RARRES1 knockdown in PWR-1E cells was validated using western blot that shows the significant decrease in RARRES1 expression as a result of RARRES1 siRNA nucleofection when compared to RARRES1 expression in the scrambled siRNA nucleofected cells. Relative protein expression change that resulted from RARRES1 knockdown in PWR-1E cells was assessed by DIGE. A total of 8121 spots were detected on the 2-dimensional gels (Figure ) of which 97 spots were consistently differentially-expressed with a p < 0.05 across the 4 biological replicates. An advantage of DIGE over regular 2-D gel techniques is the elimination of gel to gel variations since both sample and control are run on the same gel and normalized based on the internal standard pool which is also run on the same gel. Only spots with level changes of 10% or more were considered. These spots were matched and excised from the pick gel (Figure ) and processed for MALDI-MS/MS analysis 21
that allowed for the identification of 50 proteins. Differentially-expressed proteins that were identified included highly abundant proteins (e.g.
tubulin), heat shock proteins (e.g
. HSP 90) in addition to other proteins that are repeatedly identified as being differentially-expressed (e.g.
peroxiredoxins, enolases, piruvate kinases) 22
. These proteins were not considered in the study. Emphasis was given to proteins that are involved in the regulation of cell cycle. Of particular interest were Disks large homolog 2 (Dlg-2), Serine/threonine-protein phosphatase (PP2A), and Valosin containing protein (VCP) (Figure ).
Figure 1 DIGE data: Representation set of one of the 4 biological replicates. 25 µg of control PWR-1E cells lysates labeled with 200 pmoles of Cy3 (green); 25 µg of RARRES1 knock-down PWR-1E cells labeled with 200 pmoles of Cy5 (red) and a normalization (more ...)
Figure 2 Sypro Ruby Pick gel: loaded with 300µg of PWR-1E RARRES1 knock-down protein extracts and 300µg of control sample. The green dots represent the proteins that were detected by the Decyder software. Annotated proteins are those that were (more ...)
Figure 3 Differentially expressed proteins: a) 2- Cropped DIGE; b) three-dimensional view, and c) logarithmic representation of the 4 four biological replicates of Dlg-2 upregulation, PP2A and VCP down-regulation as a result of RARRES1 knock-down in PWR-1E cells. (more ...)
Retinoic Acid Receptor Responder 1 (RARRES1) is a putative carboxypeptidase inhibitor and tumor suppressor which gene is frequently silenced in cancer and aging normal cells. RARRES1 knock-down in PWR-1E cells resulted in the upregulation of disks large 2 (Dlg2), a neoplastic tumor suppressor that acts as a scaffold at cell-cell junctions 23-25
. Dlg2 consists exclusively of protein-protein interaction domains and PDZ domains implicated in cell polarity control and is localized basal to the adherens junctions 26, 27
. Dlg2 homologue was found to be upregulated in oncocytoma, a benign tumor of the kidney and is therefore considered an oncogene 28
. RARRES1 knock-down resulted also in the down-regulation of serine/threonine-protein phosphatase 2A (PP2A) in addition to its catalytic subunit. PP2A has broad substrate specificity. It targets proteins of oncogenic signaling cascades including Raf, Mek, and Akt 29
. PP2A is a negative regulator of the growth hormone stimulated signal transduction pathways. Mutation of this enzyme has been identified in several types of cancer including lung 30, 31
and breast 32-36
. Its down-regulation secondary to RARRES1 knock-down results in a decreased targeting of Raf, Mek, and Akt. Furthermore Valosin-containing Protein (VCP) was also down-regulated as a result of RARRES1 knock-down. Mutation of VCP causes inappropriate activation of NFκB signaling cascade 37
as well as impaired autophagy 38
. End-bindin protein-1 (EB1) was found to be down-regulated as a result of RARRES1 knock-down. EB1 is mainly involved in the regulation of spindle dynamics and chromosome alignment during mitosis 39
and promotes microtubule growth by suppressing catastrophes 40
. It was recently shown to promote colony formation and enhancing tumor growth in nude cells while its knock-down resulted in the inhibition of cancer cell proliferation suggesting an oncogenic role 41
. As for Ankrd26, it belongs to the POTE family of genes containing ankyrin repeat and coiled coil domains 42
. Ankrd1 has recently been found to be expressed in the majority of ovarian adenocarcimoas and found at high levels in patients with worse outcome 43
. Western blotting was performed to validate the upregulation of Dlg-2 and down-regulation of PP2A and VCP (Figure ) as a result of RARRES1 knock-down.
Western Blot Validation: of a) RARRES1 knock-down, b) Dlg2 homologue, c) PP2A, d) VCP and e) GAPDH loading control. Lane 1 scrambled siRNA nucleofected PWR-1E lysates; lane 2 RARRES1 siRNA nucleofected PWR-1E lysates.
While the down-regulation of VCP and PP2A and the up-regulation of Ankrd26 are consistent with the knock-down of tumor suppressor RARRES1, oncogenic EB1 down-regulation and tumor suppressor dlg2 up-regulation following RARRES1 knock-down are thought to have occurred to compensate for the loss of RARRES1 expression.