TGR5 as a marker for advanced gatsric cancer-Fiorucci Stefano poster presented at the DDW 2016- San Diego (CA) May 21-25, 2016

1. GPBAR1(TGR5) is highly expressed in human gastric cancers and its activation by selective or
GPBAR1/FXR dual ligands promotes epithelial mesenchymal transition and tumor spreading
Barbara Renga*, Sabrina Cipriani #
, Adriana Carino * , Silvia Marchianò * , Angela Zampella †
and Stefano Fiorucci *
*Dipartimento di Scienze Chirurgiche e Biomediche, Università degli Studi di Perugia, Nuova Facoltà di Medicina e Chirurgia , Sant’Andrea delle Fratte,Perugia, Italy
#
Dipartimento di Medicina, Università degli Studi di Perugia ,Nuova Facoltà di Medicina e Chirurgia, Perugia, Italy
†
Dipartimento di Farmacia, Università di Napoli Federico II, Napoli, Italy
Background. Gastritis associated with bile acids reflux is a risk factor in the development of intestinal metaplasia in the stomach and is believed to function as an initiator of gastric carcinogenesis. Bile acids activate nuclear and G-protein coupled receptors. GPBAR1 (also
known as TGR5) is expressed in esophageal adenocarcinoma cell lines and its activation by TDCA increases cell proliferation. Whether GPBAR1 is expressed in gastric cancers and exerts a functional role in disease initiation or progression is controversial
Aim of the study. In this study, we have examined the expression of GPBAR1 in human gastric adenocarcinomas and examined whether its activation modulate the phenotypes of gastric cancer cell lines in vitro and in vivo in a model of mice peritoneal implantation and
dissemination.
Material and methods. 35 patients with gastric adenocarcinoma were included in this study. Authorization for samples collection was granted to SF by the CEAS, Ethical committee heal System of Umbria region (Italy). An informed written consent was obtained by each
patient before surgery. Gastric cell lines used in this study were MKN45 (intestinal type cancer) and MKN74 (undifferentiated gastric cancer).
Results. GPBAR1 staining was found in all patients with intestinal metaplasia and in patients intestinal but in only 20% of the diffuse subtype of adenocarcinomas. GPBAR1 expression was found to be associated with advanced cancer (stage III and IV) and was associated
with lymphatic spreading. Expression of FXR, mRNA , and GPBAR1,mRNA and protein, was detected in several cancer cell lines, with MKN45 (intestinal cell type) having the higher expression of GPBAR1 in comparison to MKN74 (indifferentiated cell type). Exposure of
MKN45 cells to TLCA, oleanoic acid (selective GPBAR1 ligand) or 6-ECDCA (a dual FXR and GPBAR1 ligand) increased the expression of several genes usually associated with a metastatic phenotype including KDKN2A, HRAS, IGB3, MMP10 and MMP13 and
downregulated the expression of CD44 and FAT1 (among other) (p<0.01 versus untreated cells). Exposure of MKN45 to GPBAR1 ligands, including selective GPBAR1 ligands TLCA,TDCA and oleanoic acid, or dual FXR/GPBAR1 ligands, 6-ECDA and CDCA, increased cell
migration, enhanced cell adhesion to the mouse peritoneum and wound healing. These effects correlated with increased expression of N-cadherin and vimentin and were blocked by cetuximab and by MEK inhibition. By using a model of peritoneum metastasis, we found that
pretreating MKN45 cells with TLCA or 6-ECDCA increases the number of peritoneal metastasis by 30% and that this effect was abrogated by cetuximab. Because cetuximab is an EGF receptor(R) inhibitor, we have then examined molecular basis for interaction of GPBAR1
with EGFR and found that exposure of MKN45 cells to TLCA, increases the phosphorylation of EGFR in a time and concentration-dependent manner.
Conclusions. GPBAR1 is highly expresses in gastric cancers. Activation of GPBAR1 in gastric epithelial cells promotes the epithelial mesenchymal transition. Taken together these data support a potential role for GPBAR1 antagonists in the treatment of gastric cancers.
