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. 2008 Dec;19(12):2437-46.
doi: 10.1681/ASN.2008040394. Epub 2008 Oct 2.

Calcineurin inhibitors modulate CXCR3 splice variant expression and mediate renal cancer progression

Dipak Datta  1 , Alan G Contreras, Martin Grimm, Ana Maria Waaga-Gasser, David M Briscoe, Soumitro Pal
Affiliations

Calcineurin inhibitors modulate CXCR3 splice variant expression and mediate renal cancer progression

Dipak Datta et al. J Am Soc Nephrol. 2008 Dec.

Abstract

Calcineurin inhibitors (CNI) are used to prevent inflammatory diseases and allograft rejection. However, little is known about the mechanism(s) underlying their ability to promote the development and recurrence of cancer. Recent studies suggested that the chemokine receptor CXCR3 may play important roles in tumorigenesis. CXCR3 has two splice variants with opposite functions: CXCR3-A promotes cell proliferation, and CXCR3-B inhibits cell growth. Here, we explored the effects of CNI on the expression and function of CXCR3 splice variants. Compared with normal renal tissues and renal epithelial cells, human renal cancer tissues and renal cancer cell lines demonstrated higher expression of CXCR3-A and markedly lower expression of CXCR3-B. In human renal cancer cells (786-0 and Caki-1) and renal epithelial cells, CNI markedly downregulated the expression of CXCR3-B, whereas expression of CXCR3-A was unchanged. This CNI-mediated downregulation of CXCR3-B resulted in increased proliferation and migration of renal cancer cells; CNI-mediated cell proliferation involved signaling through G(i) proteins, perhaps via CXCR3-A. Finally, it was observed that CNI treatment increased the growth of human renal tumors in vivo, and the expression of CXCR3-B was significantly decreased in these tumors. In summary, these observations suggest that CNI may mediate the progression of human renal cancer by downregulating CXCR3-B and by promoting proliferative signals, likely through CXCR3-A. Targeting CXCR3 splice variants or the signaling pathways downstream of CXCR3 receptors may provide a therapeutic strategy for the prevention of CNI-mediated renal cancer progression.

