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

No potential conflicts of interest were disclosed

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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 ?

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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

Regional expression of transcription factors in the developing gut

Yuasa Y. Nat Rev Cancer. 2003.

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

Staining of IHC

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

mRNA

Protein

HOXC6/HOXC8 Knock-down at mRNA and Protein level

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

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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.