The colon adenocarcinoma proteome TCGA data analysis Unfavorable prognostic genes in colon adenocarcinoma Favorable prognostic genes in colon adenocarcinoma CPTAC relative protein expression data The colon adenocarcinoma transcriptome Additional information Relevant links and publications

The colon adenocarcinoma proteome

Colorectal cancer is the third most common cancer in the world and the fifth leading cause of cancer-related mortality. Environmental factors, including meat consumption, have been identified as important risk factors. The overall mortality is approximately 50%. The surgical stage at diagnosis is the most important factor for predicting prognosis and the survival rate varies greatly depending on the stage. The 5-year survival rate is more than 90% for stage I and less than 10% for stage IV. Most colorectal cancer cases are detected at an advanced stage. Bleeding and hematochezia are two of the most common symptoms associated with rectal lesions.

Colorectal cancer is considered to originate from normal colon epithelium that develops into precursor lesions termed adenomas that subsequently may progress to invasive colorectal adenocarcinomas with metastatic potential. Colorectal cancer is divided into two subtypes, colon adenocarcinomas (COAD) and rectum adenocarcinomas (READ), depending on the site of the tumor.

Here, we explore the colon adenocarcinoma proteome using TCGA transcriptomics data and antibody-based protein data. 606 genes are suggested as prognostic based on transcriptomics data from 254 patients; 287 genes are associated with unfavorable prognosis and 321 genes are associated with favorable prognosis.

TCGA data analysis

In this metadata study, we used data from TCGA where transcriptomics data was available from 254 patients in total, 111 females and 143 males. A majority of the patients (193 patients) were still alive at the time of data collection. The stage distribution was stage i) 39 patients, stage ii) 98 patients, stage iii) 72 patients, stage iv) 35 patients and 10 patients with missing stage information.

Unfavorable prognostic genes in colon adenocarcinoma

For unfavorable genes, higher relative expression levels at diagnosis give significantly lower overall survival for the patients. There are 287 genes associated with an unfavorable prognosis in colon adenocarcinoma, among these potential prognostic genes there are 2 genes that were validated in a separate study. In Table 1, the top 20 most significant genes related to an unfavorable prognosis are listed.

ARHGAP4 is a gene associated with unfavorable prognosis in colon adenocarcinoma. The best separation is achieved by an expression cutoff at 23 TPM which divides the patients into two groups with 41% 5-year survival for patients with high expression versus 78% for patients with low expression, p-value: 1.15e-4. Immunohistochemical staining using an antibody targeting ARHGAP4 (HPA001012) shows a differential expression pattern in colon adenocarcinoma samples.

p<0.001
ARHGAP4 - survival analysis
ARHGAP4 - high expression
ARHGAP4 - low expression

DPP7 is another gene associated with an unfavorable prognosis incolon adenocarcinoma in two separate independent cohorts. The best separation is achieved by an expression cutoff at 64 TPM which divides the patients into two groups with 44% 5-year survival for patients with high expression versus 73% for patients with low expression, p-value: 4.71e-5. The TCGA data analysis was validated in a separate study with the p-value: 2.34e-4. Immunohistochemical staining using an antibody targeting DPP7 (HPA021282) shows a differential expression pattern in colon adenocarcinoma samples.

p<0.001
DPP7 - survival analysis
DPP7 - high expression
DPP7 - low expression

Table 1. The 20 genes with highest significance associated with an unfavorable prognosis in colon adenocarcinoma.

