The liver hepatocellular carcinoma proteome

Liver cancer is the 6th most common cancer and the third leading in cancer death worldwide. It is associated with a poor prognosis due to the lack of early detection.

Hepatocellular carcinoma is the most common type of primary liver cancer and accounts for more than 80% of cases, followed by cholangiocarcinomas (intrahepatic bile duct cancers) that account for 10-20%. The overall median survival of patients with hepatocellular carcinoma is 4 months and the overall 5-year survival rate is 3%. The tumor predominantly affects males who are over 50 years of age. Risk factors include infection with hepatotropic viruses such as hepatitis B and C viruses, liver cirrhosis, liver cell dysplasia, exposure to aflatoxins and inborn errors of metabolism. Serum elevation of α-fetoprotein occurs in a large proportion of patients (up to 75%) with hepatocellular carcinoma. Most tumors are detected at an advanced stage are not suitable for liver transplantation.

Here, we explore the liver hepatocellular carcinoma proteome using TCGA transcriptomics data and antibody-based protein data. 4360 genes are suggested as prognostic based on transcriptomics data from 362 patients; 3664 genes are associated with unfavorable prognosis and 702 genes are associated with favorable prognosis.

TCGA data analysis

In this metadata study, we used data from TCGA where transcriptomics data was available from 362 patients in total with 118 female and 244 male patients. A majority of patients (233 patients) were still alive at the time of data collection. The stage distribution was stage i) 169 patients, stage ii) 84 patients, stage iii) 81 patients, stage iv) 4 patients and 24 patients with missing stage information.

Unfavorable prognostic genes in liver hepatocellular carcinoma

For unfavorable genes, higher relative expression levels at diagnosis give significantly lower overall survival for the patients. There are 3664 genes associated with an unfavorable prognosis in liver hepatocellular carcinoma, among these potential prognostic genes there are 939 genes that were validated in a separate study. In Table 1, the top 20 most significant genes related to an unfavorable prognosis are listed.

PES1 is a gene associated with an unfavorable prognosis in liver hepatocellular carcinoma in two separate independent cohorts. The best separation is achieved by an expression cutoff at 48 TPM which divides the patients into two groups with 28% 5-year survival for patients with high expression versus 61% for patients with low expression, p-value:5.86e-10. The TCGA data analysis was validated in a separate study with the p-value: 2.19e-7. Immunohistochemical staining using an antibody targeting PES1 (HPA062439) shows a differential expression pattern in liver hepatocellular carcinoma samples.

p<0.001
PES1 - survival analysis
PES1 - high expression
PES1 - low expression

MCM2 is another gene associated with an unfavorable prognosis in liver hepatocellular carcinoma in two separate independent cohorts. The best separation is achieved by an expression cutoff at 15 TPM which divides the patients into two groups with 31% 5-year survival for patients with high expression versus 55% for patients with low expression, p-value: 1.54e-6. The TCGA data analysis was validated in a separate study with the p-value: 2.17e-9. Immunohistochemical staining using an antibody targeting MCM2 (HPA031496) shows a differential expression pattern in liver hepatocellular carcinoma samples.

p<0.001
MCM2 - survival analysis
MCM2 - high expression
MCM2 - low expression

Table 1. The 20 genes with highest significance associated with an unfavorable prognosis in liver hepatocellular carcinoma.

Gene	Description	Predicted location	mRNA (cancer)	p-value	Prognostic
INTS13	Integrator complex subunit 13	Intracellular	9.3	2.04e-13	potential
FARSB	Phenylalanyl-tRNA synthetase subunit beta	Intracellular	5.9	4.60e-13	validated
TCOF1	Treacle ribosome biogenesis factor 1	Intracellular	10.2	6.41e-13	validated
CCT4	Chaperonin containing TCP1 subunit 4	Intracellular	62.2	9.30e-13	validated
CBX2	Chromobox 2	Intracellular	1.5	9.98e-13	validated
WDR77	WD repeat domain 77	Intracellular	13.6	1.52e-12	validated
GNL2	G protein nucleolar 2	Intracellular	23.2	2.47e-12	validated
POC5	POC5 centriolar protein	Intracellular	3.2	3.32e-12	potential
DDX55	DEAD-box helicase 55	Intracellular	6.5	3.85e-12	potential
HILPDA	Hypoxia inducible lipid droplet associated	Intracellular	6.9	5.46e-12	potential
MRTO4	MRT4 homolog, ribosome maturation factor	Intracellular	18.5	1.01e-11	validated
UBE2E1	Ubiquitin conjugating enzyme E2 E1	Intracellular	41.4	1.17e-11	potential
NAE1	NEDD8 activating enzyme E1 subunit 1	Intracellular	20.3	1.35e-11	validated
CAD	Carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase	Intracellular	17.6	1.51e-11	validated
HDAC2	Histone deacetylase 2	Intracellular	24.4	1.81e-11	validated
PSRC1	Proline and serine rich coiled-coil 1	Intracellular	4.1	2.57e-11	validated
CDCA8	Cell division cycle associated 8	Intracellular	5.1	4.71e-11	validated
NOP56	NOP56 ribonucleoprotein	Intracellular	28.7	4.94e-11	validated
TTK	TTK protein kinase	Intracellular	2.4	5.17e-11	validated
UTP11	UTP11 small subunit processome component	Intracellular	13.5	5.27e-11	potential
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Favorable prognostic genes in liver hepatocellular carcinoma

