasis. The serum levels of interleukin (IL)-6, IL-1β, and tumor necrosis factor-alpha (TNF-α) are significantly elevated in patients with renal cell carcinoma (RCC). However, the mechanisms of how these cytokines participate in the progression of RCC remains unknown. In the present study, we investigated the effects of tumor-derived cytokines on invasion and the epithelial-mesenchymal transition (EMT) of RCC cells. We found that expression of IL-1β, IL-6, TNF-α, hypoxia-inducible factor-alpha (HIF-1α), and matrix metalloproteinase-2 (MMP2) were significantly elevated in high malignancy A498 cells compared to low malignancy 786-O cells. The invasion ability of A498 was three-fold higher than that of 786-O cells. The invasiveness of 786-O cells was markedly enhanced by adding conditioned medium derived from A498 cells. This phenomenon was significantly inhibited by immunodepletion of TNF-α followed by MMP2, IL-6, or IL-1β from A498 conditioned medium. Synergistic inhibition was also noted after simultaneous immunodepletion of TNF-α, IL-1β, and IL-6. RCC cell lines with higher malignancy produced more TNF-α, which was correlated with their stronger invasive ability. The invasiveness of 786-O cells was significantly promoted by TNF-α in a dose-dependent manner. Moreover, TNF-α induced the EMT of 786-O cells by repressing E-cadherin, promoting vimentin expression, and activating MMP9 activity. Our findings demonstrate that pro-inflammatory cytokines, especially TNF-α, can enhance invasion and the EMT of renal cancer cells, which provides a therapeutic target to prevent and treat advanced RCC. (Cancer Sci 2008; 99: 905–913)
Renal cell carcinoma (RCC) arises from the renal tubular epithelium and account for 85% of renal cancers.(1) Four types of RCC have been delineated clinically and the genes responsible for them have been characterized. Overall, 75% of RCC are the clear cell type (ccRCC), while the other 25% are made up of the papillary, chromophobe, and oncocytic types.(2) Defects in the von Hippel–Lindau (VHL) gene appear to be responsible for 60% of sporadic ccRCC. A quarter of patients present with locally invasive or metastatic RCC. A third of the patients after resection of the tumor will have a recurrence. Moreover, systemic theraputic treatments of advanced RCC are largely ineffective and do not improve patient survival.(1,2) Therefore, defining the factors involved in disease progression and metastasis will provide molecular targets for the development of effective therapies.
A complex network of pro-inflammatory cytokines, chemokines, and their receptors influences the development of primary tumors and metastasis.(3,4) CXCR4 is most commonly found in malignant cells from different cancer types.(4) In addition to CXCR4, CCR3 has been found to be up-regulated in 28% RCC tissues and correlates with a higher grade of malignancy.(5) Furthermore, the levels of IL-6, IL-1β, and TNF-α in serum are significantly higher and correlated with tumor size in RCC.(6) IL-6 blood level is significantly higher in patients with lymph node invasion and distant metastases, while TNF-α level is significantly higher as the stage of the RCC increases.(6,7) Therefore, IL-6 may be one of the factors associated with poor prognosis of patients with RCC. In addition, TNF-α may be useful as marker for the early diagnosis of RCC.(6) However, little is known about how these cytokines affect RCC migration and progression.
The epithelial-mesenchymal transition (EMT) is a process in which polarized epithelial cells are converted into motile mesenchymal cells. Alterations in adhesion, morphology, cellular architecture, and migration capacity are the major events that occur during this process.(8) Common molecular markers for the EMT include increased expression of vimentin, nuclear localization of β-catenin, and increased production of transcriptional factors that inhibit E-cadherin production. Phenotypic markers for EMT comprise an increased capacity for migration and three-dimensional invasion as well as resistance to apoptosis.(9) The EMT occurs during critical phases of normal embryonic development in many animal species. However, this important developmental program has a more sinister role in tumor progression. EMT is involved in the progression of non-invasive adenomas into malignant metastatic carcinomas.(8–10)
In this study, an invasion assay and two human ccRCC cell lines with a VHL mutation and different levels of malignancy were used to define the tumor-derived factors involved in the progression of renal cancer. TNF-α, IL-1, IL-6, and matrix metalloproteinase-2 (MMP2), which are secreted by advanced RCC cells, were shown to promote invasion of RCC. Furthermore, tumor-derived TNF-α was the major factor correlated with malignancy and is involved in the EMT of kidney cancer.