The colorectal cancer 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 colorectal cancer proteome using TCGA transcriptomics data and antibody-based protein data. 603 genes are suggested as prognostic based on transcriptomics data from 597 patients; 243 genes are associated with unfavorable prognosis and 360 genes are associated with favorable prognosis.

TCGA data analysis

In this metadata study, we focus on combined colorectal cancer data from TCGA, separated data from colon (COAD) and rectum (READ) adenocarcinomas is also available and presented. The transcriptomics data was available from 597 patients in total, 438 COAD (204 female and 234 male) and 159 READ (71 female and 88 male). Most of the patients (473 patients) were still alive at the time of data collection. The stage distribution was stage i) 103 patients, stage ii) 215 patients, stage iii) 174 patients, stage iv) 85 patients and 20 patients with missing stage information.

Unfavorable prognostic genes in colorectal cancer

For unfavorable genes, higher relative expression levels at diagnosis give significantly lower overall survival for the patients. There are 243 genes associated with an unfavorable prognosis in colorectal cancer. 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 colorectal cancer. The best separation is achieved by an expression cutoff at 16.8 fpkm which divides the patients into two groups with 44% 5-year survival for patients with high expression versus 67% for patients with low expression, p-value: 1.19e-5. A survival analysis in the different subtypes showed significant association only in colon adenocarcinoma. Immunohistochemical staining using an antibody targeting ARHGAP4 (HPA001012) shows a differential expression pattern in colorectal cancer samples.

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

JDP2 is another gene associated with an unfavorable prognosis in colorectal cancer. The best separation is achieved by an expression cutoff at 4.5 fpkm which divides the patients into two groups with 53% 5-year survival for patients with high expression versus 64% for patients with low expression, p-value: 8.58e-5. A survival analysis in the different subtypes showed significant association only in colon adenocarcinoma. Immunohistochemical staining using an antibody targeting JDP2 (HPA059511) shows a differential expression pattern in colorectal cancer samples.

p<0.001
JDP2 - survival analysis
JDP2 - high expression
JDP2 - low expression

Table 1. The 20 genes with highest significance associated with an unfavorable prognosis in colorectal cancer.

Gene	Description	Predicted location	mRNA (cancer)	p-value
LRCH4	leucine rich repeats and calponin homology domain containing 4	Membrane, Intracellular	2.5	1.61e-7
POFUT2	protein O-fucosyltransferase 2	Intracellular	4.8	4.55e-7
CLK3	CDC like kinase 3	Intracellular	2.2	2.05e-6
EGFL7	EGF like domain multiple 7	Secreted	6.9	4.22e-6
DPP7	dipeptidyl peptidase 7	Secreted	40.4	6.07e-6
HSH2D	hematopoietic SH2 domain containing	Intracellular	6.0	9.46e-6
ASB6	ankyrin repeat and SOCS box containing 6	Intracellular	6.7	1.06e-5
SPAG4	sperm associated antigen 4	Membrane, Intracellular	3.2	1.09e-5
EXOC3L4	exocyst complex component 3 like 4	Intracellular	1.6	1.43e-5
HSPA1A	heat shock protein family A (Hsp70) member 1A	Intracellular	15.7	1.54e-5
PAQR6	progestin and adipoQ receptor family member 6	Membrane, Intracellular	1.2	1.60e-5
DIPK1B	divergent protein kinase domain 1B	Membrane, Intracellular	1.1	1.77e-5
CRACR2B	calcium release activated channel regulator 2B	Intracellular	13.3	1.81e-5
ARHGAP4	Rho GTPase activating protein 4	Intracellular	10.3	1.97e-5
NPDC1	neural proliferation, differentiation and control 1	Membrane, Intracellular	51.5	2.01e-5
DAPK1	death associated protein kinase 1	Membrane	2.4	2.48e-5
CNPY3	canopy FGF signaling regulator 3	Intracellular	47.8	3.03e-5
ARL8A	ADP ribosylation factor like GTPase 8A	Intracellular	23.4	3.08e-5
INAFM1	InaF motif containing 1	Intracellular	7.6	3.20e-5
RHBDD2	rhomboid domain containing 2	Membrane	37.0	3.31e-5
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Favorable prognostic genes in colorectal cancer

