Journal of Proteome Research ARTICLE Figure 2. Proteomic and transcriptomic proﬁle comparison of SW480APC and SW480Null. Heat map shows supervised clustering analysis of shotgun proteomics data and transcriptomic data for 111 diﬀerentially expressed proteins (adjusted quasi p-value <0.1) (red, increased expression; green, decreased expression). Clustering was performed on log2 transformed spectral counts from 6 replicate analyses for proteomics and on log2 transformed RMA values for microarray. (e.g., microRNA (miRNA), mRNA processing) contribute to the observed discordance. Interestingly, DICER1 which plays a key role in RNA-induced silencing is up-regulated in SW480APC, presumably resulting in altered microRNA biosynthesis and expression. Altered DICER1 expression is a feature of several cancers including ovarian, pancreatic and lung cancer.35 37 Pre- vious studies have shown conﬂicting results for DICER1 expres- sion levels in CRC, with some noting decreased expression in colon adenocarcimonas, while others demonstrated that over- expression is associated with poor prognosis.38,39 The data here suggest that restoration of wildtype APC correlates with en- hanced DICER1 expression which may result in altered miRNA expression proﬁles. Further inspection of 26 discordant proteins revealed enrichment for several miRNA targets, including miR- 338 (adjusted p-value =0.01) and miR-193A (adjusted p-value = 0.07) (Webgestalt enrichment analysis, see Experimental Meth- ods). miR-338 is down-regulated in hepatocellular carcinoma, while overexpression of miR-193A inhibits tumor growth in colon xenografts by targeting KRAS and PLAU.40,41 These data suggest that changes to speciﬁc miRNA levels might result in discordant protein/transcript expression.42 Therefore, restora- tion of APC may aﬀect miR-338 and miR-193A expression levels, thereby giving rise to discordant transcript/protein levels in spe- ciﬁc targets, e.g. PLD3, SIX4. Protein Networks and Pathways Affected by Reintroduction of APC Using quasi-likelihood modeling of protein spectral counts we identiﬁed a set of 155 proteins showing diﬀerential expression be- tween SW480APC and SW480Null (adjusted quasi p-value <0.1) (97 down in SW480Null, 57 up in SW480Null). Within this set of 155 proteins we carried out a systematic search for enrichment of functionally related protein classes using the Webgestalt software tool.28 Table 1 shows the top ﬁve sets of proteins (ranked by adjusted p-value) from multiple enrichment analyses (GO, KEGG, etc). This approach identiﬁed groups of proteins that were down-regulated in SW480Null cells (>75% proteins down in SW480Null, red arrows alone) including cell adhesion (GO:0007155, adjusted p-value = 0.005) and type II interferon signaling (WP619, adjusted p-value = 0.031). In- creased expression of proteins involved in protein kinase C bind- ing (GO:0005080, adjusted p-value = 0.133) and DNA topo- isomerase activity (GO:0003916, adjusted p-value = 0.133) was observed in SW480Null (>75% proteins up in SW480Null, green arrows alone). Interestingly, some groups enriched for diﬀeren- tially expressed proteins contained proteins that were either up- regulated or down-regulated in SW480Null (<75% proteins up or <75% down in SW480Null, red and green arrows), including negative regulation of the cell cycle (GO:0045786, adjusted p-value = 0.015) and terpenoid backbone biosynthesis (KEGG ID 900, adjusted p-value = 0.003). Pathways and protein functional classes aﬀected by APC restoration (derived from Webgestalt enrichment analysis) are depicted schematically in Figure 3A. A major proteomic feature of SW480Null cell line is the down-regulation cell adhesion proteins. Loss of epithelial cadherin (CDH1) and the attendant abrogation of adherens junctions have been previously reported in SW480Null cells.11 CDH1 was shown by Western blot to have 2-fold higher expression in SW480APC cells.11 Here we ob- served 2-fold higher expression of CDH1 in SW480APC by 1188 dx.doi.org/10.1021/pr2009109 |J. Proteome Res. 2012, 11, 1184–1195