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Figures

Figure 1.
Figure 1.
Differential expression of CXCR3-A and CXCR3-B in human renal cancer tissues. (A and B) Total RNA was isolated from renal cancer and normal renal tissues and reverse-transcribed. Either the fold increase of CXCR3-A (A) or the percentage decrease of CXCR3-B (B) mRNA expression in renal cancer tissues versus the mean level of expression of each gene in normal renal tissues (n = 4) was measured by real-time PCR. S1 through S6 represent low-stage (Robson stages I and II), whereas S7 through S12 represent high-stage (Robson stages III and IV) renal tumor tissues. Columns are the average of triplicate readings of the sample; bars are ±SD.
Figure 2.
Figure 2.
Expression pattern of CXCR3 splice variants and CXCR3-binding ligands in human renal cancer and renal epithelial cell lines. (A through C) Total RNA was isolated from REC, 786-0, and Caki-1 cells and reverse-transcribed. Fold changes in mRNA expression of CXCR3-A (A), CXCR3-B (B), or CXCR3-binding ligands (C) was measured by real-time PCR. Data reflect three independent experiments. Columns are average of triplicate readings of the sample; bars are ±SD. In A and B, **P < 0.01 versus REC; in C, **P < 0.01 and *P < 0.05 versus REC.
Figure 3.
Figure 3.
CNI downregulate the expression of CXCR3-B in human renal cancer cells. (A through C) REC, 786-0, and Caki-1 cells were treated with CsA (0.1 and 1.0 μg/ml), FK506 (0.01 and 0.10 μg/ml), or vehicle alone and incubated for 18 h. Total RNA was isolated from these cells and reverse-transcribed. Fold changes in mRNA expression of CXCR3-B in response to CsA (A) or FK506 (B) was measured by real-time PCR. Fold changes in mRNA expression of CXCR3-A in response to CsA was also measured by real-time PCR (C). Data reflect three independent experiments. Columns are average of triplicate readings of two different samples; bars are ±SD. In A and B, *P < 0.05 versus vehicle-treated cells.
Figure 4.
Figure 4.
Inhibition of CXCR3-B expression is associated with increased proliferation of human renal cancer cells. (A) Caki-1 cells were transfected with either the control or the CXCR3-B siRNA (25 and 50 nM). (B) Caki-1 cells were transfected with either CXCR3-B overexpression plasmid (1.0 and 1.5 μg/ml) or the empty expression vector. All of the cells in A and B were incubated for 72 h and subjected to cell proliferation assay by measuring [3H]thymidine incorporation within the cells as described in the Concise Methods section. Data reflect three independent experiments. Columns are average of triplicate readings (cpm) of the samples; bars are ±SD. In A and B, *P < 0.05 versus either control siRNA–or empty vector–transfected cells.
Figure 5.
Figure 5.
CNI-mediated downregulation of CXCR3-B is associated with increased proliferation and migration of human renal cancer cells. (A and B) REC and 786-0 cells were treated with either different concentrations (0.01 to 1.00 μg/ml) of CsA or vehicle alone for 72 h. Some cells from both CsA-treated and the vehicle-treated group were incubated with CXCL4 (1.0 μg/ml) in the last 24 h. All of the cells in A and B were subjected to cell proliferation assay by measuring [3H]thymidine incorporation within the cells as described in the Concise Methods section. Data reflect three independent experiments. Columns are average of triplicate readings (cpm) of the samples; bars are ±SD. (C) The effects of CsA and CXCL4 on renal cancer cell migration were tested by in vitro wound-healing assay, as described in the Concise Methods section. Confluent monolayers of 786-0 cells were scarred, and repair was monitored microscopically after 36 h of treatment with CsA (0.1 and 1.0 μg/ml), or CXCL4 (1.0 μg/ml), CsA (1.0 μg/ml) + CXCL4 (1.0 μg/ml), or vehicle alone. The right panel represents the quantification of distance migrated by the cells in 36 h relative to vehicle-treated control cells in 0 h. Representative and average readings of three independent experiments; bars are ±SD. In *P < 0.05 versus vehicle-treated cells.
Figure 6.
Figure 6.
CNI-mediated proliferation of renal cancer cells involves Gi proteins. (A) Caki-1 cells were transfected with either the CXCR3-B or control siRNA (50 nM) for 48 h, incubated with PTX (1.0 μg/ml)/vehicle for 16 h, and then either untreated or treated with CXCL10 (20 ng/ml) for 24 h. (B) 786-0 cells were incubated with PTX (1.0 μg/ml)/vehicle for 16 h and then treated with either CsA (1.0 μg/ml) or vehicle alone for 72 h. All of the cells in A and B were subjected to cell proliferation assay by measuring [3H]thymidine incorporation within the cells as described in the Concise Methods section. Data reflect three independent experiments. Columns are average of triplicate readings (cpm) of the samples; bars are ±SD. In A and B, *P < 0.05 versus either control siRNA-transfected or vehicle-treated cells; in A, +P < 0.05 versus CXCR3-B siRNA-transfected but PTX-untreated cells and #P < 0.05 versus control siRNA-transfected untreated cells; in B, +P < 0.05 versus CsA-treated but PTX-untreated cells.
Figure 7.
Figure 7.
CNI CsA promotes the growth of human renal tumors in vivo with decreased expression of CXCR3-B in tumor tissues. (A) Human renal cancer cells (786-0; 1.0 × 106) were injected subcutaneously into immunodeficient (nu/nu) mice, which (n = 8 in each group) were treated either with CsA (10 mg/kg per d) or with vehicle. Treatment was continued for 30 d, and tumor volume was monitored at regular intervals using a digital caliper. Tumors were harvested 30 d after tumor injection. The data reflect three independent experiments. Columns are average value of tumor volume; bars are ±SD. *P < 0.05 versus vehicle-treated controls. (B) Total RNA was isolated from harvested tumor tissues (day 30) and reverse-transcribed. Fold change in CXCR3-B and CXCR3-A mRNA expression was measured by real-time PCR. Data reflect three independent experiments resulting from duplicate readings of three different samples. Columns are average value of CXCR3-B and CXCR3-A mRNA expression, bars are ±SD. *P < 0.05 versus vehicle-treated cells.
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