The Notch and TGF-β Signaling Pathways Contribute to the Aggressiveness of Clear Cell Renal Cell Carcinoma
Figure 5
Notch inhibition attenuates CCRCC cell migration and invasion.
(A) [3H]thymidine incorporation of 786-O and SKRC-10 cells grown for indicated hours (h) in the presence of 1.0 ng/ml TGF-β1 or treated with vehicle control (c). Data represents mean+95% confidence intervals of three separate experiments. Data were normalized to vehicle control treated cells. (B) Cell migration assessed by Boyden chamber assays of 786-O and SKRC-10 cells treated with vehicle control (c) or DAPT. The cells were allowed to migrate towards the lower compartment for 4 h (786-O) or 5 h (SKRC-10). Data represents mean+95% confidence intervals of three separate experiments. Data were normalized to vehicle control treated cells. (C) Cell migration as determined by Boyden chamber assays of 786-O and SKRC-10 cells transfected with non-specific control (c-si) or Notch1 (siN-1) specific siRNAs. After 24 h of transfection, cells were counted and seeded into the Boyden chamber. The cells were allowed to migrate towards the lower compartment for 4 h (786-O) or 5 h (SKRC-10). Data represents mean+95% confidence intervals of three separate experiments. Data were normalized to vehicle control treated cells. (D) Cell migration as determined by Boyden chamber assays of SKRC-10 cells treated with vehicle control (c), 2 µM SB431542, 10 µM DAPT and control. The cells were allowed to migrate towards the lower compartment for 5 h. Data represents mean+95% confidence intervals of three separate experiments. Data were normalized to vehicle control treated cells. (E) Cell migration analyses of SKRC-10 cells treated with vehicle control (c) or treated with 0.25 ng/ml TGF-β1 and vehicle (c) or 0.25 ng/ml TGF-β1 and DAPT. The cells were allowed to migrate towards the lower compartment for 5 h. Data represents mean+95% confidence intervals of three separate experiments. Data were normalized to vehicle control treated cells. (F) Cell invasion as determined by Matrigel coated Boyden chamber assays of 786-O and SKRC-10 cells treated with vehicle control (c) or DAPT. The cells were allowed to invade and migrate towards the lower compartment containing 10% FCS for 16 h (786-O) or 21 h (SKRC-10). Data represents mean+95% confidence intervals of three separate experiments. Data were normalized to vehicle control treated cells. ***, ** and * indicates statistical significant changes (two-sided Student's t-test, p<0.001, p<0.01 and p<0.05 respectively). (G) TGF-β pathway activity correlates to metastatic spread of primary CCRCCs. CCRCCs from patients with either metastatic disease at diagnosis or that later developed metastasis (Metastatic, n = 13) showed a significantly elevated TGF-β signaling activity based on the 145-gene TGF-β signature as compared to tumors from patients with a localized disease and with no documented metastases during follow-up (Localized, n = 9) (two-sided Student's t-test, p = 0.044).