For favorable genes, higher relative expression levels at diagnosis give significantly higher overall survival for the patients. There are 702 genes associated with a favorable prognosis in liver hepatocellular carcinoma, among these potential prognostic genes there are 113 genes that were validated in a separate study. In Table 2, the top 20 most significant genes related to a favorable prognosis are listed.

CPS1 is a gene associated with a favorable prognosis in liver hepatocellular carcinoma in two separate independent cohorts. The best separation is achieved by an expression cutoff at 26 TPM which divides the patients into two groups with 63% 5-year survival for patients with high expression versus 33% for patients with low expression, p-value: 1.61e-4. The TCGA data analysis was validated in a separate study with the p-value: 9.60e-5. Immunohistochemical staining using an antibody targeting CPS1 (HPA021400) shows a differential expression pattern in liver hepatocellular carcinoma samples.

p<0.001
CPS1 - survival analysis
CPS1 - high expression
CPS1 - low expression

TTC36 is a gene associated with a favorable prognosis in liver hepatocellular carcinoma 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 65% 5-year survival for patients with high expression versus 39% for patients with low expression, p-value: 7.11e-5. The TCGA data analysis was validated in a separate study with the p-value: 1.07e-4. Immunohistochemical staining using an antibody targeting TTC36 (HPA038731) shows a differential expression pattern in liver hepatocellular carcinoma samples.

p<0.001
TTC36 - survival analysis
TTC36 - high expression
TTC36 - low expression

Table 2. The 20 genes with highest significance associated with a favorable prognosis in liver hepatocellular carcinoma.

Gene	Description	Predicted location	mRNA (cancer)	p-value	Prognostic
BTNL9	Butyrophilin like 9	Membrane	2.5	4.61e-10	validated
CLEC3B	C-type lectin domain family 3 member B	Secreted, Intracellular	26.2	9.15e-9	validated
ST20-MTHFS	ST20-MTHFS readthrough	Intracellular	56.0	1.25e-8	potential
KNG1	Kininogen 1	Secreted	1355.5	7.27e-8	validated
SOCS2	Suppressor of cytokine signaling 2	Intracellular	11.2	9.13e-8	validated
APOC1	Apolipoprotein C1	Secreted	13713.0	1.08e-7	validated
RBP4	Retinol binding protein 4	Secreted, Intracellular	6778.1	1.16e-7	validated
LCAT	Lecithin-cholesterol acyltransferase	Secreted, Intracellular	56.1	1.68e-7	validated
CFHR4	Complement factor H related 4	Secreted	30.7	3.05e-7	validated
RAMP3	Receptor activity modifying protein 3	Membrane	12.6	3.56e-7	validated
DNASE1L3	Deoxyribonuclease 1 like 3	Secreted, Intracellular	15.2	4.43e-7	validated
ALDH2	Aldehyde dehydrogenase 2 family member	Intracellular	149.8	4.95e-7	validated
APOH	Apolipoprotein H	Secreted, Intracellular	4417.2	5.45e-7	potential
CYP2C9	Cytochrome P450 family 2 subfamily C member 9	Intracellular	165.5	8.94e-7	validated
SPP2	Secreted phosphoprotein 2	Secreted, Intracellular	110.2	9.13e-7	potential
TRIM21	Tripartite motif containing 21	Intracellular	13.3	1.19e-6	potential
KLF2	KLF transcription factor 2	Intracellular	6.7	1.23e-6	validated
APOC4-APOC2	APOC4-APOC2 readthrough (NMD candidate)	Membrane	147.9	1.64e-6	potential
ADH4	Alcohol dehydrogenase 4 (class II), pi polypeptide	Intracellular	353.8	1.89e-6	potential
SLC22A7	Solute carrier family 22 member 7	Membrane	164.0	2.89e-6	validated
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CPTAC relative protein expression data

Proteins that are significantly down- or upregulated in liver hepatocellular carcinoma 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 165 tumor samples and 165 normal samples.

In liver hepatocellular carcinoma, 2812 and 2952 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 liver hepatocellular carcinoma compared to normal tissue.

