Inhibitor of DNA binding 1 (Id1) mediates stemness of colorectal cancer cells through the Id1-c-Myc-PLAC8 axis via the Wnt/β-catenin and Shh signaling pathways

Figures & data

Table 1 Demographic and clinical characteristics of the study subjects

Characteristics		Number of patients (%)
Age (years)	≤58	39 (52.0)
	>58	36 (48.0)
Gender	Males	47 (62.7)
	Females	28 (32.3)
Tumor size (cm)	≤5	39 (52.0)
	>5	36 (48.0)
T category	T1	3 (4)
	T2	10 (13.3)
	T3	27 (36.0)
	T4	35 (46.6)
N category	N0	35 (46.7)
	N1	20 (26.7)
	N2	20 (26.7)
M category	M0	68 (90.7)
	M1	7 (9.3)
AJCC stage	I	9 (12.0)
	II	23 (30.7)
	III	37 (49.3)
	IV	6 (8.0)
Lymph node metastasis	No	36 (48.0)
	Yes	39 (52.0)
Differentiation status	Poor	29 (38.6)
	Moderate	43 (57.3)
	High	3 (4)
CEA (ng/ml)	≤5	43 (58.1)
	>5	31 (41.9)
CA199 (U/ml)	≤37	60 (82.2)
	>37	13 (17.8)

Notes: Data are presented as number (%); a patient had no CEA data, and two patients had no CA199 data.

Abbreviation: AJCC, American Joint Committee on Cancer.

Table 2 Sequences of short-hairpin RNAs targeting inhibitor of DNA binding 1

Short-hairpin RNAs	Sequence
ShId1-1	5′-AGTCTCTGGTGACTAGTAG-3′
ShId1-2	5ʹ-TGAGGCGTGAGTAACAGCC-3’
ShId1-3	5ʹ-ACCTGCTGCTCGTCCAGCA-3’
ShId1-4	5ʹ-CATGTCGTAGAGCAGCACG-3’

Table 3 Primers for genes of interest

Gene	Primer sequence
Id1	F: 5′-CGTGCTGCTCTACGACATGA-3′, R: 5′-GCTCCAACTGAAGGTCCCTG-3′
CD24	F: 5′-GGTTGGCCCCAAATCCAACT-3′, R: 5′-GACCACGAAGAGACTGGCTG-3′
CD44	F: 5′-GACAGCACAGACAGAATC-3′, R: 5′-AGTAGTTGAGCCTTCAGAA-3′
CD133	F: 5′-CAACGAGTCCTTCCTATA-3′, R: 5′-CTCTCCAACAATCCATTC-3′
CD166	F: 5′-ACCTCAGAATCTCATGTTTGG-3′, R: 5′-ATCACTGATCCTTGCATTACTG-3′
EpCAM	F: 5′-TCGTCAATGCCAGTGTACTTC-3′, R: 5′-ATCGCAGTCAGGATCATAAAG-3′
EZH2	F: 5′-TGATGACGATGATGATGATGGAGAC-3′, R: 5′-TGTGCCCTTATCTGGAAACATTGAG
β-catenin	F: 5′-TTAAGTCTGGAGGCATTC-3′, R: 5′-GGTTGTGGAGAGTTGTAA-3′
OCT4	F: 5′-ATACACTTCAGATAACTAC-3′, R: 5′-TAAGAAGATGATGGAGTA-3′
CXCR4	F: 5′-ATACACTTCAGATAACTAC-3′, R: 5′-TAAGAAGATGATGGAGTA-3′
Twist	F: 5′-GGGAGTCCGCAGTCTTACG-3′, R: 5′-CTTGAGGGTCTGAATCTTGCT-3′
Snail	F: 5′-TCCAGACCCACTCAGATGT-3′, R: 5′-CGGACTCTTGGTGCTTGTG-3′
E-cadherin	F: 5′-GAACGCATTGCCACATAC-3′, R: 5′-CACCTTCCATGACAGACCC-3′
Survivin	F: 5′-TCTCTACATTCAAGAACT-3′, R: 5′-TTGAAGCAGAAGAAACAC-3′
CyclinD1	F: 5′-CCATGAACTACCTGGACCG-3′, R: 5′-GATGGAGTTGTCGGTGTAGAT-3′
β-actin	F: 5′-TGGCACCACACCTTCTACA-3′, R: 5′-AGCACAGCCTGGATAGCA-3′

