Klotho-beta expression is frequently up-regulated in hepatocellular carcinoma and its inhibition may induce adaptive de-differentiation response in liver cancer cellsWeijie Poh1,2, Han Kiat Ho1, Shazib Pervaiz2, Axel Ullrich1 1Singapore OncoGenome Laboratory, Institute of Medical Biology, A *STAR, 8A Biomedical Grove, #06-06 Immunos, Singapore 138648 2Department of Physiology, Yong Loo Lin School of Medicine; NUS Graduate School for Integrative Sciences and Engineering,; Duke-NUS Graduate Medical School; Singapore-MIT Alliance, National University of Singapore, Singapore 117597

IntroductionHepatocellular carcinoma (HCC) HCC is the dominant histological form among primary liver cancer types and presents in 70%-85% of patients1 Most HCC patients are diagnosed at advanced stages and HCC tumours are resistant to chemotherapy, thus patient survival rates are less than 11% at 5 years2

ResultsGenotype analysis of KLB gene in HCC patients revealed two novel SNPs and Q1020K distribution significantly different from normal Asian populationTable 1. Distribution of genetic mutations in HCC patients and the NCBI SNP database

Results (contd)KLB expression changes alters FGFR4, AFP, CYP7A1 genes and FGFR4 protein expressionKLB knockdown (HuH7)FGFR4 gene expression (% control)

A60 50 40 43.5% 52.2%HCC patients Normal Asian population

B44.4% 37.8%

KLB over-expression (Sk-Hep1) FGFR4FGFR4 gene expression (% control)

A

160 140 120 100 80 60 40 20 0 Control siRNA

*140.4%100%

B

120

100%

100

Fibroblast growth factor receptor 4 (FGFR4) FGFR4 is the dominant isotype in mature FGFR4 gene expression is frequently up-regulated in HCC patients; its inhibition reduced secretion of HCC marker alpha-fetoprotein (AFP), suppressed proliferation and lowered invasiveness of HCC cells4 hepatocytes3

80

* 84.9%

%

30 20 10 0 CC (Wild-type) CA AA (Mutant) 17.8% 4.3%

60

40

20

0 Klotho-beta siRNA Empty vector ESK-KLB

AFP gene expression (% control)

160 140 120 100 80 60 40 20 0 Control siRNA

Klotho-beta (KLB) KLB belongs to the klotho family within the glycosidase family 1 superfamily and encodes a 130 kDa type 1 single-pass transmembrane protein expressed on cell surfaces in the adipose, liver and pancreatic tissues5 KLB was identified as a FGFR4 co-receptor required for FGF19 binding and activation of FGFR4 signalling6

population. (A) Proportion of HCC patients carrying homozygous wild*novel type genotype CC was more than 2-fold of that in a typical Asian population (B) DNA chromatograms of Q1020K SNP found

AFP gene expression (% control)

*novel Figure 1. Q1020K genotype distribution in HCC patients and typical Asian

C

200 180

*161.9%100%

DAFP

120

100%

100

80

* 80.2%

60

40

20

0

Klotho-beta siRNA

Empty vector

ESK-KLB

KLB gene expression is frequently up-regulated in HCC tissues in patients and significant KLB gene up-regulation correlates with multiple tumour development7.0

CYP7A1 gene expression (% control)

350 300 250 200 150 100 50 0 Control siRNA Klotho-beta siRNA

CYP7A1 gene expression (% control)

E

450 400

*364.1%

FCYP7A1

120

100%80

100

60

* 62.4%

100%

40

20

Relative fold change in KLB gene expression in tumour relative to matched normal tissue

0 Empty vector ESK-KLB

G

ObjectivesThe clinical relevance and functional role of KLB in HCC progression was investigated through the following : Using matched tumour and non-tumourigenic liver tissues from 56 HCC patients Conduct mutation analysis to uncover genetic alterations and compare distribution with published databases Detect KLB gene expression changes and correlate with clinical characteristics Perturbation of KLB expression by silencing and over-expression in liver cancer cells Detect changes in cell viability Measure changes in expression of FGFR4, AFP, CD133 and CYP7A1 genes

6.0

5.0

Fold change

Significantly down-regulated (2-fold)

4.0

Figure 6. With KLB silencing in HuH7, (A) FGFR4 mRNA and (G) FGFR4 protein levels increased. Also, (C) AFP and (E) CYP7A1 gene expression were up-regulated. Overexpression of KLB in Sk-Hep1 decreased mRNA expression of (B) FGFF4, (D) AFP and (F) CYP7A1. Error bars represent SD. * p 2-fold

70

n (%)%

60 50

Materials & MethodsHCC patients (n=56) Liver cancer cell lines

18 (32.1%) 38 (67.9%) 10 (17.6%) 32 (57.1%) 14 (25.0%)

40 30 20% 20 10 0 Down-regulated gene expression Up-regulated gene expression 28.6%

Figure 3. KLB mRNA levels are (A) significantly up-regulated in HCC tissues and (B) correlate with development of multiple tumours. **p