Colorectal Cancer

This vignette from the R package canprot reproduces calculations of compositional oxidation state and hydration state and thermodynamic potential that are described in two papers published in PeerJ (Dick, 2016 and Dick, 2017).

The sections below give the Abbreviations used here, a Summary Table comparing the chemical compositions of groups of human proteins that are relatively down- and up-expressed (n1 and n2, respectively) in colorectal cancer (CRC), the Data Sources for protein expression in CRC, a plot of the Mean Differences of average oxidation state of carbon (ZC) and water demand per residue (nH2O), Stability Plots showing the weighted rank difference of chemical affinity between down- and up-expressed groups, and References.

Abbreviations

T (tumor), N (normal), C (carcinoma or adenocarcinoma), A (adenoma), CM (conditioned media), AD (adenomatous colon polyps), CIS (carcinoma in situ), ICC (invasive colonic carcinoma), NC (non-neoplastic colonic mucosa).

Summary Table

library(canprot)
data(canprot)

Identify the datasets for protein expression.

datasets <- pdat_CRC()

Get the amino acid compositions of the up- and down-expressed proteins (get_pdat) and make comparisons of indicators of oxidation and hydration state (ZC_nH2O).

comptab <- lapply_canprot(datasets, function(dataset) {
  pdat <- get_pdat(dataset)
  ZC_nH2O(pdat, plot.it=FALSE)
})

Generate the HTML table with xsummary, which adds bold and underline formatting to the output of xtable. The columns show the difference in means (DM), common language effect size (ES), and p-value (p) for comparisons between groups of the average oxidation state of carbon (ZC) and water demand per residue (nH2O).

library(xtable)
xsummary(comptab)
ZC nH2O
set reference (description) n1 n2 MD ES p-value MD ES p-value
a WTK+08 (T / N) 57 70 0.018 55 3e-01 0.006 52 7e-01
b AKP+10 (CRC nuclear matrix C / A) 101 28 -0.012 47 7e-01 -0.009 48 8e-01
c AKP+10 (CIN nuclear matrix C / A) 87 81 -0.031 40 3e-02 0.006 48 7e-01
d AKP+10 (MIN nuclear matrix C / A) 157 76 -0.002 52 7e-01 -0.013 45 3e-01
e JKMF10 (serum biomarkers up / down) 43 56 -0.007 46 5e-01 0.056 67 4e-03
f XZC+10 (stage I / normal) 48 166 0.008 52 7e-01 0.025 56 2e-01
g XZC+10 (stage II / normal) 77 321 0.021 60 7e-03 0.018 54 3e-01
h ZYS+10 (microdissected T / N) 61 57 0.020 59 1e-01 0.021 58 2e-01
i BPV+11 (adenoma / normal) 71 92 -0.023 40 4e-02 0.004 49 8e-01
j BPV+11 (stage I / normal) 109 72 -0.007 47 5e-01 0.005 50 9e-01
k BPV+11 (stage II / normal) 164 140 0.031 62 3e-04 0.006 51 7e-01
l BPV+11 (stage III / normal) 63 131 0.025 62 9e-03 -0.005 47 5e-01
m BPV+11 (stage IV / normal) 42 26 -0.010 44 4e-01 0.005 52 8e-01
n JCF+11 (T / N) 72 45 0.032 63 2e-02 -0.003 49 8e-01
o MRK+11 (adenoma / normal) 335 288 0.011 54 1e-01 0.058 68 2e-15
p MRK+11 (adenocarcinoma / adenoma) 373 257 0.034 65 1e-10 -0.009 47 1e-01
q MRK+11 (adenocarcinoma / normal) 351 232 0.034 63 4e-08 0.035 61 8e-06
r KKL+12 (poor / good prognosis) 75 61 0.026 64 5e-03 -0.002 48 7e-01
s KYK+12 (MSS-type T / N) 73 175 0.024 61 9e-03 0.023 56 1e-01
t WOD+12 (T / N) 79 677 0.016 54 2e-01 0.026 58 2e-02
u YLZ+12 (CM T / N) 55 68 0.026 62 2e-02 0.008 53 6e-01
v MCZ+13 (stromal T / N) 33 37 0.047 74 5e-04 -0.034 42 2e-01
w KWA+14 (chromatin-binding C / A) 51 55 -0.039 29 2e-04 -0.010 48 7e-01
x UNS+14 (epithelial adenoma / normal) 58 65 0.001 49 8e-01 0.032 61 4e-02
y WKP+14 (tissue secretome T / N) 44 210 0.006 53 6e-01 0.057 68 1e-04
z STK+15 (membrane enriched T / N) 113 66 0.005 52 6e-01 0.025 55 2e-01
A WDO+15 (adenoma / normal) 1061 1254 0.030 64 6e-33 0.023 58 7e-11
B WDO+15 (carcinoma / adenoma) 772 1007 -0.013 42 2e-08 -0.003 50 7e-01
C WDO+15 (carcinoma / normal) 879 1281 0.014 57 9e-08 0.024 58 1e-10
D LPL+16 (stromal AD / NC) 123 75 -0.039 32 2e-05 0.037 60 2e-02
E LPL+16 (stromal CIS / NC) 125 60 -0.007 46 4e-01 -0.001 52 7e-01
F LPL+16 (stromal ICC / NC) 99 75 0.001 47 6e-01 -0.021 48 7e-01
G LXM+16 (biopsy T / N) 191 178 0.005 50 9e-01 0.028 57 2e-02
H PHL+16 (AD / NC) 113 86 0.011 54 4e-01 0.037 60 2e-02
I PHL+16 (CIS / NC) 169 138 0.019 59 5e-03 0.001 49 7e-01
J PHL+16 (ICC / NC) 129 100 0.016 57 5e-02 -0.007 46 3e-01