Figure 1 Id1 and CD133 expression in human colorectal cancer specimens and normal mucosal specimens. (A and B) higher Id1 (A) and CD133 (B) mRNA expression is detected in human colorectal cancer specimens than in matched normal mucosal specimens (P<0.05). The data are presented as mean ± SD. *P<0.05 vs the normal mucosal specimens; (C) Spearman correlation analysis reveals a positive correlation between Id1 and CD133 mRNA expression; (D) representative immunohistochemical staining of Id1 and CD133 expression (400×); (E) Spearman correlation analysis reveals a positive correlation between Id1 and CD133 protein expression in colorectal cancer patients.

Abbreviation: Id1, inhibitor of DNA binding 1.

Table 4 Associations of Id1 and CD133 expression with clinicopathologic characteristics in colorectal cancer patients

Characteristics		No. of cases	Id1 expression		P-value	CD133 expression		P-value
Low (n=40)	High (n=35)	Low (n=53)	High (n=22)
Gender	Male	47	25	22	0.975	31	16	0.246
	Female	28	15	13		22	6
	≤58	39	22	17	0.578	27	12	0.776
Age (years)	>58	36	18	16		26	10
Tumor size (cm)	≤5	39	20	19	0.711	29	10	0.465
	>5	36	20	16		24	12
T category	T1–T2	13	6	7	1	13	0	0.002
	T3–T4	62	34	28		40	22
N category	N0	35	19	16	0.877	24	11	0.709
	N1–N2	40	21	19		29	11
M category	M0	68	34	34	0.071	46	22	0.073
	M1	7	6	1		7	0
AJCC stage	0–II	32	18	14	0.662	22	10	0.753
	III–IV	43	22	21		31	12
Lymph node metastasis	No	36	19	17	0.926	25	11	0.823
	Yes	39	21	18		28	11
Differentiation	Poor	29	11	18	0.034	21	8	0.794
	Moderate and high	46	29	17		32	4
CEA (ng/ml)	≤5	43	25	18	0.27	30	13	0.911
	>5	31	14	17		22	9
CA199 (U/ml)	≤37	60	31	29	0.518	41	19	0.541
	>37	13	8	5		10	3

Abbreviations: AJCC, American Joint Committee on Cancer; ID1, inhibitor of DNA binding 1.

Figure 2 Effects of inhibitor of DNA binding 1 (Id1) knockdown on cancer-initiating cell features in HCT116 cells. (A) Id1 knockdown efficiency is checked by Western blotting assay; (B) real-time cell analysis (RTCA) shows greater inhibition of Id1 knockdown HCT116 cells than control cells 48 h post-transfection; (C) fluorescence-activated cell sorting (FACS) analysis reveals a significant reduction in the number of Id1 knockdown HCT116 cells at S phase and a remarkable rise in the number of Id1 knockdown HCT116 cells at G0/G1 phase relative to controls cells (P<0.05); (D) cell colony-forming assay shows lower numbers of cell colonies in Id1 knockdown HCT116 cells than in control cells. *P<0.05 vs the control cells.