Gene	Description	Predicted location	Log2 fold change	p-value	Regulation
ITGA9	Integrin subunit alpha 9	Membrane, Intracellular	-1.3	3.86e-54	Downregulated
STEAP4	STEAP4 metalloreductase	Membrane	-1.65	5.94e-54	Downregulated
EHD3	EH domain containing 3	Intracellular	-1.26	1.38e-53	Downregulated
PDE2A	Phosphodiesterase 2A	Intracellular	-1.16	2.04e-53	Downregulated
CLEC4M	C-type lectin domain family 4 member M	Membrane, Intracellular	-2.78	2.07e-53	Downregulated
TNS2	Tensin 2	Intracellular	-0.97	2.23e-53	Downregulated
OIT3	Oncoprotein induced transcript 3	Secreted	-1.65	7.08e-53	Downregulated
CLEC4G	C-type lectin domain family 4 member G	Membrane, Intracellular	-2.18	7.60e-53	Downregulated
STAB2	Stabilin 2	Membrane, Intracellular	-1.83	1.39e-52	Downregulated
FCN2	Ficolin 2	Secreted	-1.74	1.54e-52	Downregulated
COL6A6	Collagen type VI alpha 6 chain	Secreted, Intracellular	-1.98	2.01e-52	Downregulated
LYVE1	Lymphatic vessel endothelial hyaluronan receptor 1	Membrane	-2.21	2.20e-52	Downregulated
GMPS	Guanine monophosphate synthase	Intracellular	0.67	2.91e-52	Upregulated
DDX23	DEAD-box helicase 23	Intracellular	0.57	4.68e-52	Upregulated
AGL	Amylo-alpha-1, 6-glucosidase, 4-alpha-glucanotransferase	Membrane	-1.63	5.39e-52	Downregulated
ANGPTL6	Angiopoietin like 6	Secreted	-1.71	5.49e-52	Downregulated
AOC3	Amine oxidase copper containing 3	Intracellular	-1.34	7.02e-52	Downregulated
PRPF6	Pre-mRNA processing factor 6	Intracellular	0.56	7.02e-52	Upregulated
EML1	EMAP like 1	Intracellular	-0.98	1.69e-51	Downregulated
ASPDH	Aspartate dehydrogenase domain containing	Intracellular	-1.54	1.78e-51	Downregulated
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The liver hepatocellular carcinoma transcriptome

The transcriptome analysis shows that 64% (n=12934) of all human genes (n=20162) are expressed in liver hepatocellular carcinoma. All genes were classified according to the liver hepatocellular carcinoma-specific expression into one of five different categories, based on the ratio between mRNA levels in liver hepatocellular carcinoma 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 liver hepatocellular carcinoma as well as in all other cancer tissues.

497 genes show some level of elevated expression in liver hepatocellular carcinoma 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 liver hepatocellular carcinoma.

		Distribution in the 31 cancers
		Detected in single	Detected in some	Detected in many	Detected in all	Total
Specificity	Cancer enriched	48	84	79	20	231
	Group enriched	0	46	52	12	110
	Cancer enhanced	8	28	67	53	156
	Total	56	158	198	85	497

Additional information

Liver transplantation is considered to be the best treatment for hepatocellular carcinoma and also for the underlying cirrhosis. Tumors of late stage are however not eligible for transplantation. Other therapeutic options include surgical resection, targeted systemic chemotherapy and radiotherapy. Early stages of the disease have a much better prognosis with various treatment options.

Histologically, hepatocellular carcinomas present a range of appearances. A common feature is the presence of fibrosis and inflammation as hepatocellular carcinoma often develops in the liver of patients with late stages of chronic hepatitis. Well-differentiated tumors may be difficult to discriminate from normal liver tissues since tumor cells have a similar appearance to normal hepatocytes. The more poorly differentiated tumors display marked pleomorphism, with tumor giant cells showing little resemblance to normal hepatocytes. Characteristic features of hepatocellular carcinomas include a sinusoidal growth pattern and absence of intracellular mucin and wells as a lack of bile production. Immunohistochemically, hepatocellular carcinomas are immunoreactive for α-fetoprotein, various keratins, α-1 antitrypsin, various integrins, villin and CD15.

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

Kampf C et al., The human liver-specific proteome defined by transcriptomics and antibody-based profiling. FASEB J. (2014)
PubMed: 24648543 DOI: 10.1096/fj.14-250555

Raza A et al., Hepatocellular carcinoma review: current treatment, and evidence-based medicine. World J Gastroenterol. (2014)
PubMed: 24764650 DOI: 10.3748/wjg.v20.i15.4115

Van Beers BE., Diagnosis of cholangiocarcinoma. HPB (Oxford). (2008)
PubMed: 18773062 DOI: 10.1080/13651820801992716

Zhao DY et al., Current biologics for treatment of biliary tract cancers. J Gastrointest Oncol. (2017)
PubMed: 28736630 DOI: 10.21037/jgo.2017.05.04

El-Serag HB., Epidemiology of viral hepatitis and hepatocellular carcinoma. Gastroenterology. (2012)
PubMed: 22537432 DOI: 10.1053/j.gastro.2011.12.061

Histology dictionary - Liver cancer