For favorable genes, higher relative expression levels at diagnosis give significantly higher overall survival for the patients. There are 360 genes associated with a favorable prognosis in colorectal cancer. 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 colorectal cancer. The best separation is achieved by an expression cutoff at 6.9 fpkm which divides the patients into two groups with 71% 5-year survival for patients with high expression versus 36% for patients with low expression, p-value: 1.16e-5. A survival analysis in the different subtypes showed significant association only in colon adenocarcinoma. Immunohistochemical staining using an antibody targeting ABCD3 (HPA032026) shows a differential expression pattern in colorectal cancer samples.

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

MCM4 is another gene associated with a favorable prognosis in colorectal cancer. The best separation is achieved by an expression cutoff at 16.5 fpkm which divides the patients into two groups with 65% 5 year survival for patients with high expression versus 44% for patients with low expression, p-value: 2.19e-4. A survival analysis in the different subtypes showed significant association only in colon adenocarcinoma. Immunohistochemical staining using an antibody targeting MCM4 (HPA004873) shows a differential expression pattern in colorectal cancer samples.

p<0.001
MCM4 - survival analysis
MCM4 - high expression
MCM4 - low expression

Table 2. The 20 genes with highest significance associated with a favorable prognosis in colorectal cancer.

Gene	Description	Predicted location	mRNA (cancer)	p-value
RBM3	RNA binding motif protein 3	Intracellular	85.3	3.07e-7
NOL11	nucleolar protein 11	Intracellular	12.8	1.00e-6
USP53	ubiquitin specific peptidase 53	Intracellular	4.1	3.15e-6
TEX2	testis expressed 2	Membrane, Intracellular	5.7	3.90e-6
HOOK1	hook microtubule tethering protein 1	Intracellular	7.8	4.53e-6
ZYG11B	zyg-11 family member B, cell cycle regulator	Intracellular	3.4	5.42e-6
HSPA8	heat shock protein family A (Hsp70) member 8	Intracellular	327.4	6.14e-6
DLAT	dihydrolipoamide S-acetyltransferase	Intracellular	13.2	6.19e-6
SORT1	sortilin 1	Membrane, Intracellular	15.7	7.74e-6
DDX46	DEAD-box helicase 46	Intracellular	7.9	9.01e-6
FBXO7	F-box protein 7	Intracellular	11.2	1.08e-5
ABCD3	ATP binding cassette subfamily D member 3	Membrane	9.3	1.16e-5
NGLY1	N-glycanase 1	Membrane, Intracellular	4.2	1.17e-5
PARS2	prolyl-tRNA synthetase 2, mitochondrial	Intracellular	5.1	1.26e-5
CLCC1	chloride channel CLIC like 1	Membrane, Intracellular	4.1	1.33e-5
AP3B1	adaptor related protein complex 3 subunit beta 1	Intracellular	9.5	1.37e-5
PRPSAP1	phosphoribosyl pyrophosphate synthetase associated protein 1	Intracellular	6.5	1.39e-5
PSMA5	proteasome 20S subunit alpha 5	Intracellular	24.7	1.45e-5
GRSF1	G-rich RNA sequence binding factor 1	Intracellular	17.3	1.47e-5
CD274	CD274 molecule	Membrane	1.1	1.64e-5
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The colorectal cancer transcriptome

The transcriptome analysis shows that 68% (n=13806) of all human genes (n=20162) are expressed in colorectal cancer. All genes were classified according to the colorectal cancer-specific expression into one of five different categories, based on the ratio between mRNA levels in colorectal cancer compared to the mRNA levels in the other 16 analyzed cancer tissues.

Figure 1. The distribution of all genes across the five categories based on transcript abundance in colorectal cancer as well as in all other cancer tissues.

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

Table 3. The number of genes in the subdivided categories of elevated expression in colorectal cancer.

		Distribution in the 17 cancers
		Detected in single	Detected in some	Detected in many	Detected in all	Total
Specificity	Cancer enriched	7	11	13	0	31
	Group enriched	0	30	60	2	92
	Cancer enhanced	4	26	57	15	102
	Total	11	67	130	17	225

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