Possible role of sonic hedgehog and epithelial-mesenchymal transition in renal cell cancer progression

Abstract

Purpose: Sonic hedgehog (Shh) signaling and epithelial-mesenchymal transition (EMT) are both known to relate to cancer progression. The purpose of this study was to investigate the role of Shh signaling and EMT in renal cell carcinoma (RCC).

Materials and methods: Cell proliferation was assayed in RCC cell lines in the presence or absence of a Shh signaling stimulator, recombinant Shh (r-Shh) protein, or a Shh signaling inhibitor, cyclopamine. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was performed to study the expression of EMT markers (E-cadherin, N-cadherin, and vimentin) and osteonectin. The expression of Ki-67, Gli-1, osteonectin, and EMT markers in nephrectomy specimens from RCC patients was also measured by immunohistochemical (IHC) staining.

Results: RCC cells showed enhanced cell proliferation by r-Shh protein, whereas cell proliferation was suppressed by the addition of cyclopamine in RenCa cells. Real-time RT-PCR showed that r-Shh suppressed the expression of E-cadherin and that this suppression was partly blocked by cyclopamine alone in RenCa cells. In the IHC results, osteonectin significantly correlated with vein sinus invasion (p=0.0218), and the expression of vimentin significantly correlated with lymphatic invasion (p=0.0392).

Conclusions: Shh signaling and EMT play roles in RCC progression, and the Shh signaling inhibitor cyclopamine might be a possible molecular targeted therapeutic strategy for RCC.

Keywords: Epithelial-mesenchymal transition; Hedgehogs; Renal cell carcinoma.

FIG. 1

in vitro relative cell proliferation assay in renal cell carcinoma cell lines in RenCa (Panel A) and ACNH cells (Panel B) treated with dimethyl sulfoxide. Only RenCa cells showed that Cy blocked cell proliferation enhanced by r-Shh treatment. ACNH cells did not show any inhibiting effect of Cy on cell proliferation. Cell proliferation of V was set as 1.0. The treatments were performed for 48 hours. Each point represents triplicate averages±standard deviation or ON. Asterisks show significant cell proliferations compared with V. V, vehicle-treated control; S, recombinant sonic hedgehog (r-Shh) protein (1 µg/mL); Cy, cyclopamine (5 µM).

FIG. 2

in vitro relative cell proliferation assay of cyclopamine (Cy) doses (0.1, 0.5, 1, 5 and 10 µM) in RenCa cells. Cy 0.5, 1, 5, and 10 µM (p=0.005, p=0015, p=0.0002, and p=0.0079, respectively) significantly inhibited cell proliferation compared with the vehicle-treated control (V), and Cy at 5 µM significantly inhibited RenCa cell growth most. Asterisks show significant inhibition of cell proliferation compared with V.

FIG. 3

Expression of osteonectin (ON) and epithelial-mesenchymal transition (EMT) markers (E-cadherin, N-cadherin, and vimentin) in the presence of recombinant sonic hedgehog (r-Shh) protein or cyclopamine (Cy) in RenCa cells. r-Shh protein suppressed E-cadherin (×0.266 compared with vehicle control) and this suppression was partly blocked by addition of Cy alone. However, r-Shh protein did not show induction of vimentin or ON. Asterisks show significant cell proliferation or inhibition compared with vehicle-treated control (V). S, r-Shh protein.

FIG. 4

Typical findings of immunohistochemical staining of renal cell carcinoma (RCC) specimens with Gli-1, osteonectin (ON), E-cadherin, N-cadherin, and vimentin antibodies are shown. Panels A and B show strong (A) and weak (B) cytoplasmic expression of Gli-1 in the RCC area (scale bars, 100.0 µm; ×200, respectively). Panels C and D show strong (C) and weak (D) cytoplasmic expression of ON in the RCC area. (scale bars, 100.0 µm; ×200, respectively). Panels E and F showed strong (E) and weak (F) membrane expression of E-cadherin in the RCC area (scale bars, 100.0 µm; ×200, respectively). Panels G and H show strong (G) and weak (H) membrane expression of N-cadherin in the RCC area (scale bars, 100.0 µm; ×200, respectively). Panels I and J show strong (I) and weak (J) cytoplasmic expression of vimentin in the RCC area (scale bars, 100.0 µm; ×200, respectively).