Figure 1: (A-D)The expression of GPBAR1 was examinated in surgical samples, obtained from patients who
underwent surgery for the treatment of gastric cancer, first by immunohistochemistry. We found that GPBAR1 was
expressed either in non-neoplastic and neoplastic tissues, but the expression resulted slight up-regulated,
although not significantly, in the neoplastic tissue in comparison to healthy gastric mucosa. (E-G) This finding
was further confirmed by quantitative RT-PCR.The expression of the receptor was significantly higher in
patients with advanced disease; (F) patients with stage III and IV had significantly higher expression in
comparison with patients in stage I-II (p<0.05). Such difference was not observed in non-neoplastic areas (E) and
that there was no difference among the two main histological phenotypes (i.e. intestinal vs diffuse) (G). Values are
normalized to GAPDH and are expressed relative to those of positive controls(non-neoplastic tissues); The
relative mRNA expression is expressed as 2(-ΔΔCt). These data support the concept that development of more
advanced disease associated with higher expression of GPBAR1
Figure 2: Expression levels of GPBAR1and FXR were evalueted both by ReaL-Time PCR (mRNA) (A-B) and Western Blot analysis (protein) (C) in MKN45 and
MKN74 human cell lines. MKN45 cells were characterized by a slightly higher GPBAR1 mRNA expression than MKN74, FXR resulted express at very low levels
in both gastric cell lines. Values are normalized to GAPDH, the relative mRNA expression is expressed as 2(-ΔΔCt). Western Blot data was normalized with α-
Tubulin expression. (D-E) Transwell migration assay: MKN45 were seeded in a 6-well plate, starved and then primed with 10µM of Cholic Acid,
Chenodeoxycholic Acid, Ursodeoxycholic Acid, Taurolithocholic Acid, Taurodeoxycholic Acid and 6-ECDCA (D). In an another experimental setting, cells were
treated with Taurolithocholic Acid (1, 10 and 100µM), Taurodeoxycholic Acid (1, 10 and 100µM), 6-ECDCA (1, 10 and 50µM) for 72 hours (E)After the incubation
period 200 µl of cell suspension (2,5x105
cells/ml) were seeded in the upper chamber of the transwell. The lower chamber was filled with culture medium
containing 10% FBS as a chemoattractant. The invaded cells were fixed with methanol and stained with 0.1 % Toluidine blue solution and counted under a light
microscope (200×/10 fields for well). Experiments were performed in triplicate. All three ligands increased, in a dose-response manner, migration activity of
gastric cells in comparison with control cells . (F) MKN45 cells were plated and treated as described and used in a cell adhesion to peritoneum experiment. On
day 5, excised parietal peritoneum ( 1.6 cm2) was placed in a 24-well culture plate, filled with 1.0 ml of 1% BSA/RPMI 1640. Gastric cancer cells were∼
detached, fluorescently labeled with BCECF-AM (5 µM). 500µl of a suspension cells (5 x 105
cells/ml) were overlaid on the peritoneum in a 24-well plate, and
incubated at 37°C for 60 minutes. The cells adherent to the peritoneum were lysed with 1.0 ml of TRIS 50 mM plus SDS 1%; fluorescence intensity was
measured with a fluorescence spectrophotometer (Ex = 490 nm and Em = 520 nm). Experiments were conducted in triplicate. TLCA treatment of MKN45 cells
significantly increased cellular adhesiveness of gastric tumor cells to murine parietal peritoneum in a dose dependent manner.
Figure 3: Scratch wound healing was used to determine migration behavior in MKN45 cell line left untreated or stimulated
with CA, CDCA, TLCA, TDCA and 6E-CDCA 100 or 50 μM (A-B) In an another experimental setting, cells were treated
with an increasing dose of the same compounds (1,10 and 100μM) (C). After treatments, the cell monolayers were
scraped in a straight line using a p200 pipette tip in order to create a “scratch”. A wound was generated and imaged at 0
an 24 hours with a phase-contrast. Images obtained from each samples at both time points were analyzed using Image J
software and migration areas were expressed in pixels. All experiments were performed at least in triplicate. Wound
closure was significantly higher in treated cells, primarly with TLCA, TDCA and 6E-CDCA, and the migration increased in a
dose-response manner.
Figure 4: To further characterize the effects of GPBAR1 ligation in MKN45
cells, was used a gene array designed to analyze the expression of genes
related to tumor metastasis. The cDNA used for the array was obtained from
MKN45 cells (1x106
) plated, serum starved for 24 hours and then treated for 72
hours with 6-ECDCA 50µM, TLCA 100µM or OA 25µM. A comparative analysis
of effects exerted by the three GPBAR1 agonists, demonstrated that the three
agents shared a regulatory effect on a small number of genes. Array analysis
was carried out with the online software RT2 Profiler PCR Array Data Analysis (
http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php).
Up-regulated/down-regulated genes were those genes whose expressions had
been altered by more than 1,9 fold.
Non tumor tissue
A. Metaplastic area high expression
Low/absent expression
Tumor with high expression
Tumor with low expression
Non-neoplastic
area
I-II III-IV
0
5
10
15
20
25
30
35
40
p=0.06
Cancer staging
GPBAR1mRNA
I-II III-IV
-1
0
1
2
3
4
5
6
7
8
9
10 Primary tumor
*p<0,5
Cancer staging
GPBAr1expression
With Without
0
10
20
30
Intestinal metaplasia
p=0.3
GPBAR1mRNA
C.
D.
E.
F.
