1. HOXC6 and HOXC8 are potentially novel prognosis predictors of esophageal squamous cell carcinoma
Keneng Chen, M.D., PhD, RCSF
Luyan 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

2. No potential conflicts of interest were disclosed

3. Several issues with the clinical practice of
ESCC remain unsolved
prognosis
goal
Chemo response
The morbidity and mortality remains high 01
long-term survival remains unsatisfied 02
模板来自于
Lack of reports with long-term follow-up data from prospective database 03
Lack of efficient biomarkers for helping to guide the clinical practice 04 3

4. Why we choose HOX genes ?
There is some similarity between embryogenesis and tumorigenesis 01
AFP in liver cancer and CEA in colorectal cancer 02
HOX gene family regulates embryogenesis 03
HOX genes are deregulated in hematologic malignancy and many solid tumors 04

5. Regional expression of transcription factors in the developing gut
Yuasa Y. Nat Rev Cancer

6. potential biomakers Expression of 11 HOX genes is deregulated in ESCC
HOX genes were abnormally expressed in ESCC (n=36)
Gene
Positive cases in ESCC(%)
Positive cases in noncancerous mucosa(%)
HOXA10
3(8.3)
0(0)
HOXA13
27(75)
HOXB7
21(58.3)
HOXC4
12(33.3)
HOXC8
15(41.7)
HOXD9
6(16.7)
HOXD10
HOXD13
HOXA7
30(83.3)
HOXA9
24(66.7)
9(25)
HOXC6
We detected the expression pattern of 39 HOX genes in ESCC tissue and noncancerous mucosa by using RT-PCR, and then found that eight HOX genes were expressed only in some ESCC tissues but in any of the noncancerous mucosa samples. Spectically, the positive rate of HOXA13 was the highest, in 36 cases of ESCC samples, 27 presented positive.
potential biomakers
Chen KN, et al. Clin Cancer Res

7. Study design and work flow diagram FFPE tissue specimen
IHC for HOXC6、HOXC8 protein detection
FFPE tissue specimen
Prognosis
S Group (138 )
NC+S Group (136)
S+AC Group (148)
Lentivirus
infection
CCK8 Assay
Proliferation
Annexin v/PI
Cell cycle & Apoptosis
KYSE-450
Soft agar colony formation assay
Ability of colony formation
Knockdown of
HOXC6/HOXC8
Tumor formation in nude mice
Ability of tumorigenesis

8. Staining of IHC

9. Survival analysis in naive subgroup Median survival，mo(95%CI)
Surgery only
p = 0.070
p = 0.005
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( )
51.5
0.070
High expression
55(39.9)
35.0( )
32.9
HOXC8
85(61.6)
110.3( )
51.9
0.005
53(38.4)
30.3( )
30.6
Du YB, et al. J Surg Res, 2014.

10. Survival analysis in neoadjuvant chemotherapy subgroup
NC+S
p = 0.002
p = 0.004
The association of HOXC6/HOXC8 expression with overall survival in neoadjuvant subgroup (n=136)
Item
No.(%)
Median survival
mo(95%CI)
5-y survival
(%) p
HOXC6
Low expression
31(22.8)
82.7(-) 60
0.002
High expression
105(77.2)
27.8( ) 33
HOXC8
73(53.7)
110.3(-)
49.9
0.004
63(46.3)
21.2( ) 25
Du YB, et al. J Surg Res, 2014.

11. Survival analysis in adjuvant chemotherapy subgroup
S+AC
p = 0.002
p = 0.001
The association of HOXC6/HOXC8 expression with overall survival in neoadjuvant subgroup (n=148)
Item
No.(%)
Median survival
mo(95%CI)
5-y survival
(%) p
HOXC6
Low expression
81(54.7)
61.0( )
53.8
0.002
High expression
67(45.3)
33.4( )
30.3
HOXC8
99(66.9)
62.3( )
53.0
0.001
49(33.1)
31.3( )
21.7

12. HOXC6/HOXC8 Knock-down at mRNA and Protein level

13. Tumor growth in nude mice
Down-regulation affects cell proliferation in vitro & in vivo
CCK8 assay
Tumor growth in nude mice
shNC
shHOXC6
shHOXC8
Soft agar
Colony formation
HOXC6(-)
HOXC8(-)
Control

14. Down-regulation affects cell apoptosis in vitro
Apoptosis rate variation when HOXC6 or HOXC8 was knock-down
Apoptosis rate（%）
ShHOXC6
32.8±0.29*
ShHOXC8
36.1±0.35*
ShNC
12.7±0.31
* p ＜ 0.050

15. Down-regulation affects cell cycle in vitro
HOXC6(-)
HOXC8(-)
Control
Knockdown of HOXC6 and HOXC8 Affects the Cell Cycle
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
* p ＜ 0.050

16. Conclusion
HOXC6, HOXC8 might be a biomarker for evaluating prognosis
and chemo response of ESCC 01
Down-regulation of HOXC6/HOXC8 inhibited the cancer cell progression 02

17. Acknowledgment Dr. Chen’s LAB: Yabing Du Bin Dong Wanpu Yan Liang Dai
Xiaozheng Kang
Zhen Liang
Hongchao Xiong

19. HOX genes present the expression pattern
of temporal and spatial colinearity
A A11 A10 A B A6 A5 A4 A3 A2 A1
7p15.3
B B B8 B B6 B5 B4 B3 B2 B1
17p21.3
C13 C12 C11 C10 C C C6 C5 C4
12q13.3
D13 D12 D11 D10 D D D4 D D1
As we know, many molecular pathways underlying carcinogenesis represents aberrations of normal processes that control embyogenesis. There are many examples, the abnormal expression of these genes which regulate growth and development were implicated in carcinogenesis. Among these examples are HOX genes, which encode transcript regulatory proteins that are widely expressed in development and aberrantly expressed in cancers. There are 39 HOX genes organised in four genomic clusters,each localised on a different chromosome (HOX A HOXB, HOXC, and HOX D) and comprising from 9 to 11 genes arranged in a homologous sequence organisation. During mammalian development, according to the rules of temporal and spatial colinearity, with 3' Hox genes expressed early in development and controlling anterior regions, followed by progressively more 5' genes expressed later in development and controlling more posterior regions. For example, HOXA13 located at 5' terminal, primarily control the development of lumbo-scaral region, including In particular, 3' Hox genes in groups 1 to 4 primarily control the development of the branchial area and the rhombencephalon, the embryonic region corresponding to the hindbrain. Central Hox genes in groups 5 to 8 control the thoracic portion of the body, whereas 5' Hox genes in groups 9 to 13 control the lumbo-sacral region, including hindgut, urinary system and genitalia.
2q31
lumbo-sacral
thoracic
cervical
Posterior
Late 5' 3'
Anterior
Early
Schematics representation of four HOX loci
Cillo C, et al. J cell physiol, 2001

20. HOXC6/HOXC8 expression was related with TNM stage
Association between HOXC6 or HOXC8 expression and clinical characteristics (n=274)
Clinicopathologic
Characteristics
HOXC6 expression No.(%) P
HOXC8 expression No.(%)
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
0.524 L1
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)
<0.001 T2
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)
0.004 N1
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)
59(34.1)
114(65.9)
0.001 II
41(73.2)
15(26.8)
28(50.0)
III
41(87.2)
6(12.8)
29(61.7)
18(38.3)

21. Tumor Regression Grade (TRG)
NC+S
The association of HOXC6 and HOXC8 expression with TRG (n=136)
HOXC6 expression No.(%) p
HOXC8 expression No.(%)
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)
Du YB, et al. J Surg Res, 2014.