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. 2012 Mar 15;103(6):1099–1104. doi: 10.1111/j.1349-7006.2012.02246.x

Orally active lysophosphatidic acid receptor antagonist attenuates pancreatic cancer invasion and metastasis in vivo

Mayumi Komachi 1,†, Koichi Sato 1,†, Masayuki Tobo 1, Chihiro Mogi 1, Takayuki Yamada 1, Hideo Ohta 2, Hideaki Tomura 1, Takao Kimura 1, Dong‐Soon Im 3, Keisuke Yanagida 4, Satoshi Ishii 5, Izumi Takeyoshi 6, Fumikazu Okajima 1,✉
PMCID: PMC7685076  PMID: 22348348

Abstract

Pancreatic cancer is highly metastatic and has a poor prognosis. However, there is no established treatment for pancreatic cancer. Lysophosphatidic acid (LPA) has been shown to be present in effluents of cancers and involved in migration and proliferation in a variety of cancer cells, including pancreatic cancer cells, in vitro. In the current study, we examined whether an orally active LPA antagonist is effective for pancreatic cancer tumorigenesis and metastasis in vivo. Oral administration of Ki16198, which is effective for LPA 1 and LPA 3, into YAPC‐PD pancreatic cancer cell‐inoculated nude mice significantly inhibited tumor weight and remarkably attenuated invasion and metastasis to lung, liver, and brain, in association with inhibition of matrix metalloproteinase (MMP) accumulation in ascites in vivo. Ki16198 inhibited LPA‐induced migration and invasion in several pancreatic cancer cells in vitro, which was associated with the inhibition of LPA‐induced MMP production. In conclusion, Ki16198 is a promising orally active LPA antagonist for inhibiting the invasion and metastasis of pancreatic cancer cells. The inhibitory effects of the antagonist on invasion and metastasis in vivo may be partially explained by the inhibition of motility activity and MMP production in cancer cells. (Cancer Sci 2012; 103: 1099–1104)

Pancreatic cancer is the fourth leading cause of cancer death, although it is the 10th cause of new cancer, in 2010 in the USA.1 The principle reason for the poor prognosis is the high frequency of peritoneal dissemination and/or extensive invasion into surrounding tissues and metastasis to distant organs even at an early stage. Unfortunately, the efficacy of systemic chemotherapy against invasion to surrounding tissues and metastasis to distant organs is insufficient. Therefore, the development of an effective modality of treatment is necessary in order to regulate the invasion and metastasis of pancreatic cancer.1, 2

In ascites and effluent of cancer patients, including pancreatic cancer and ovarian cancer, an enormous amount of lysophosphatidic acid (LPA) exists.3, 4 In pancreatic cancer cells, LPA1 and LPA2 are more highly expressed than LPA3 and LPA4.4 However, the role of LPA receptor subtype in motility is totally different: LPA1 is implicated in cell motility and invasive activities,4 while LPA2 has shown an inhibitory migratory activity.5 In cancer cells other than pancreatic cancer cells, LPA1 is overexpressed in comparison to LPA2 and LPA3 and has been implicated in the motility, invasion, and metastasis of lung, breast, and colon cancer cells.6, 7, 8 However, ovarian cancer shows significantly decreased LPA1 expression in comparison to normal tissues.9 Moreover, LPA2 and LPA3 have been shown to be aberrantly overexpressed and to determine the aggressiveness of the cancer, i.e., enhancement of invasion, metastasis, and tumorigenicity.9, 10 Thus, the role of LPA receptor subtypes differs among caner cell types.

We have shown that Ki16425 is an LPA antagonist for LPA1 and LPA3 11 and that it effectively inhibited the migration and invasion of pancreatic cancer cell line in vitro.4 Since then, the inhibitory effects of Ki16425 on migration and invasion have been reported in a variety of cancer cell lines, including glioma, gastric cancer cells, colon cancer cells, prostate cancer cells, and breast cancer cells in vitro.7, 8, 12, 13 However, the effectiveness of Ki16425 to combat the aggressiveness of pancreatic cancer, including peritoneal dissemination, ascites formation, and invasion and metastasis to local and distal tissues, has not been examined in vivo. For this purpose, we prepared Ki16198, a methylated form of Ki16425, to make the drug suitable for oral administration. The oral administration of Ki16198 effectively attenuated the invasion and metastasis of pancreatic cancer cells in association with a significant inhibition of MMP accumulation in ascites. Thus, interference with LPA receptors, especially LPA1, may provide a novel approach for the treatment of pancreatic cancer.

Materials and Methods

The detailed protocol is described in Data S1.