Data Sources

a. Watanabe et al. (2008) used 2-nitrobenzenesulfenyl labeling and MS/MS analysis to identify 128 proteins with differential expression in paired CRC and normal tissue specimens from 12 patients. The list of proteins used in this study was generated by combining the lists of up- and down-regulated proteins from Table 1 and Supplementary Data 1 of Watanabe et al. (2008) with the Swiss-Prot and UniProt accession numbers from their Supplementary Data 2. b. c. d. Albrethsen et al. (2010) used nano-LC-MS/MS to characterize proteins from the nuclear matrix fraction in samples from 2 patients each with adenoma (ADE), chromosomal instability CRC (CIN+) and microsatellite instability CRC (MIN+). Cluster analysis was used to classify proteins with differential expression between ADE and CIN+, MIN+, or in both subtypes of carcinoma (CRC). Here, gene names from Supplementary Tables 5–7 of Albrethsen et al. (2010) were converted to UniProt IDs using the UniProt mapping tool. e. Jimenez et al. (2010) compiled a list of candidate serum biomarkers from a meta-analysis of the literature. In the meta-analysis, 99 up- or down-expressed proteins were identified in at least 2 studies. The list of UniProt IDs used in this study was taken from Table 4 of Jimenez et al. (2010). f. g. Xie et al. (2010) used a gel-enhanced LC-MS method to analyze proteins in pooled tissue samples from 13 stage I and 24 stage II CRC patients and pooled normal colonic tissues from the same patients. Here, IPI accession numbers from Supplemental Table 4 of Xie et al. (2010) were converted to UniProt IDs using the DAVID conversion tool. h. Zhang et al. (2010) used acetylation stable isotope labeling and LTQ-FT MS to analyze proteins in pooled microdissected epithelial samples of tumor and normal mucosa from 20 patients, finding 67 and 70 proteins with increased or decreased expression (ratios ≥2 or ≤0.5). Here, IPI accession numbers from Supplemental Table 4 of Zhang et al. (2010) were converted to UniProt IDs using the DAVID conversion tool. i. j. k. l. m. Besson et al. (2011) analyzed microdissected cancer and normal tissues from 28 patients (4 adenoma samples and 24 CRC samples at different stages) using iTRAQ labeling and MALDI-TOF/TOF MS to identify 555 proteins with differential expression between adenoma and stage I, II, III, IV CRC. Here, gene names from supplemental Table 9 of Besson et al. (2011) were converted to UniProt IDs using the UniProt mapping tool. n. Jankova et al. (2011) analyzed paired samples from 16 patients using iTRAQ-MS to identify 118 proteins with >1.3-fold differential expression between CRC tumors and adjacent normal mucosa. The protein list used in this study was taken from Supplementary Table 2 of Jankova et al. (2011). o. p. q. Mikula et al. (2011) used iTRAQ labeling with LC-MS/MS to identify a total of 1061 proteins with differential expression (fold change ≥1.5 and false discovery rate ≤0.01) between pooled samples of 4 normal colon (NC), 12 tubular or tubulo-villous adenoma (AD) and 5 adenocarcinoma (AC) tissues. The list of proteins used in this study was taken from from Table S8 of Mikula et al. (2011). r. Kim et al. (2012) used difference in-gel electrophoresis (DIGE) and cleavable isotope-coded affinity tag (cICAT) labeling followed by mass spectrometry to identify 175 proteins with more than 2-fold abundance ratios between microdissected and pooled tumor tissues from stage-IV CRC patients with good outcomes (survived more than five years; 3 patients) and poor outcomes (died within 25 months; 3 patients). The protein list used in this study was made by filtering the cICAT data from Supplementary Table 5 of Kim et al. (2012) with an abundance ratio cutoff of >2 or <0.5, giving 147 proteins. IPI accession numbers were converted to UniProt IDs using the DAVID conversion tool. s. Kang et al. (2012) used mTRAQ and cICAT analysis of pooled microsatellite stable (MSS-type) CRC tissues and pooled matched normal tissues from 3 patients to identify 1009 and 478 proteins in cancer tissue with increased or decreased expression by higher than 2-fold, respectively. Here, the list of proteins from Supplementary Table 4 of Kang et al. (2012) was filtered to include proteins with expression ratio >2 or <0.5 in both mTRAQ and cICAT analyses, leaving 175 up-expressed and 248 down-expressed proteins in CRC. Gene names were converted to UniProt IDs using the UniProt mapping tool. t. Wiśniewski et al. (2012) used LC-MS/MS to analyze proteins in microdissected samples of formalin-fixed paraffin-embedded (FFPE) tissue from 8 patients; at P < 0.01, 762 proteins had differential expression between normal mucosa and primary tumors. The list of proteins used in this study was taken from Supplementary Table 4 of Wiśniewski et al. (2012). u. Yao et al. (2012) analyzed the conditioned media of paired stage I or IIA CRC and normal tissues from 9 patients using lectin affinity capture for glycoprotein (secreted protein) enrichment by nano LC-MS/MS to identify 68 up-regulated and 55 down-regulated differentially expressed proteins. IPI accession numbers listed in Supplementary Table 2 of Yao et al. (2012) were converted to UniProt IDs using the DAVID conversion tool. v. Mu et al. (2013) used laser capture microdissection (LCM) to separate stromal cells from 8 colon adenocarcinoma and 8 non-neoplastic tissue samples, which were pooled and analyzed by iTRAQ to identify 70 differentially expressed proteins. Here, gi numbers listed in Table 1 of Mu et al. (2013) were converted to UniProt IDs using the UniProt mapping tool; FASTA sequences of 31 proteins not found in UniProt were downloaded from NCBI and amino acid compositions were added to human_extra.csv. w. Knol et al. (2014) used differential biochemical extraction to isolate the chromatin-binding fraction in frozen samples of colon adenomas (3 patients) and carcinomas (5 patients), and LC-MS/MS was used for protein identification and label-free quantification. The results were combined with a database search to generate a list of 106 proteins with nuclear annotations and at least a three-fold expression difference. Here, gene names from Table 2 of Knol et al. (2014) were converted to UniProt IDs. x. Uzozie et al. (2014) analyzed 30 samples of colorectal adenomas and paired normal mucosa using iTRAQ labeling, OFFGEL electrophoresis and LC-MS/MS. 111 proteins with expression fold changes (log2) at least +/- 0.5 and statistical significance threshold q < 0.02 that were also quantified in cell-line experiments were classified as “epithelial cell signature proteins”. UniProt IDs were taken from Table III of Uzozie et al. (2014). y. Wit et al. (2014) analyzed the secretome of paired CRC and normal tissue from 4 patients, adopting a five-fold enrichment cutoff for identification of candidate biomarkers. Here, the list of proteins from Supplementary Table 1 of Wit et al. (2014) was filtered to include those with at least five-fold greater or lower abundance in CRC samples and p < 0.05. Two proteins listed as “Unmapped by Ingenuity” were removed, and gene names were converted to UniProt IDs using the UniProt mapping tool. z. Sethi et al. (2015) analyzed the membrane-enriched proteome from tumor and adjacent normal tissues from 8 patients using label-free nano-LC-MS/MS to identify 184 proteins with a fold change > 1.5 and p-value < 0.05. Here, protein identifiers from Supporting Table 2 of Sethi et al. (2015) were used to find the corresponding UniProt IDs. A. B. C. Wiśniewski et al. (2015) analyzed 8 matched formalin-fixed and paraffin-embedded (FFPE) samples of normal tissue (N) and adenocarcinoma (C) and 16 nonmatched adenoma samples (A) using LC-MS to identify 2300 (N/A), 1780 (A/C) and 2161 (N/C) up- or down-regulated proteins at p < 0.05. The list of proteins used in this study includes only those marked as having a significant change in SI Table 3 of Wiśniewski et al. (2015). D. E. F. Li et al. (2016) used iTRAQ and 2D LC-MS/MS to analyze pooled samples of stroma purified by laser capture microdissection (LCM) from 5 cases of non-neoplastic colonic mucosa (NC), 8 of adenomatous colon polyps (AD), 5 of colon carcinoma in situ (CIS) and 9 of invasive colonic carcinoma (ICC). A total of 222 differentially expressed proteins between NC and other stages were identified. Here, gene symbols from Supplementary Table S3 of Li et al. (2016) were converted to UniProt IDs using the UniProt mapping tool. G. Data were extracted from SI Table S3 of Liu et al. (2016), including proteins with p-value <0.05. H. I. J. Peng et al. (2016) used iTRAQ 2D LC-MS/MS to analyze pooled samples from 5 cases of normal colonic mucosa (NC), 8 of adenoma (AD), 5 of carcinoma in situ (CIS) and 9 of invasive colorectal cancer (ICC). A total of 326 proteins with differential expression between two successive stages (and, for CIS and ICC, also differentially expressed with respect to NC) were detected. The list of proteins used in this study was generated by converting the gene names in Supplementary Table 4 of Peng et al. (2016) to UniProt IDs using the UniProt mapping tool.

