The breast cancer proteome

Breast cancer is the most common invasive cancer form in women worldwide and the leading cause of cancer-related mortality in women. The global age-adjusted incidence rate for breast cancer is 124 per 100,000 women per year. Male breast cancer is exceedingly rare and accounts for around 1% of cases. Although the rate of breast cancer diagnosis increased during the 1990's, it has decreased since the year 2000 and the overall breast cancer death rate has dropped steadily in the western world. The majority of breast cancers develop sporadically, but for 5-10% of patients there is a hereditary component. The most well known genes associated with increased breast cancer risk are BRCA1 and BRCA2. Women with abnormal BRCA1 or BRCA2 experience up to a 60% risk to develop breast cancer by the age of 90. Other risk factors include early menarche and late menopause. Pregnancy has been reported to decrease risk, probably due to the changes in breast tissue.

Breast cancer forms in tissues of the breast, usually the ducts (tubes that carry milk to the nipple) and lobules (glands where the milk is produced). Based on the presumed site of origin and morphology, breast cancer is broadly classified as ductal or lobular cancers.

Here, we explore the breast cancer proteome using TCGA transcriptomics data and antibody-based protein data. 656 genes are suggested as prognostic based on transcriptomics data from 1075 patients; 210 genes are associated with unfavorable prognosis and 446 genes are associated with favorable prognosis.

TCGA data analysis

In this metadata study, we used data from TCGA where transcriptomics data was available from 1075 patients in total. The dataset included 1063 females and 12 males. Most of the patients (923 patients) were still alive at the time of data collection. The stage distribution was stage i) 180 patients, stage ii) 609 patients, stage iii) 243 patients, stage iv) 20 patients, and 11 patients with missing stage information.

Unfavorable prognostic genes in breast cancer

For unfavorable genes, higher relative expression levels at diagnosis give significantly lower overall survival for the patients. There are 210 genes associated with an unfavorable prognosis in breast cancer. In Table 1, the top 20 most significant genes related to an unfavorable prognosis are listed.

CBX3 is a gene associated with an unfavorable prognosis in breast cancer. The best separation is achieved by an expression cutoff at 59.2 fpkm which divides the patients into two groups with 78% 5-year survival for patients with high expression versus 84% for patients with low expression, p-value: 7.92e-4. Protein DNA-binding Chromobox protein homolog 3 is encoded by the CBX3 gene and is a component of the heterochromatin, thus important in transcriptional silencing. Immunohistochemical staining using an antibody targeting CBX3 (HPA004902) shows a differential expression pattern in breast cancer samples.

p<0.001
CBX3 - survival analysis
CBX3 - high expression
CBX3 - low expression

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

Gene	Description	Predicted location	mRNA (cancer)	p-value
LRP11	LDL receptor related protein 11	Intracellular,Membrane	18.6	2.31e-8
PGK1	phosphoglycerate kinase 1	Intracellular	86.5	7.05e-8
PCMT1	protein-L-isoaspartate (D-aspartate) O-methyltransferase	Intracellular,Membrane	30.9	4.02e-7
ATPSCKMT	ATP synthase c subunit lysine N-methyltransferase	Intracellular,Membrane	6.2	1.32e-6
MAL2	mal, T cell differentiation protein 2	Membrane	98.0	4.57e-6
LIMCH1	LIM and calponin homology domains 1	Intracellular	7.6	5.55e-6
ATP5F1B	ATP synthase F1 subunit beta	Intracellular	236.9	7.17e-6
RTN4IP1	reticulon 4 interacting protein 1	Intracellular	4.6	9.18e-6
TPD52	tumor protein D52	Intracellular	35.6	1.11e-5
SPDYC	speedy/RINGO cell cycle regulator family member C	Intracellular	6.5	1.20e-5
PDSS2	decaprenyl diphosphate synthase subunit 2	Intracellular	5.7	1.34e-5
ATG4A	autophagy related 4A cysteine peptidase	Intracellular	5.9	1.36e-5
TRMT2B	tRNA methyltransferase 2 homolog B	Intracellular	6.0	1.58e-5
DCTPP1	dCTP pyrophosphatase 1	Intracellular	36.3	1.64e-5
XRCC4	X-ray repair cross complementing 4	Intracellular	3.2	1.80e-5
TIMM17A	translocase of inner mitochondrial membrane 17A	Membrane	23.6	1.98e-5
TAGLN2	transgelin 2	Intracellular	318.4	2.06e-5
PTGES3	prostaglandin E synthase 3	Intracellular	102.0	2.15e-5
RAB5B	RAB5B, member RAS oncogene family	Intracellular	35.9	2.24e-5
RTL8B	retrotransposon Gag like 8B	Intracellular	10.6	2.41e-5
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Favorable prognostic genes in breast cancer

For favorable genes, higher relative expression levels at diagnosis give significantly higher overall survival for the patients. There are 446 genes associated with a favorable prognosis in breast cancer. In Table 2, the top 20 most significant genes related to a favorable prognosis are listed.

