Journal of Proteome Research ARTICLE Figure 3. Proteomic changes associated with APC restoration. (A) Schematic ﬁgure represents a subset of enriched classes from 155 diﬀerentially expressed proteins (<0.1 adjusted quasi p-value) between SW480Null and SW480APC (see Table 2 for details on all enriched classes). APCnull status results in decreased expression (red symbols) of proteins associated with cell adhesion and the actin cytoskeleton and up-regulation (green symbols) of proteins involved in cell cycle control cholesterol biosynthesis. (B) SW480APC cells display redistribution of CTNNB1 from cytoplasm to nucleus. Cells were fractionated and nuclear and cytosolic fractions were analyzed by LC MRM for CTNNB1 (top panel) and HIST1H4A (nuclear protein marker) (bottom panel). LC MS/MS and 3-fold higher expression by LC MRM (see Table 2). SW480Null cells had decreased levels of many other proteins involved in adherens junction formation and cell adhe- sion, including CDH3, CTTNA1, CTTND1, NCAM1, EVPL, DSG2, CECAM1, and EPCAM (Figure 3A). Another group of proteins down-regulated in SW480Null cells includes regulators of the actin cytoskeleton,11 which undergoes dramatic reorgani- zation associated with the APCnull phenotype. Levels of the actins ACTB and ACTG1 were unchanged in the SW480Null cells, but levels of the actin regulators FARPB2, EPPK1, VIL1, ACTN1 and GSN all were decreased. Thus, proteomic ﬁndings conﬁrm a previously identiﬁed phenotypic feature of SW480Null cells. The eﬀect of APC loss on the Wnt signaling pathway itself is relatively modest and appears to reﬂect adaptation to APC loss. The negative Wnt regulators DKK4 and CLU were decreased. CACYBP, which participates in CTTNB1 degradation and USP15, a deubiquitylation enzyme which prevents APC degrada- tion, both were increased in SW480ull cells (Figure 3A). We observed a trend of elevated CTNNB1 in SW480APC, although this trend did not meet the 0.1 adjusted quasi p-value threshold (adjusted quasi p-value = 0.18, log2 SW480APC/SW480Null rate ratio = 0.52). However, measurement of cytosolic and nuclear CTNNB1 by MRM revealed a 3-fold increase in cytosolic CTNNB1 in SW480APC (p < 0.001, unpaired t test), suggesting that restoration of APC results in reduced Wnt activation (Figure 3B, top panel). MRM analysis conﬁrmed that total CTNNB1 (cytosolic and nuclear) is higher in SW480APC (log2 SW480APC/ SW480Null = 0.81, p = 0.001, unpaired t test). Changes to proteomic expression proﬁles in APC restored cells may be the result of altered transcription factor activities. Enrichment analysis revealed that targets of the transcription repressor ZEB1 (hsa_CAGGTA_V$AREB6_01, adjusted p-value = 0.022) were measured at lower levels in SW480Null, implying elevated ZEB1 activity in this cell line. Activation of ZEB1 is thought to play a key role in epithelial to mesenchymal transition (EMT), a process characterized by loss of cell polarity and adhesion and increased motility. A prominent feature of EMT is loss of CDH1 expression. ZEB1 represses CDH1 by binding to E-box elements in the promoter region. Therefore, the observed decrease in CDH1 expression in SW480Null may be a direct result of ZEB1 activation.43 Furthermore, the ZEB1 core- pressor SMARCA4 is up-regulated in SW480Null (Figure 3A). Others have shown that disruption of the ZEB1/SMARCA4 binding causes an increase in CDH1 expression and a decrease in the mesenchymal marker VIM, trends seen here in SW480APC.44 Therefore, SMARCA4-dependent activation of ZEB1 may play a role in conferring key phenotypic features in SW480Null. Functional class enrichment analysis identiﬁed several groups with both up-regulated and down-regulated proteins in SW480Null (Table 1, red and green arrows). This result suggests that APC restoration may aﬀect expression of proteins in a common pathway, but changes do not necessarily occur in a uniform direction. For instance, 2/6 proteins (PDCD4, NGFR) were down in SW480- Null and 4/6 proteins (SMARCA4, RB1, EGFR, CENPF) were up in SW480Null for the GO category negative regulation of the cell cycle (GO:0045786, adjusted p-value = 0.014). This ob- servation may reﬂect the multiple roles played by individual pro- teins in the cell cycle. Similarly, for α6β4 integrin signaling (WP244, adjusted p-value = 0.004) 5/7 proteins (LAMA3, LAMB1, DSP, EIF4E, SFN) were down in SW480Null and 2/7 proteins (VIM, EGFR) were up in SW480Null. Elevated VIM and EGFR levels in SW480Null may result in changes to integrin-mediated cell 1190 dx.doi.org/10.1021/pr2009109 |J. Proteome Res. 2012, 11, 1184–1195