Background:
Clear-cell renal cell carcinoma (ccRCC) is the sixth most common malignancy in men in North America with an incidence that has been steadily increasing at a rate of 3% yearly over the last three decades. Patients with ccRCC can be treated with surgery; however, 30% of first-time diagnosed patients present with metastatic disease. Since ccRCC is a malignancy dependent on neovascularization, current first line systemic therapies target the formation of new vessels allowing nutrient depravation and cell death. A family of drugs that uses this mechanism is called Tyrosine kinase inhibitors (TKI), and one of the members of this family is Sunitinib. Sunitinib is the first-line of therapy currently used in patients with ccRCC. However, recent studies have shown that after approximately 1 year of treatment patients develop resistance, showing disease progression while on therapy. Therefore in this study we propose to identify the protein(s) responsible for increase migration and invasion with the aim of developing a new therapy that will target the identified protein and potentially slow down the progression of the disease.
Methods:
Human renal cancer cell lines were used (CAKI-1, CAKI-2, ACHN) and treated with Sunitinib at increasing doses to develop a Sunitinib conditioned renal cell carcinoma cell line. A mRNA microarray was performed to compare the differences in gene expression between CAKI-1 Sunitinib conditioned cell line and CAKI-1 non-conditioned cell line. Moreover, we have successfully established an orthotopic renal cell carcinoma Sunitinib resistant animal model. Western blots and q-PCR were used to confirm microarray results and to evaluate the expression of Netrin-1 in-vitro. XCELLigence system was used to evaluate migration and invasion of CAKI-1 Sunitinib conditioned cell lines. Immunostaining of our in vivo Sunitinib resistant model confirms the in vitro findings.
Results:
Human renal cell carcinoma Sunitinib conditioned cell lines showed a highly upregulated expression of Netrin-1 in the microarray results as well as in Western blotting and q-PCR when compared to the un-treated renal cell carcinoma cell lines. XCELLigence system demonstrated an increased migration in CAKI-1 Sunitinib conditioned cell lines when compared to the non-treated ones as well as, increased endothelial cell migration when co-cultured with CAKI-1 Sunitinib conditioned cell line. Our results also demonstrated an increase expression of UNC5B and DCC Netrin-1 receptors in CAKI-1 Sunitinib conditioned cell line. Silencing of Netrin-1 in CAKI-1 Sunitinib conditioned cell line demonstrated a significant reduction in cell migration and invasion. Interestingly, our orthotopic renal cell carcinoma Sunitinib resistance animal model as well as human renal cell carcinoma Sunitinib resistant tumors confirmed a high expression of Netrin-1 when compared to those of control.
Data Summary:
Quantitative results showed a highly upregulated expression of Netrin-1 in Sunitinib conditioned cell lines by microarray, Western blotting, and q-PCR. XCELLigence demonstrated increased migration in CAKI-1 Sunitinib conditioned versus non-treated cells, and increased endothelial cell migration in co-culture. Silencing of Netrin-1 led to a significant reduction in cell migration and invasion. Expression of UNC5B and DCC Netrin-1 receptors was increased. Both orthotopic animal model and human Sunitinib resistant tumors confirmed high Netrin-1 expression compared to controls.
Conclusions:
In conclusion we have found that Netrin-1 plays a key role in the migration and progression of renal cell carcinoma. Development of therapies targeting Netrin-1 suggests to be a promising approach to suppress renal cell carcinoma tumor progression and potentially prolong patients’ survival.
Practical Significance:
Development of therapies targeting Netrin-1 suggests to be a promising approach to suppress renal cell carcinoma tumor progression and potentially prolong patients’ survival, addressing the need for new treatments after Sunitinib resistance develops.