Gene	Description	Predicted location	mRNA (cancer)	p-value	Prognostic
DPP7	Dipeptidyl peptidase 7	Secreted	60.0	4.71e-5	validated
NUDT1	Nudix hydrolase 1	Intracellular	48.9	6.81e-5	potential
PCBP3	Poly(rC) binding protein 3	Intracellular	1.8	3.44e-4	potential
LZTS1	Leucine zipper tumor suppressor 1	Intracellular	1.5	5.08e-4	validated
AIMP2	Aminoacyl tRNA synthetase complex interacting multifunctional protein 2	Intracellular	45.9	6.75e-4	potential
NUMBL	NUMB like endocytic adaptor protein	Intracellular	6.5	7.25e-4	potential
RP9	RP9 pre-mRNA splicing factor	Intracellular	17.5	8.53e-4	potential
KDSR	3-ketodihydrosphingosine reductase	Membrane, Intracellular	7.8	1.45e-1	unprognostic
PELI2	Pellino E3 ubiquitin protein ligase family member 2	Intracellular	2.4	2.78e-1	unprognostic
CPEB3	Cytoplasmic polyadenylation element binding protein 3	Intracellular	0.8	0.00e+0	unprognostic
PDCL	Phosducin like	Intracellular	11.0	7.97e-2	unprognostic
KIDINS220	Kinase D interacting substrate 220	Membrane, Intracellular	11.1	1.35e-1	unprognostic
NECAP1	NECAP endocytosis associated 1	Intracellular	12.1	5.89e-2	unprognostic
UBE2R2	Ubiquitin conjugating enzyme E2 R2	Intracellular	26.4	3.10e-1	unprognostic
TSC1	TSC complex subunit 1	Membrane, Intracellular	3.7	9.47e-2	unprognostic
RAB14	RAB14, member RAS oncogene family	Intracellular	32.9	3.29e-1	unprognostic
SAV1	Salvador family WW domain containing protein 1	Intracellular	10.6	1.32e-1	unprognostic
PTPN9	Protein tyrosine phosphatase non-receptor type 9	Intracellular	14.3	1.94e-1	unprognostic
WDR20	WD repeat domain 20	Intracellular	8.9	6.57e-2	unprognostic
FKBP4	FKBP prolyl isomerase 4	Intracellular	81.1	3.60e-1	unprognostic
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Favorable prognostic genes in colon adenocarcinoma

For favorable genes, higher relative expression levels at diagnosis give significantly higher overall survival for the patients. There are 321 genes associated with a favorable prognosis in colon adenocarcinoma, among these potential prognostic genes there are 3 genes that were validated in a separate study. In Table 2, the top 20 most significant genes related to a favorable prognosis are listed.

ABCD3 is a gene associated with a favorable prognosis in colon adenocarcinoma. The best separation is achieved by an expression cutoff at 15 TPM which divides the patients into two groups with 74% 5-year survival for patients with high expression versus 28% for patients with low expression, p-value: 6.83e-9. Immunohistochemical staining using an antibody targeting ABCD3 (HPA032026) shows a differential expression pattern in colon adenocarcinoma samples.

p<0.001
ABCD3 - survival analysis
ABCD3 - high expression
ABCD3 - low expression

DSC2 is another gene associated with a favorable prognosis in colon adenocarcinoma in two separate independent cohorts. The best separation is achieved by an expression cutoff at 12 TPM which divides the patients into two groups with 68% 5 year survival for patients with high expression versus 45% for patients with low expression, p-value: 8.02e-4. The TCGA data analysis was validated in a separate study with the p-value: 2.92e-4. Immunohistochemical staining using an antibody targeting DSC2 (HPA011911) shows a differential expression pattern in colon adenocarcinoma samples.

p<0.001
DSC2 - survival analysis
DSC2 - high expression
DSC2 - low expression

Table 2. The 20 genes with highest significance associated with a favorable prognosis in colon adenocarcinoma.

Gene	Description	Predicted location	mRNA (cancer)	p-value	Prognostic
SORT1	Sortilin 1	Membrane, Intracellular	22.1	9.78e-6	potential
ARFIP1	ADP ribosylation factor interacting protein 1	Intracellular	20.8	1.74e-5	potential
USP53	Ubiquitin specific peptidase 53	Intracellular	7.7	2.41e-5	potential
SHQ1	SHQ1, H/ACA ribonucleoprotein assembly factor	Intracellular	9.3	3.68e-5	potential
ZNF207	Zinc finger protein 207	Intracellular	78.7	6.06e-5	potential
TAF13	TATA-box binding protein associated factor 13	Intracellular	13.5	6.10e-5	potential
UBE2N	Ubiquitin conjugating enzyme E2 N	Intracellular	92.0	7.49e-5	potential
SFPQ	Splicing factor proline and glutamine rich	Intracellular	68.0	7.64e-5	potential
UBE2D2	Ubiquitin conjugating enzyme E2 D2	Intracellular	93.5	8.30e-5	potential
PSMB5	Proteasome 20S subunit beta 5	Intracellular	142.8	1.31e-4	potential
AMD1	Adenosylmethionine decarboxylase 1	Intracellular	38.9	1.52e-4	potential
RFT1	RFT1 homolog	Membrane	21.4	1.78e-4	potential
LYPD6	LY6/PLAUR domain containing 6	Secreted	2.1	2.48e-4	validated
ATP8B1	ATPase phospholipid transporting 8B1	Membrane, Intracellular	20.1	3.38e-4	potential
MAGI3	Membrane associated guanylate kinase, WW and PDZ domain containing 3	Intracellular	4.4	3.52e-4	potential
NUDT9	Nudix hydrolase 9	Intracellular	21.5	3.57e-4	potential
ORC1	Origin recognition complex subunit 1	Intracellular	5.9	3.71e-4	potential
SNX7	Sorting nexin 7	Intracellular	24.1	4.06e-4	potential
TXNDC12	Thioredoxin domain containing 12	Intracellular	86.9	4.28e-4	potential
SRSF3	Serine and arginine rich splicing factor 3	Intracellular	128.8	4.47e-4	potential
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CPTAC relative protein expression data