Figure 3 Inhibitor of DNA binding 1(Id1) promotes colorectal cancer cell stemness and tumor-initiating capability. (A) sphere-forming assay reveals higher sphere-forming rate of Id1 knockdown HCT116 cells than that of control cells (37% ±2.4% vs 14% ±1.8%) and larger Id1 knockdown HCT116 cells than controls cells (median diameter, 115.3±5.24 vs 41.67±3.48 μm), *P<0.01 vs the control cells; (B) fluorescence-activated cell sorting (FACS) analysis shows lower proportions of CD24⁺, CD24⁺/CD133⁺ and CD133⁺ cells in Id1 knockdown HCT116 cells than in control cells; (C) Western blotting determines lower CD24, CD133, EpCAM, Oct4, EZH2, Lgr5 and β-catenin expression in Id1 knockdown HCT116 cells than in controls cells, and no significant differences are seen in CD44 or CD166 expression between in Id1 knockdown HCT116 cells and control cells; (D) Western blotting determines significantly higher Id1 expression in sphere-forming HCT116 cells than in HCT116 cells; (E) slower growth of the xenograft tumor is seen from Id1 knockdown HCT116 cells than from HCT116 cells at a same cell dosing; (F) smaller xenograft tumors are measured from Id1 knockdown HCT116 cells than from HCT116 cells at a same cell dosing. *P<0.05 vs the control cells.

Table 5 Tumor incidence of inhibitor of DNA binding 1 (Id1) knocked down (KD) HCT116 and control HCT116 cells injected into nude mice

Cells injected	Tumor incidence per number of mice
1×10^Citation4 cells	1×10^Citation5 cells	5×10^Citation5 cells	1×10^Citation6 cells
Control HCT116 cells	0/6	1/6	5/6	6/6
Id1 KD HCT116 cells	0/6	0/6	2/6	4/6

Figure 4 Inhibitor of DNA binding 1 (Id1) knockdown suppresses epithelial-mesenchymal transition (EMT) and increases chemosensitivity in HCT116 cells. (A) HCT116 cells have scattered, spindle- and fibroblastic-like shapes, and Id1 knockdown HCT116 cells have tightly packed, cuboid, epithelial-like appearance; (B) Western blotting analysis determines lower Twist, Snail, Vimentin and Slug expression and higher E-cadherin, ZO-1 and Claudin-1 expression in Id1 knockdown HCT116 cells than in HCT116 cells; (C) MTS assay measures a lower viability of Id1 knockdown HCT116 cells than that of control cells following 5-FU treatment, and the 5-FU IC₅₀ values are 5.2±0.3 and 8.4±0.4 μM for the Id1 knockdown HCT116 cells and control cells, respectively; (D) flow cytometry detects a significantly higher apoptotic rate in Id1 knockdown HCT116 cells than in control cells following treatment with 5-FU at a dose of 10 μM. *P<0.05 vs the control cells.

Figure 5 Inhibitor of DNA binding 1 (Id1) maintains the stemness of colorectal cancer cells through the Id1-c-Myc-PLAC8 axis via the Wnt/β-catenin and Sonic Hedgehog (Shh) signaling pathways. (A) Western blotting determines lower CyclinD1, Survivin, C-myc and cytoplasmic and nuclear β-catenin expression in Id1 knockdown HCT116 cells than in control cells, and lower relative TCF/LEF activity is measured in Id1 knockdown HCT116 cells than in control cells. *P<0.05; (B) Western blotting determines higher PTCH2 and SUFU expression and lower GLI2 and Shh expression in Id1 knockdown HCT116 cells than in control cells; (C) Western blotting determines lower C-myc expression in HCT116 cells exposed to FH535 and HPI-1 than in cell treated with FH535 or HPI-1 alone; (D) higher C-myc expression is seen in Id1 knockdown HCT116 cell overexpressing C-myc than in Id1 knockdown HCT116 cells; (E) Western blotting determines higher C-myc, EZH2, CD133, CD24, Oct4, EPCAM and Bmi1 expression in Id1 knockdown HCT116 cell overexpressing C-myc than in Id1 knockdown HCT116 cells; (F) lower PLAC8 expression is detected in Id1 knockdown HCT116 cells than in control cells; (G) fluorescence-activated cell sorting (FACS) analysis reveals a significant rise in the number of CD24⁺ and CD133⁺/CD24⁺ cell populations in Id1 knockdown HCT116 cells overexpressing PLAC8 than in control cells; (H) Western blotting determines higher C-myc, EZH2, CD133, CD24, Oct4, EPCAM and Bmi1 expression in Id1 knockdown HCT116 cells overexpressing PLAC8 relative to Id1 knockdown HCT116 cells.

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