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HOXC6 and HOXC8 are potentially novel prognosis predictors of esophageal squamous cell carcinoma
Keneng Chen, M.D., PhD, RCSFLuyan Shen, M.D., PhD
2015/4/28
Department of Thoracic Surgery I, Key Laboratory of Carcinogenesis and Translational Research (Ministry of
Education), Beijing Cancer Hospital, Peking University School of Oncology, Beijing, China
02
03
04
01 The morbidity and mortality remains high
long-term survival remains unsatisfied
Lack of reports with long-term follow-up data from prospective database
Lack of efficient biomarkers for helping to guide the clinical practice
Several issues with the clinical practice of ESCC remain unsolved
prognosis
Why we choose HOX genes ?
02
03
04
01 There is some similarity between embryogenesis and tumorigenesis
AFP in liver cancer and CEA in colorectal cancer
HOX gene family regulates embryogenesis
HOX genes are deregulated in hematologic malignancy and many solid tumors
Gene Positive cases in ESCC(%)
Positive cases in noncancerous mucosa(%)
HOXA10 3(8.3) 0(0)
HOXA13 27(75) 0(0)
HOXB7 21(58.3) 0(0)
HOXC4 12(33.3) 0(0)
HOXC8 15(41.7) 0(0)
HOXD9 6(16.7) 0(0)
HOXD10 15(41.7) 0(0)
HOXD13 15(41.7) 0(0)
HOXA7 30(83.3) 12(33.3)
HOXA9 24(66.7) 9(25)
HOXC6 24(66.7) 9(25)
HOX genes were abnormally expressed in ESCC (n=36)
Chen KN, et al. Clin Cancer Res. 2005.
potential biomakers
Expression of 11 HOX genes is deregulated in ESCC
CCK8 Assay
Annexin v/PI Cell cycle & Apoptosis
Proliferation
Soft agar colony formation assay
Ability of colony formation
KYSE-450
IHC for HOXC6 、HOXC8 protein detection
Prognosis FFPE tissue specimen
Lentivirus infection
Knockdown of HOXC6/HOXC8
Tumor formation in nude mice
Ability of tumorigenesis
S Group (138 )NC+S Group (136)S+AC Group (148)
Study design and work flow diagram
The association of HOXC6/HOXC8 expression with overall survival in naive subgroup (n=138)
Item No. (%) Median survival , mo(95%CI)
5-y survival (%)
p
HOXC6 Low expression 83(60.1) 70.1(34.2-105.9) 51.5 0.070
High expression 55(39.9) 35.0(23.2-46.8) 32.9
HOXC8 Low expression 85(61.6) 110.3(35.5-185.3) 51.9 0.005
High expression 53(38.4) 30.3(17.7-42.9) 30.6
Du YB, et al. J Surg Res, 2014.
p = 0.070 p = 0.005
Surgery only
Survival analysis in surgery subgroup
The association of HOXC6/HOXC8 expression with overall survival in neoadjuvant subgroup (n=136)
Item No.(%) Median survivalmo(95%CI)
5-y survival(%)
p
HOXC6
Low expression 31(22.8) 82.7(-) 60 0.002
High expression 105(77.2) 27.8(16.7-38.8) 33
HOXC8
Low expression 73(53.7) 110.3(-) 49.9 0.004
High expression 63(46.3) 21.2(16.5-38.9) 25
NC+Sp = 0.002 p = 0.004
Survival analysis in neoadjuvant chemotherapy subgroup
Du YB, et al. J Surg Res, 2014.