Proteins that are significantly down- or upregulated in colon adenocarcinoma compared to normal tissue is illustrated in a vulcano plot using tandem mass tag (TMT) mass spectrometry data from the CPTAC dataset based on the analysis of 97 tumor samples and 100 normal samples.

In colon adenocarcinoma, 1923 and 1887 genes are down- (blue) and upregulated (red) compared to normal tissue, respectively. In Table 3, the top 20 most significant genes are listed.

Figure 1. Proteins highlighted in blue are significantly downregulated in cancer tissue, while those in red are significantly upregulated when compared to normal tissue. Gray points represent non-significant proteins based on the log2 (fold change). Wilcox rank test with adjusted p values.

Table 3. The 20 genes with the highest significance associated with a downregulated or upregulated protein expression in colon adenocarcinoma compared to normal tissue.

Gene	Description	Predicted location	Log2 fold change	p-value	Regulation
NCAM1	Neural cell adhesion molecule 1	Secreted, Membrane, Intracellular	-2.08	8.09e-34	Downregulated
SCGN	Secretagogin, EF-hand calcium binding protein	Intracellular	-2.34	8.60e-34	Downregulated
CADM3	Cell adhesion molecule 3	Membrane, Intracellular	-1.78	8.60e-34	Downregulated
ANK2	Ankyrin 2	Intracellular	-1.2	8.60e-34	Downregulated
DDX18	DEAD-box helicase 18	Intracellular	0.81	8.87e-34	Upregulated
GNL3	G protein nucleolar 3	Intracellular	0.89	1.03e-33	Upregulated
L1CAM	L1 cell adhesion molecule	Membrane, Intracellular	-1.95	1.06e-33	Downregulated
NOP2	NOP2 nucleolar protein	Intracellular	0.78	1.06e-33	Upregulated
GTPBP4	GTP binding protein 4	Intracellular	0.85	1.10e-33	Upregulated
RPL7L1	Ribosomal protein L7 like 1	Intracellular	0.95	1.20e-33	Upregulated
PDCD11	Programmed cell death 11	Intracellular	0.71	1.24e-33	Upregulated
EPB41L3	Erythrocyte membrane protein band 4.1 like 3	Membrane, Intracellular	-0.95	1.40e-33	Downregulated
ADH1B	Alcohol dehydrogenase 1B (class I), beta polypeptide	Intracellular	-2.32	1.53e-33	Downregulated
NEGR1	Neuronal growth regulator 1	Membrane	-2.04	1.53e-33	Downregulated
NOC2L	NOC2 like nucleolar associated transcriptional repressor	Intracellular	0.75	1.84e-33	Upregulated
UTP4	UTP4 small subunit processome component	Intracellular	0.84	1.90e-33	Upregulated
ADH1A	Alcohol dehydrogenase 1A (class I), alpha polypeptide	Intracellular	-2.22	2.01e-33	Downregulated
ABCA8	ATP binding cassette subfamily A member 8	Membrane	-1.94	2.01e-33	Downregulated
METTL7A	Methyltransferase like 7A	Membrane	-1.04	2.08e-33	Downregulated
NUP93	Nucleoporin 93	Membrane, Intracellular	0.49	2.42e-33	Upregulated
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The colon adenocarcinoma transcriptome

The transcriptome analysis shows that 67% (n=13454) of all human genes (n=20162) are expressed in colon adenocarcinoma. All genes were classified according to the colon adenocarcinoma-specific expression into one of five different categories, based on the ratio between mRNA levels in colon adenocarcinoma compared to the mRNA levels in the other 16 analyzed cancer tissues.

