Limits...
Common reduction of the Raf kinase inhibitory protein in clear cell renal cell carcinoma.

Hill B, De Melo J, Yan J, Kapoor A, He L, Cutz JC, Feng X, Bakhtyar N, Tang D - Oncotarget (2014)

Bottom Line: Collectively, the magnitude of RKIP reduction and the levels of its downregulation match those of VHL.While modulation of RKIP expression did not affect the proliferation of A498 and 786-0 ccRCC cells and neither their ability to form xenograft tumors in NOD/SCID mice, ectopic expression or knockdown of RKIP inhibited or enhanced A498 and 786-0 ccRCC cell invasion, respectively.This was associated with robust changes in vimentin expression, a marker of EMT.

View Article: PubMed Central - PubMed

Affiliation: Division of Nephrology, Department of Medicine, McMaster University, Hamilton, Ontario, Canada, Hamilton, Ontario, Canada. Father Sean O'Sullivan Research Institute, Hamilton, Ontario, Canada. The Hamilton Center for Kidney Research, St. Joseph's Hospital, Hamilton, Ontario, Canada.

ABSTRACT
Despite the recent progress in our understanding of clear cell renal cell carcinomas (ccRCCs), the etiology of ccRCC remains unclear. We reported here a prevailing reduction of the raf kinase inhibitory protein (RKIP) in ccRCC. In our examination of more than 600 ccRCC patients by western blot and immunohistochemistry, RKIP was significantly reduced in 80% of tumors. Inhibition of RKIP transcription in ccRCC occurs to greater levels than VHL transcription based on the quantification analysis of their transcripts in six large datasets of DNA microarray available in Oncomine™ with the median rank of suppression being 582 and 2343 for RKIP and VHL, respectively. Collectively, the magnitude of RKIP reduction and the levels of its downregulation match those of VHL. Furthermore, RKIP displays tumor suppressing activity in ccRCC. While modulation of RKIP expression did not affect the proliferation of A498 and 786-0 ccRCC cells and neither their ability to form xenograft tumors in NOD/SCID mice, ectopic expression or knockdown of RKIP inhibited or enhanced A498 and 786-0 ccRCC cell invasion, respectively. This was associated with robust changes in vimentin expression, a marker of EMT. Taken together, we demonstrate here that downregulation of RKIP occurs frequently at a rate that reaches that of VHL, suggesting RKIP being a critical tumor suppressor for ccRCC. This is consistent with RKIP being a tumor suppressor for other cancers.

Show MeSH

Related in: MedlinePlus

Modulations of RKIP affect A498 ccRCC cell invasion(A and B) The indicated A498 cell lines were assayed for their invasion ability using either a control membrane or 8 μM matrigel membranes. Typical image of cells that have passed through either membrane are shown (left panels). Quantification of three independent repeats (means ± SE) is also graphed (right panels). * p < 0.01 in comparison to the respective controls (2-tailed student t-test). (C and D) A498 EV, RKIP, Ctrl shRNA, and shRKIP cells were examined for the expression of vimentin and actin by western blot (see the insets for typical results). Vimentin in individual cell lines were normalized to the respective actin. Experiments were repeated in triplicate. Means ± SE are graphed. * p < 0.05 in comparison to either A498 EV or Ctrl shRNA cells (2-tailed student t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4202132&req=5

Figure 6: Modulations of RKIP affect A498 ccRCC cell invasion(A and B) The indicated A498 cell lines were assayed for their invasion ability using either a control membrane or 8 μM matrigel membranes. Typical image of cells that have passed through either membrane are shown (left panels). Quantification of three independent repeats (means ± SE) is also graphed (right panels). * p < 0.01 in comparison to the respective controls (2-tailed student t-test). (C and D) A498 EV, RKIP, Ctrl shRNA, and shRKIP cells were examined for the expression of vimentin and actin by western blot (see the insets for typical results). Vimentin in individual cell lines were normalized to the respective actin. Experiments were repeated in triplicate. Means ± SE are graphed. * p < 0.05 in comparison to either A498 EV or Ctrl shRNA cells (2-tailed student t-test).

Mentions: RKIP has been shown to inhibit breast and prostate cancer metastasis [26,41]. Since the invasion ability of a cell is closely related to cancer's metastatic potential, we have determined the impact of RKIP on ccRCC cell invasion. Overexpression of RKIP in A498 and 786-0 cell lines reduced their invasion ability in comparison to the EV cells (Fig. 6A, Supplementary Fig. 4A). Conversely, knockdown of RKIP in both lines robustly enhanced their invasion capacity (Fig. 6B, Supplementary Fig. 4B).