The association of HOXC6/HOXC8 expression with overall survival in neoadjuvant subgroup (n=148)
Item No.(%) Median survivalmo(95%CI)
5-y survival(%)
p
HOXC6
Low expression 81(54.7) 61.0(21.8-112.2) 53.8 0.002
High expression 67(45.3) 33.4(20.5-46.3) 30.3
HOXC8
Low expression 99(66.9) 62.3(41.5-83.0) 53.0 0.001
High expression 49(33.1) 31.3(16.9-45.8) 21.7
S+ACp = 0.002 p = 0.001
Survival analysis in adjuvant chemotherapy subgroup
ControlHOXC8(-)HOXC6(-)
shNC
shHOXC6
shHOXC8
Tumor growth in nude mice
Soft agar Colony formation
CCK8 assay
Down-regulation inhibited cell proliferation in vitro & in vivo
Apoptosis rate( %)shHOXC6 32.8±0.29*
shHOXC8 36.1±0.35*
ShNC 12.7±0.31
Apoptosis rate variation when HOXC6 or HOXC8 was knock-down
* p < 0.050
Down-regulation induced cell apoptosis
G2+S(%) G1(%)
shHOXC6 45.38±2.29* 54.62±2.29*
shHOXC8 28.04±3.1* 71.9±3.1*
shNC 64.87±3.17 35.1±3.17
Knockdown of HOXC6 and HOXC8 Affects the Cell Cycle
* p < 0.050
HOXC6(-) HOXC8(-) Control
Down-regulation arrested cell cycle in G1 phase
01
02
HOXC6, HOXC8 might be biomarker for evaluating prognosis of ESCC
HOXC6/HOXC8 promotes the esophageal cancer cell proliferation
Conclusion
Acknowledgment
Dr. Chen’s LAB:
Yabing Du
Bin Dong
Wanpu Yan
Liang Dai
Xiaozheng Kang
Zhen Liang
Hongchao Xiong
13 12 11 10 9 8 7 6 5 4 3 2 1
D13 D12 D11 D10 D9 D8 D4 D3 D1
C13 C12 C11 C10 C9 C8 C6 C5 C4
B13 B9 B8 B7 B6 B5 B4 B3 B2 B1
A13 A11 A10 A9 B9 A6 A5 A4 A3 A2 A1
cervicalthoraciclumbo-sacral 3'5' AnteriorEarly
PosteriorLate
Schematics representation of four HOX loci
7p15.3
17p21.3
12q13.3
2q31
HOX genes present the expression pattern of temporal and spatial colinearity
Cillo C, et al. J cell physiol, 2001
ClinicopathologicCharacteristics
HOXC6 expression No.(%)
P HOXC8 expression No.(%)
P
High Low High Low
Age ≦60 74(62.7) 44(37.3) 0.219 53(44.9) 65(55.1) 0.309
> 60 87(55.0) 71(45.0) 63(39.9) 95(60.1)
Sex Male 121(59.0) 84(41.0) 0.891 88(42.9) 117(57.1) 0.608 Female 40(56.3) 31(43.7) 28(39.4) 43(60.6) G stage G1 10(41.7) 14(58.3) 0.010 8(33.3) 16(66.7) 0.222 G2 25(39.0) 39(61.0) 29(45.3) 35(54.7) G3 20(40.0) 30(60.0) 16(32.0) 34(68.0) Tumor location(L)
L0 15(62.5) 9(37.5) 0.101 9(37.5) 15(62.5) 0.524L1 38(67.9) 18(32.1) 26(46.4) 30(53.6) L2 75(59.1) 52(40.9) 57(44.9) 70(55.1) L3 30(46.2) 35(53.8) 23(35.4) 42(64.6)
Tumor invasion(T)
T1 33(47.1) 37(52.9) 0.011 19(27.1) 51(72.9) <0.001T2 33(50.0) 33(50.0) 22(34.9) 41(65.1) T3 68(63.0) 40(37.0) 52(48.1) 56(51.9) T4 26(78.8) 7(21.2) 23(69.7) 10(30.3)
Lymph node metastasis (N)
N0 96(53.9) 82(46.1) 0.031 65(36.5) 113(63.5) 0.004N1 33(58.9) 23(41.1) 32(57.1) 24(42.9) N2 20(71.4) 8(28.6) 10(35.7) 18(64.3) N3 11(91.7) 1(8.30) 9(75.0) 3(25.0)
TNMG stage
I 79(45.7) 94(54.3) <0.001 59(34.1) 114(65.9) 0.001II 41(73.2) 15(26.8) 28(50.0) 28(50.0) III 41(87.2) 6(12.8) 29(61.7) 18(38.3)
Association between HOXC6 or HOXC8 expression and clinical characteristics (n=274)
HOXC6/HOXC8 expression was related with TNM stage
HOXC6 expression No.(%) p HOXC8 expression No.(%) p
Low High Low High
TRG
TRG1/2 15(48.4) 27(30.9 ) 0.016 35(47.9) 7(11.1) <0.001
TRG3/4 16(51.6) 78(69.1) 38(52.1) 56(88.9)
The association of HOXC6 and HOXC8 expression with TRG (n=136)
NC+S
Tumor Regression Grade (TRG)
Du YB, et al. J Surg Res, 2014.