G.
h M Φ MKN45 MKN74
0.0
0.5
1.0
GPBAR1
mRNArel.expression
HepG2 MKN45 MKN74
0.025
0.000
0.5
1.0
FXR
mRNArel.expression
GPBAR1
α-Tubulin
MKN45 MKN74
1 10 100 1 10 100 1 10 50
0
100
200
300
400
NT TLCA TDCA 6-ECDCA
*
*
*
*
*
*∆%vsNTcells
NT CA CDCA 6-ECDCA UDCA TLCA TDCA
0
100
200
300
*
*
*
*
*
(10µm)
∆%vsNTcells
1 10 100
0
10
20
30
40
50
60
70
NT TLCA
*
*
∆%vsNTcells
NT CA CDCA 6-ECDCA UDCA TLCA TDCA
0
10
20
30
40
50
(10µM)
*
*
*
*
∆%vsNTcells
1 10 100 1 10 100 1 10 100 1 10 100 1 10 50
0
10
20
30
40
50
NT CA CDCA TLCA TDCA 6E-CDCA
*
*
*
*
*
*
∆%vsNTcells
NT CA CDCA 6-ECDCA UDCA TLCA TDCA
0
10
20
30
40
50
(10µM)
*
*
∆%vsNTcells
TLCA
6-ECDCA
OA
U:1
D:3
U:0
D:1U:5
D:6
U:2
D:0
U:1
D:0
U:11
D:5
U:1
D:0
U:18; D:11
U:7 ; D:10
U:8; D:7
CDKN2A
HRAS
ITGB3
MMP10
MMP3
CD44
FAT1
MMP13
MMP7
MTSS1
RPSA
WB: P-EGFR
WB: EGFR
NT 30’15’ 18h60’
TLCA
Figure 8: Effects of TLCA on EGFR phosphorylation in MKN45 cells. Cells
treated with 100 µM of TLCA were harvested at the indicated time. Cell lysates
were immunoblotted with antibodies against phosphorylated EGFR (pEGFR)
and total EGFR. The exposure of MKN45 cells to TLCA, increases the
phosphorylation of EGFR in a time-dependent manner.
B.
NT TLCA TDCA
0.0
0.5
1.0
1.5
(100 µM)
Vimentin
mRNArel.expression
NT TLCA TDCA
0.0
0.5
1.0
1.5
(100 µM)
*
E-caderin
mRNArel.expression
NT TLCA TDCA
0
1
2
3
(100 µM)
*
N-caderin
mRNArel.expression
p < 0.05p < 0.05
Figure 5: We have then investigatedby a RT-PCRanalysis, whether GPBAR1 activation induces phenotypic changes consistent with
acquisition of a mesenchymal phenotype. MKN45 cells were plated and after 24 hours of starvation were stimulated for 72 hours with TLCA
and TDCA (100 µM). We found that exposing MKN45 cells to TLCA resulted in 50% percent reduction of E-cadherin mRNA (n= 4; P<0.05)
and ≈ 100% increase of N-cadherin(n=4; P<0.05) (A-B). No change was observed in the expression of vimentin (C).
NT TLCA - TLCA - TLCA
0
10
20
30
*
*
#
*
#
Cetuximab MEK Inhib
Cell/field20X
Figure 6: Blockade of EGFR signaling abrogate the increased MKN45 migration activity TLCA-
induced. MKN45 cells were treated with TLCA, the EFGR inhibitor cetuximab (alone or in
combination with TLCA) and the MAPK inhibitor U0126. Data obtained are reported as ∆%
compared to NT cells (* vs NT, # vs TLCA). (Figure 6).
Control TLCA - TLCA
0
5
10
15
20
25
**
*
Cetuximab
NumberofNodules
Control TLCA - TLCA
0
5
10
15
20
25
**
*
Cetuximab
NumberofNodules
Control TLCA - TLCA
-4
-3
-2
-1
0
Cetuximab
∆Weight(g)
Figure 7: We have investigated the role in cancer progression of the GPBAR1/EGFR signaling,in a murine model of peritoneal carcinomatosis.
MKN45 cells were left untreated or pretreated for with TLCA, Cetuximab or with the combination of both for 3 days and then implanted in the
peritoneum of NOD-SCID mice. (A-C) MKN45 cells stimulated with TLCA significantly increased the number and the volume of peritoneal nodules in
comparison to control cells, whereas the inhibition of EGF-R pathway by cetuximab almost completely abrogated MKN45 potential to form peritoneal
nodules . Cetuximab reverted the increased MKN45 cells metastatic ability induced by TLCA priming
NT CA CDCA TLCA TDCA 6-ECDCA
100μM 100μM 100μM 100μM 50μM
24h0h
C.
B.
A.
D.
E.
F.
NT 15' 30' 60' 18H
0
1
2
3
TLCA (100µM)
P-EGFR/EGFR
B.
A.
C.
A. B. C. A. B. C.