S100A4 over-expression underlies lymph node metastasis and poor prognosis in colorectal cancer

Table 1 Clinicopathological characteristics of colorectal cancer patients included in screening and confirmation study

Patient No.	TNM	Sex	Age (yr)	Location
Non-LNM CRC
1	T2N0M0	Male	73	Colon
2	T4N0M0	Male	44	Rectum
3	T2N0M0	Male	49	Colon
4	T3N0M0	Female	53	Colon
5	T3N0M0	Male	54	Colon
6	T4N0M0	Female	52	Colon
7	T3N0M0	Male	69	Rectum
8	T3N0M0	Female	59	Colon
9	T4N0M0	Male	66	Colon
10	T2N0M0	Male	73	Colon
11	T2N0M0	Female	72	Rectum
12	T3N0M0	Male	68	Rectum
13	T2N0M0	Male	71	Colon
14	T4N0M0	Male	68	Colon
15	T3N0M0	Male	59	Rectum
16	T2N0M0	Female	43	Rectum
LNM CRC
1	T4N2M0	Male	41	Colon
2	T3N1M0	Male	60	Rectum
3	T2N1M0	Male	63	Rectum
4	T2N2M0	Female	37	Rectum
5	T4N1M0	Female	40	Colon
6	T4N2M0	Male	80	Colon
7	T4N1M0	Male	32	Colon
8	T4N2M0	Female	65	Colon
9	T3N1M0	Female	49	Colon
10	T2N1M0	Female	47	Rectum
11	T2N2M0	Female	55	Rectum
12	T4N1M0	Male	86	Colon
13	T3N1M0	Female	71	Colon
14	T3N2M0	Male	79	Colon
15	T3N1M0	Male	70	Colon
16	T4N1M0	Male	56	Colon

LNM: Lymph node metastasis; CRC: Colorectal cancer.

Table 2 Protein expression upregulated and downregulated at least 2.5-fold in lymph node metastasis colorectal cancer compared with non-lymph node metastasis colorectal cancer

Accession No.	Protein name	Protein ratio1	SD	Peptide	Coverage rate (%)
Upregulated
IPI00171494	Isoform 2 of cytoplasmic dynein 2 heavy chain 1	21.8	0	1	2.4
IPI00103253	Isoform 5 of pyrin and hin domain-containing protein 1	8.68	2.85	19	4.9
IPI00298520	Putative uncharacterized protein dkfzp686m09245	7.39	0	1	2.5
IPI00027194	Syntaxin-18	6.30	0	1	3.9
IPI00410639	Isoform 2 of fch and double sh3 domains protein 2	5.25	1.03	5	2.1
IPI00300631	Scaffold attachment factor B1	4.69	0	1	1.51
IPI00009236	Isoform α of caveolin-1	4.42	3.75	2	3.0
IPI00216654	Isoform β of nucleolar phosphoprotein P130	4.35	0	1	1.6
IPI00004273	RNA binding motif protein 25	3.87	0	1	1.5
IPI00239077	Histidine triad nucleotide-binding protein 1	3.73	0.38	5	7.91
IPI00010320	Chromobox protein homolog 1	3.56	1.90	4	1.8
IPI00010274	Isoform 1 of tryptase α-1 precursor	3.52	0.09	2	9.0
IPI00010414	Pdz and lim domain protein 1	3.41	0	1	4.0
IPI00014852	Isoform 1 of phosphoglucomutase-like protein 5	3.24	1.07	4	11.1
IPI00000156	Ligase III, DNA, ATP-dependent isoform β precursor	3.10	0.60	2	2.1
IPI00102821	Isoform 1 of proapoptotic caspase adapter protein precursor	3.09	0.40	2	36.1
IPI00032313	Protein S100-A4	3.04	0.69	2	7.9
IPI00008750	Metallothionein-1H	2.95	0.11	2	27.9
IPI00216153	40S Ribosomal protein S15	2.94	0.57	4	9.0
IPI00654777	Eukaryotic translation initiation factor 3 subunit 5	2.90	0.24	2	5.1
IPI00024933	60S Ribosomal protein L12	2.86	1.02	11	14.4
IPI00025366	Citrate synthase, mitochondrial precursor	2.85	0.47	4	5.9
IPI00007928	Pre-mRNA-processing-splicing factor 8	2.81	0	1	1.3
IPI00024976	Mitochondrial import receptor subunit tom22 homolog	2.78	0	1	8.5
IPI00577039	Annexin A2	2.77	0.32	2	3.6
IPI00062151	Similar to 60s ribosomal protein L15	2.70	0.37	6	6.5
IPI00219757	Glutathione S-transferase P1	2.55	0.37	2	9.1
IPI00396437	Isoform 2 of drebrin-like protein	2.51	0.45	5	2.8
Downregulated
IPI00023673	Galectin-3-binding protein precursor	0.09	0	1	2.2
IPI00011062	Isoform 1 of carbamoyl-phosphatesynthase , mitochondrial precursor	0.10	0	1	1.0
IPI00030279	Isoform 1 of zinc finger ran-binding domain-containing protein 3	0.11	0	1	7.6
IPI00219682	Erythrocyte band 7 integral membrane protein	0.20	0	1	9.8
IPI00433499	Rhomboid, veinlet-like 6 isoform 1	0.20	0	1	2.1
IPI00000230	Tropomyosin 1 α chain isoform 2	0.26	0.06	32	51.8
IPI00029631	Enhancer of rudimentary homolog	0.27	0	1	7.7
IPI00009950	Vesicular integral-membrane protein vip36 precursor	0.30	0	1	3.4
IPI00384603	Isoform 2 of ribonuclease p protein subunit P21	0.30	0.11	13	10.7
IPI00177543	Peptidylglycine α-amidating monooxygenase isoform A	0.31	0.08	2	8.1
IPI00000330	Isoform 1 of uncharacterized protein c9orf80	0.35	0	1	17.6
IPI00298949	Cyclin G-associated kinase	0.35	0	1	1.3
IPI00008964	RAS-related protein rab-1B	0.37	0	1	7.5
IPI00013860	3-Hydroxyisobutyrate dehydrogenase, mitochondrial precursor	0.37	0	1	5.4
IPI00296053	Isoform mitochondrial of fumarate hydratase, mitochondrial precursor	0.39	0.18	1	2.4

