Jade-1, a candidate renal tumor suppressor that promotes apoptosis

Abstract

Medical therapies are lacking for advanced renal cancer, so there is a great need to understand its pathogenesis. Most renal cancers have defects in the von Hippel-Lindau tumor suppressor pVHL. The mechanism by which pVHL protein functions in renal tumor suppression remains unclear. Jade-1 is a short-lived, kidney-enriched transcription factor that is stabilized by direct interaction with pVHL. Loss of Jade-1 stabilization by pVHL correlates with renal cancer risk, making the relationship between Jade-1 and renal cancer compelling. We report that Jade-1 expression was barely detectable in all tested renal cancer cell lines, regardless of VHL status. Strikingly, proteasome inhibitor treatment increased endogenous Jade-1 expression up to 10-fold. Jade-1 inhibited renal cancer cell growth, colony formation, and tumor formation in nude mice. Intriguingly, Jade-1 also affected the pattern of cell growth in monolayer culture and 3D culture. Jade-1 increased apoptosis by 40-50% and decreased levels of antiapoptotic Bcl-2. Antisense Jade-1-expressing cells confirmed these results. Therefore, Jade-1 may suppress renal cancer cell growth in part by increasing apoptosis. Jade-1 may represent a proapoptotic barrier to proliferation that must be overcome generally in renal cancer, perhaps initially by pVHL inactivation and subsequently by increased proteasomal activity. Therefore, Jade-1 may be a renal tumor suppressor.

Fig. 1.

Jade-1 protein expression is minimal in renal cancer cell lines and induced with proteasome inhibitors. Endogenous Jade-1 expression was examined in human cultured cell lines by Western blotting using Jade-1 anti-serum. (a) Jade-1 protein expression is increased in all lines with proteasome inhibition (PI). The proteasome inhibitor was MG132 at 10 μM for 18 h. Jade-1 expression was tested in the VHL-deficient renal cancer lines 769-P, RCC4, A704, and UMRC6 cells; wild-type VHL-containing CaKi-1, TK10, and ACHN renal cancer cells; human embryonic kidney 293 cells; and human colon cancer line SW620. The Jade-1 control includes lysates of 293 cells transfected with untagged human Jade-1. (b) VHL-stable transfection affects proteasome inhibitor response. 786-O or A498 renal cancer cell lines stably transfected with empty vector or wild-type (wt) VHL were tested for Jade-1 protein expression, with and without proteasome inhibitor MG132. (c) Proteasome inhibitors of multiple classes increase endogenous Jade-1 expression. Renal cancer line 769-P was treated with the proteasome inhibitors MG132, MG262, epoxomycin (Epoxo.), lactacystin β-lactone, or 4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-Leu-vinylsulfone (NLVS), as well as calpain inhibitor-1 (MG101), lysosomal protease inhibitor chloroquin (Chloroq.), or vehicle alone (DMSO) for 18 h. Results are representative of five experiments.

Fig. 2.

Jade-1 inhibits growth of renal cancer cells. (a) Western blots identify stable, clonal 786-O renal cancer cell lines expressing HA-tagged Jade-1 (J). The HA-J lane positive control represents lysates from 293T17 cells transiently transfected with the HA-Jade-1 expression vector. Lines J18, J22, J7, and J9 were positive for Jade-1. Jade-1/VHL (JV) lines JV10, JV12, JV37, and JV21 were positive for Jade-1 and for pVHL (data not shown). (b) Stable expression of Jade-1 slows the growth of renal cancer lines in cell-counting experiments. HA-Jade-1 786-O stable lines (▪) and Jade-1/VHL (▴) lines (Jade-1 lines) were compared with parental and pooled HA empty vector, stable 786-O cells (♦) (control lines). (Error bars, 1 SD; n = 2.) (c) Thymidine incorporation experiments in the same cell lines, represented by the same symbols as those shown in b. (Error bars, 1 SD; n = 2.)

Fig. 3.

Jade-1 alters growth of renal cancer cells in 3D culture and in vivo. (a-c) Jade-1 alters growth of renal cancer cells in a collagen gel. Jade-1-expressing renal cancer cells grown in 3D culture in a collagen gel (b) show less branching and less extraneous growth than vector control cells (a). (c) Extent of branching (gray bars) and extraneous growth (black bars) were scored as follows: 1, negligible; 2, moderate; or 3, extensive. ^*, P < 0.05, versus controls. (Error bars, SE.) (d) Jade-1 inhibits growth of renal cancer cells in nude mice. ^*, P < 0.01, versus control. (Error bars, SE.)

Fig. 4.

Jade-1 antisense promotes growth of kidney cells. (a) Jade-1 expression in 293 tet-off Jade-1 antisense lines. Western blot analysis of endogenous Jade-1 expression of antisense Jade-1 (ASJ) lines ASJ3 and ASJ2 in the presence of Dox (+Dox) or without Dox (-). In the positive control lane (Jade-1), 293 cells were transfected with an untagged Jade-1 expression vector. (b) Jade-1 antisense promotes growth of 293 cells in culture. As in Fig. 2b, cell-counting experiments were performed by using the 293 antisense lines from a.

Fig. 5.

Jade-1 antisense alters monolayer morphology of cells in culture. Representative examples of monolayer morphology noted with all 293 Jade-1 antisense lines. 293 antisense line ASJ3 in monolayer culture grown with (b and d) or without (a and c) Dox, shown at low (×40) (a and b) and high (×200) (c and d) magnification.

Fig. 6.

Jade-1 promotes apoptosis. (a) Binding of FITC-annexin V to 786-O stable renal cancer cell lines by FACS analysis. (Error bars, 1 SD.) ^*, P < 0.01 versus controls. (b) Cleavage of PARP by caspase 3 in 786-O renal cancer lines transfected with Jade-1 (J) or Jade-1 and pVHL (JV) by Western blot analysis. Representative result of four experiments. (c) Western blotting for antiapoptotic Bcl-2 in renal cancer stable lines. 786-O cells are the parental line. Shown are the representative results of three experiments. (d) Jade-1 increases apoptosis in transiently transfected 293T17 cells as assessed with fluorescent dye Hoechst 33342. n = 5. ^*, P < 0.05. (Error bars, 1 SD.) (e) Jade-1 antisense reduces the percentage of fragmented cells. Jade-1 antisense line ASJ3 was tested with and without Dox after permeabilization and propidium iodide staining by FACS analysis. Light side scatter (SSC) was compared with forward scatter (FSC). A representative result of three experiments using two antisense Jade-1 cell lines is shown. (f) Jade-1 antisense increases Bcl-2 levels. Lines ASJ2 and ASJ3 were grown with and without Dox. A representative result of four experiments is shown.