Mean Differences

Using data from the table above, this plot shows that the groups of proteins that are relatively up-expressed in colorectal cancer or more advanced cancer stages predominantly have higher ZC and/or nH2O. The datasets comparing adenoma to normal proteomes are highlighted in red.

col <- rep("black", length(datasets))
# highlight adenoma / normal datasets
col[grepl("=AD", datasets)] <- "red"
diffplot(comptab, col=col)

Stability Plots

These plots show the weighted rank-sum differences of chemical affinities of formation from basis species of proteins in different groups. A higher ranking of relatively down- or up-expressed proteins in colorectal cancer is represented by blue or red color, respectively. The last plot shows effective values of Eh (redox potential) as a function of the same variables (oxygen fugacity and water activity).

Identify the datasets for protein expression.

datasets <- c("JKMF10", "UNS+14", "WKP+14", "MRK+11_AD.NC", "MRK+11_AC.AD", "MRK+11_AC.NC", "JCF+11", "KWA+14")

Set up the diagram and make the plots.

par(mfrow=c(3, 3))
par(xaxs="i", yaxs="i", las=1, mar=c(4, 4, 2, 2), mgp=c(2.6, 1, 0), cex=1)
for(i in seq_along(datasets)) {
  pdat <- get_pdat(datasets[i], basis="QEC4")
  rankplot(pdat, res=50)
  CHNOSZ::label.figure(LETTERS[i], paren=FALSE, font=2, yfrac=0.94)
}
Ehplot()
CHNOSZ::label.figure("I", paren=FALSE, font=2, yfrac=0.94)

This is similar to Figure 6 of Dick (2016). To reduce the time needed to create the vignette, the plots here are made at lower resolution than those in the paper.

References

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