¹Protein ratio represents the abundance ratio between lymph node metastasis group and non-lymph node metastasis group. LNM: Lymph node metastasis; CRC: Colorectal cancer.

Table 3 Relationships of S100A4 expression with clinicopathological factors in colorectal cancer

Clinicopathological factors	n	S100A4 expression		P value1
		Negative	Positive
Sex
Male	59	32	27	0.252
Female	53	23	30
Age (yr)
≤ 60	76	39	37	0.497
> 60	36	16	20
Tumor size (cm)
≤ 5	74	32	42	0.083
> 5	38	23	15
Tumor location
Colon	47	23	24	0.975
Rectum	65	32	33
Tumor differentiation2
I-II	82	43	39	0.244
III-IV	30	12	18
Tumor status2
T1-2	34	18	16	0.592
T3-4	78	37	41
Lymph node metastasis2
N0	59	49	10	< 0.001
N1-2	53	9	44
TNM stage2
I-II	57	46	11	< 0.001
III-IV	55	9	46

¹Statistical analysis was estimated with χ² test, and P < 0.05 was considered statistically significant;

²Grading of differentiation status and TNM classification for colorectal cancer were based on the American Joint Committee on Cancer Cancer Staging Manual (6th edition, 2002). The tumors were classified into two groups: well differentiated (grades I and II) and poorly differentiated (grades III and IV).

Table 4 Univariate and multivariate analyses of recurrence and survival (Cox regression)

Variables	Recurrence		Survival
	HR (95% CI)	P value	HR (95% CI)	P value
Univariate analysis
Sex
Male/female	0.872 (0.490-1.549)	0.640	0.857 (0.452-1.624)	0.636
Age (yr)
≤ 60/> 60	1.517 (0.842-2.732)	0.165	1.842 (0.967-3.508)	0.063
Tumor size (cm)
≤ 5/> 5	0.881 (0.165-0.685)	0.687	0.880 (0.444-1.745)	0.715
Tumor location
Colon/rectum	0.823 (0.463-1.464)	0.508	0.920 (0.483-1.752)	0.799
Tumor differentiation
I-II/III-IV	1.234 (0.660-2.305)	0.511	1.171 (0.581-2.361)	0.659
Tumor status
T1-2/T3-4	0.886 (0.484-1.620)	0.693	1.026 (0.517-2.033)	0.942
Lymph node metastasis
N0/N1-2	2.727 (1.510-4.923)	0.001	2.852 (1.463-5.559)	0.002
TNM stage
I-II/III-IV	3.560 (1.940-6.534)	< 0.001	3.393 (1.680-6.850)	< 0.001
S100A4 expression
Negative/positive	3.666 (1.929-6.964)	< 0.001	4.154 (1.963-8.792)	< 0.001
Multivariate analysis
LNM
N0/N1-2	0.205 (0.055-0.769)	0.019	0.193 (0.044-0.857)	0.031
TNM stage
I-II/III-IV	8.915 (2.081-38.189)	0.003	9.057 (1.705-48.108)	0.010
S100A4 expression
Negative/positive	2.454 (1.056-5.705)	0.037	2.888 (1.131-7.379)	0.027

HR: Hazard ratio; CI: Confidence interval.