Figure 2. The distribution of all genes across the five categories based on transcript abundance in colon adenocarcinoma as well as in all other cancer tissues.

191 genes show some level of elevated expression in colon adenocarcinoma compared to other cancers (Figure 1). The elevated category is further subdivided into three categories as shown in Table 3.

Table 4. The number of genes in the subdivided categories of elevated expression in colon adenocarcinoma.

		Distribution in the 31 cancers
		Detected in single	Detected in some	Detected in many	Detected in all	Total
Specificity	Cancer enriched	0	0	0	0	0
	Group enriched	0	42	53	1	96
	Cancer enhanced	2	34	54	5	95
	Total	2	76	107	6	191

Additional information

Appropriate diagnosis and staging are crucial for determining the best choice of treatment. The surgical stage represents a classification system based on the extent and depth of tumor growth. Stage I colorectal cancer shows invasive growth into the anatomical layers of the the large intestine, but the tumor has not spread beyond the tissue of origin. Stage II colorectal cancer shows extended growth through the outer layer of the large intestine (peritoneum) and may have extended into nearby organs, but has not spread to any lymph node. Stage III colorectal cancer has spread to nearby lymph nodes but not yet metastasized to distant sites in the body. Finally, in Stage IV colorectal cancer the tumor has spread to distant organs such as the liver, lungs, or other sites. The Dukes classification is an older and less complicated staging system that predates the TNM system, and translates so that Duke A= Stage I, Duke B= Stage II, Duke C= Stage III and Dukes D= Stage IV.

Early colorectal cancer, where tumor spread is restricted to large intestine, is treated surgically and chemotherapy is used for more advanced stages where the tumor has spread to other organs. Anti-EGFR treatment is one recently introduced therapy. Epidermal growth factor receptor (EGFR) is commonly expressed in colorectal tumors and monoclonal antibodies inhibiting EGFR demonstrate clinical efficacy in patients with tumors that do not harbor downstream activating KRAS mutations. Today KRAS mutation status is analyzed routinely before starting anti-EGFR treatment.

The vast majority of colorectal cancer are adenocarcinomas, with less than 10% of the cancers being distinguished by an abundant secretion of mucin. The tumors are classified according to the degree of morphological differentiation into well, moderately and poorly differentiated. About 80% are well or moderately differentiated with a growth pattern consisting of tumor cells that form irregular glandular structures present at different layers of the bowel wall. Poorly differentiated colorectal cancer show no, or only slight, glandular formation. Generally poor differentiation is associated with poor prognosis, however there is no firmly established system for measuring the grade of differentiation. Therefore, treatment decisions are based on the surgical stage and not morphological features. Apart from adenocarcinomas, endocrine tumors can also arise within the colorectal mucosa. Squamous and adenosquamous tumors are exceedingly rare.

In addition to microscopical examination of biopsies, immunohistochemistry can be used to determine the colorectal origin of a metastasis or to visualize the spread of tumor cells in surrounding tissues. Tumors of colorectal origin are immunoreactive toward cytokeratin 20, CDX-2, SATB2 and cadherin-17. Chromogranin-A antibodies can be used to distinguish endocrine tumors in the bowel from adenocarcinomas.

Relevant links and publications

Uhlen M et al., A pathology atlas of the human cancer transcriptome. Science. (2017)
PubMed: 28818916 DOI: 10.1126/science.aan2507

Cancer Genome Atlas Research Network et al., The Cancer Genome Atlas Pan-Cancer analysis project. Nat Genet. (2013)
PubMed: 24071849 DOI: 10.1038/ng.2764

Uhlén M et al., Tissue-based map of the human proteome. Science (2015)
PubMed: 25613900 DOI: 10.1126/science.1260419

Gremel G et al., The human gastrointestinal tract-specific transcriptome and proteome as defined by RNA sequencing and antibody-based profiling. J Gastroenterol. (2015)
PubMed: 24789573 DOI: 10.1007/s00535-014-0958-7

Histology dictionary - Colorectal cancer