Common reduction of the Raf kinase inhibitory protein in clear cell renal cell carcinoma.

Hill B, De Melo J, Yan J, Kapoor A, He L, Cutz JC, Feng X, Bakhtyar N, Tang D - Oncotarget (2014)

Modulations of RKIP affect A498 ccRCC cell invasion(A and B) The indicated A498 cell lines were assayed for their invasion ability using either a control membrane or 8 μM matrigel membranes. Typical image of cells that have passed through either membrane are shown (left panels). Quantification of three independent repeats (means ± SE) is also graphed (right panels). * p < 0.01 in comparison to the respective controls (2-tailed student t-test). (C and D) A498 EV, RKIP, Ctrl shRNA, and shRKIP cells were examined for the expression of vimentin and actin by western blot (see the insets for typical results). Vimentin in individual cell lines were normalized to the respective actin. Experiments were repeated in triplicate. Means ± SE are graphed. * p < 0.05 in comparison to either A498 EV or Ctrl shRNA cells (2-tailed student t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4202132&req=5

Figure 6: Modulations of RKIP affect A498 ccRCC cell invasion(A and B) The indicated A498 cell lines were assayed for their invasion ability using either a control membrane or 8 μM matrigel membranes. Typical image of cells that have passed through either membrane are shown (left panels). Quantification of three independent repeats (means ± SE) is also graphed (right panels). * p < 0.01 in comparison to the respective controls (2-tailed student t-test). (C and D) A498 EV, RKIP, Ctrl shRNA, and shRKIP cells were examined for the expression of vimentin and actin by western blot (see the insets for typical results). Vimentin in individual cell lines were normalized to the respective actin. Experiments were repeated in triplicate. Means ± SE are graphed. * p < 0.05 in comparison to either A498 EV or Ctrl shRNA cells (2-tailed student t-test).
Mentions: RKIP has been shown to inhibit breast and prostate cancer metastasis [26,41]. Since the invasion ability of a cell is closely related to cancer's metastatic potential, we have determined the impact of RKIP on ccRCC cell invasion. Overexpression of RKIP in A498 and 786-0 cell lines reduced their invasion ability in comparison to the EV cells (Fig. 6A, Supplementary Fig. 4A). Conversely, knockdown of RKIP in both lines robustly enhanced their invasion capacity (Fig. 6B, Supplementary Fig. 4B).

Bottom Line: Collectively, the magnitude of RKIP reduction and the levels of its downregulation match those of VHL.While modulation of RKIP expression did not affect the proliferation of A498 and 786-0 ccRCC cells and neither their ability to form xenograft tumors in NOD/SCID mice, ectopic expression or knockdown of RKIP inhibited or enhanced A498 and 786-0 ccRCC cell invasion, respectively.This was associated with robust changes in vimentin expression, a marker of EMT.

View Article: PubMed Central - PubMed

Affiliation: Division of Nephrology, Department of Medicine, McMaster University, Hamilton, Ontario, Canada, Hamilton, Ontario, Canada. Father Sean O'Sullivan Research Institute, Hamilton, Ontario, Canada. The Hamilton Center for Kidney Research, St. Joseph's Hospital, Hamilton, Ontario, Canada.

ABSTRACT
Despite the recent progress in our understanding of clear cell renal cell carcinomas (ccRCCs), the etiology of ccRCC remains unclear. We reported here a prevailing reduction of the raf kinase inhibitory protein (RKIP) in ccRCC. In our examination of more than 600 ccRCC patients by western blot and immunohistochemistry, RKIP was significantly reduced in 80% of tumors. Inhibition of RKIP transcription in ccRCC occurs to greater levels than VHL transcription based on the quantification analysis of their transcripts in six large datasets of DNA microarray available in Oncomine™ with the median rank of suppression being 582 and 2343 for RKIP and VHL, respectively. Collectively, the magnitude of RKIP reduction and the levels of its downregulation match those of VHL. Furthermore, RKIP displays tumor suppressing activity in ccRCC. While modulation of RKIP expression did not affect the proliferation of A498 and 786-0 ccRCC cells and neither their ability to form xenograft tumors in NOD/SCID mice, ectopic expression or knockdown of RKIP inhibited or enhanced A498 and 786-0 ccRCC cell invasion, respectively. This was associated with robust changes in vimentin expression, a marker of EMT. Taken together, we demonstrate here that downregulation of RKIP occurs frequently at a rate that reaches that of VHL, suggesting RKIP being a critical tumor suppressor for ccRCC. This is consistent with RKIP being a tumor suppressor for other cancers.

Show MeSH
Related in: MedlinePlus