MVP is a gene associated with a favorable prognosis in breast cancer. The best separation is achieved by an expression cutoff at 26.9 fpkm which divides the patients into two groups with 87% 5-year survival for patients with high expression versus 75% for patients with low expression, p-value: 4.51e-4. The Major Vault Protein is encoded by MVP and is an important component of large ribonucleoprotein particles found in eukaryotic cells. The MVP protein may play a role in several cellular functions such as signaling pathway regulation of the JAK/STAT, PI3K/AKT and MAP kinase pathways. MVP also seems to be implicated in multi-drug resistance and previous reports link the expression of MVP to prognosis in several cancer types. Immunohistochemical staining using an antibody targeting MVP (HPA064740) shows a differential expression pattern in breast cancer samples.

p<0.001
MVP - survival analysis
MVP - high expression
MVP - low_expression

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

Gene	Description	Predicted location	mRNA (cancer)	p-value
IGHA2	immunoglobulin heavy constant alpha 2 (A2m marker)	Intracellular,Membrane,Secreted	155.1	1.98e-9
ZNF385B	zinc finger protein 385B	Intracellular	2.0	4.86e-8
IGKV1-9	immunoglobulin kappa variable 1-9	Secreted	39.9	2.10e-7
TFPI2	tissue factor pathway inhibitor 2	Intracellular,Secreted	7.8	8.33e-7
IGHD	immunoglobulin heavy constant delta	Intracellular,Membrane,Secreted	6.6	1.49e-6
IGKV1OR2-108	immunoglobulin kappa variable 1/OR2-108 (non-functional)	Intracellular	4.0	1.53e-6
IL27RA	interleukin 27 receptor subunit alpha	Membrane	8.3	1.74e-6
JCHAIN	joining chain of multimeric IgA and IgM	Secreted	115.8	1.81e-6
IGLV6-57	immunoglobulin lambda variable 6-57	Secreted	50.9	1.92e-6
ARID5A	AT-rich interaction domain 5A	Intracellular	11.8	2.05e-6
MAP2K6	mitogen-activated protein kinase kinase 6	Intracellular	1.1	2.27e-6
NEFH	neurofilament heavy	Intracellular	2.4	2.33e-6
IGHV3-66	immunoglobulin heavy variable 3-66	Secreted	6.3	2.43e-6
DEF6	DEF6 guanine nucleotide exchange factor	Intracellular	9.5	3.02e-6
IGLV7-46	immunoglobulin lambda variable 7-46	Secreted	14.8	3.34e-6
C6orf141	chromosome 6 open reading frame 141	Intracellular	5.6	3.92e-6
CA3	carbonic anhydrase 3	Intracellular	1.5	4.24e-6
GSTK1	glutathione S-transferase kappa 1	Intracellular	31.3	4.41e-6
PEX10	peroxisomal biogenesis factor 10	Intracellular,Membrane	4.9	4.50e-6
IGKV1D-13	immunoglobulin kappa variable 1D-13	Secreted	4.1	4.68e-6
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The breast cancer transcriptome

The transcriptome analysis shows that 72% (n=14512) of all human genes (n=20090) are expressed in breast cancer. All genes were classified according to the breast cancer-specific expression into one of five different categories, based on the ratio between mRNA levels in breast 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 breast cancer as well as in all other cancer tissues.

211 genes show some level of elevated expression in breast 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 breast cancer.

		Distribution in the 17 cancers
		Detected in single	Detected in some	Detected in many	Detected in all	Total
Specificity	Cancer enriched	15	16	12	5	48
	Group enriched	0	40	41	5	86
	Cancer enhanced	6	22	37	12	77
	Total	21	78	90	22	211

Additional information

The staging of breast cancer is based on the presence of local and/or distant spread. Localized, disease (Stage I) comprises approximately 60% of cases, while in about 5% the cancer has spread to distant organs such as the liver and bone (Stage IV). Approximately 35% are Stage II or III, indicating tumor spread to regional lymph nodes.

All breast cancers may be differentiated histologically into three grades utilizing the Nottingham Grading System (NGS), also termed the Elston-Ellis grading system, by evaluating three tumor parameters. Parameters evaluated in this system are (i) extent of tubular differentiation, (ii) nuclear pleomorphism and (iii) mitotic activity assessed by counting mitotic figures in ten high power fields. Each parameter is given a score of 1 to 3 and the score of all three components are added together to a final score e.g. 1+1+1=3. The lowest final scores of 3, 4 and 5 represent well-differentiated tumors (Grade I) associated with better survival. The highest possible score is 9 (3+3+3=9) reflecting a poorly differentiated (Grade III) tumor associated with poor overall survival. In a large proportion of breast cancer, precursor lesions such as intraductal carcinoma are present adjacent to the invasive component of the tumor. Such regions of non-invasive cancer are denoted as cancer in situ and are important to recognize in the diagnostic procedure.

Immunohistochemistry is used routinely on all breast cancers to gain important information about the prognosis as well as for predicting response to specific anticancer therapies. The most commonly used antibodies include antibodies detecting the estrogen a receptor (ER, ESR1), progesterone receptor (PR, PGR), HER-2 (ERBB2) and the proliferation marker Ki-67 (MKI67). The tumor stage and grade, as well as results from immunohistochemistry, are used to personalize treatment options.

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

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

Tao Z et al., Breast Cancer: Epidemiology and Etiology. Cell Biochem Biophys. (2015)
PubMed: 25543329 DOI: 10.1007/s12013-014-0459-6

Key TJ et al., Epidemiology of breast cancer. Lancet Oncol. (2001)
PubMed: 11902563 DOI: 10.1016/S1470-2045(00)00254-0

Histology dictionary - Breast cancer