Materials

1‐Oleoyl‐sn‐glycero‐3‐phosphate (LPA) was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA); fatty acid‐free BSA was from Calbiochem‐Novabiochem Co. (San Diego, CA, USA); and epidermal growth factor was from Sigma Aldrich (St Louis, MO, USA). Ki16425 (3‐ (4‐[4‐([1‐(2‐chlorophenyl)ethoxy]carbonyl amino)‐3‐methyl‐5‐isoxazolyl] benzylsulfonyl)propanoic acid)11 and Ki16198 (3‐(4‐[4‐([1‐(2‐chlorophenyl)ethoxy]carbonyl amino)‐3‐methyl‐5‐isoxazolyl] benzylsulfanyl) propanoic acid methyl ester) were provided by Kyowa Hakko Kirin Co., Ltd. (Tokyo, Japan). The sources of all other reagents were the same as described previously.4, 5

Cell culture

Human pancreatic cancer cell lines were obtained as follows: Panc‐1 was kindly provided by the Cancer Cell Repository, Tohoku University (Sendai, Japan), BxPC‐3 and CFPAC‐1 were purchased from the American Type Culture Collection (Rockville, MD, USA), and YAPC‐PD was established as a highly peritoneal metastatic cell line from the pancreatic cancer patient in our laboratory.4, 14 ) All pancreatic cancer cell lines were cultured in RPMI1640 containing 10% FBS as described previously.4, 5 The cells were cultured on 10‐cm dishes for migration and invasion assay and on 6‐well dishes for evaluation of MMPs activity. When cells had become 80–90% confluent, the culture medium was changed to fresh medium containing 0.1% BSA to make them quiescent overnight. RH7777 hepatoma cells expressing LPA receptor subtype, including LPA1,11 LPA2,11 LPA3,11 LPA4,15 and LPA5,16 and B103 rat neuroblastoma cells expressing LPA6 (or p2y5)16 were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum.

Inositolphosphate response

For details see Data S1.

Neurite retraction assay of B103‐LPA6 cells

For details see Data S1.

Animal experiments

Male BALB/c nude mice (6 weeks old) were purchased from Charles River Japan, Inc. (Yokohama, Japan) for the in vivo studies. All animal procedures were performed in accordance with the guidelines of the Animal Care and Experimentation Committee of Gunma University. We examined the effects of LPA and Ki16198, an LPA receptor antagonist, on peritoneal dissemination and metastases to tissues, including liver, lung, and brain, as follows. YAPC‐PD cells (1×107 in 100 μL) were injected via the right flank of a mouse at day 0 into the abdominal cavity. In the experiments with LPA, the bioactive lipid (0.4 μmol in 100 μL) was intraperitoneally injected every day from day 0 to day 7, when mice were killed. In the experiments with LPA antagonist, Ki16198 (1 mg in 500 μL of PBS/12.5% DMSO) was orally administered into the mice every day from day 0 (just before the inoculation of the cancer cell line) to day 28. For control mice, vehicles (100 μL saline for the LPA experiment and 500 μL of 12.5% DMSO for the Ki16198 experiment) were administered. Ascites were collected to determine the MMP activity and tumor volumes were determined by weighing all the visual tumor nodes. Invasive or metastasis activity was evaluated by measuring the external mRNA expression of human glyceraldehydes 3‐phosphate dehydrogenase (GAPDH) together with mouse GAPDH in isolated liver, lung, and brain.

Cell migration assay

The migration experiment was performed using a modified Boyden's chamber apparatus, as described previously.5 For details see Data S1.

Matrigel invasion assay

Cell invasion activity was assessed by using a Matrigel invasion chamber, as described previously.5 For details see Data S1.

Quantitative real‐time PCR (RT‐qPCR)

Quantitative real‐time PCR (RT‐qPCR) was performed by TaqMan technology, as described previously.5 For details see Data S1.

Gelatin zymography

Matrix metalloproteinases activity in mouse ascites and supernatants of culture medium for YAPC‐PD cells were measured with a gelatin‐zymography kit according to the manufacturer's instructions (Primary Cell, Sapporo, Japan). In brief, each sample (20 μL) together with MMP molecular weight markers (proMMP‐2, MMP‐2, proMMP‐9) was subjected to SDS‐PAGE containing gelatin. The gels were washed with 2% Triton X‐100 and incubated for 22 h at 37°C in incubation buffer. After the gels were stained with Coomassie Blue R‐250, the bands were scanned and quantified by Image J software.

Statistical analyses

The results of multiple observations are presented as the mean ± SE or as representative results, unless otherwise stated. Statistical significance was assessed by the Student's t‐test; values were considered significant at *P < 0.05 and **P < 0.01.

Results

Pharmacological specificity of Ki16198

Ki16198 (Suppl. Fig. S1), a methyl ester of Ki16425, is an orally active LPA antagonist. Ki16425 has been shown to inhibit LPA1=LPA3>LPA2 11 but not LPA4,17 LPA5,18 nor LPA6.16 We characterized the pharmacological specificity of Ki16198 using cells overexpressing each LPA receptor subtypes in comparison with Ki16425. Inositol phosphate response to LPA was used for LPA1, LPA2, LPA3, LPA4, and LPA5, and cell shape response was for LPA6. As shown in Suppl. Fig. S2, either Ki16425 or Ki16198 substantially inhibited LPA1‐ and LPA3‐mediated responses with a similar potency, but not other LPA receptor subtype‐induced responses (Suppl. Figs. S2,S3). Thus, the specificity and potency of Ki16198 were indistinguishable from those of Ki16425.

Prevention by Ki16198 of migration and invasion of pancreatic cancer cells in vitro

We compared the ability of Ki16198 with that of Ki16425 to inhibit LPA‐induced migration and invasion in pancreatic cancer cell lines. Consistent with our previous results, Ki16425 inhibited migration (Fig. 1a) and invasion (Fig. 1b) in response to LPA in YAPC‐PD cancer cell line. Ki16198 was also effective to inhibit migration and invasion responses to LPA with a potency similar to that of Ki16425 (Fig. 1a,b). The inhibitory effects of Ki16425 and/or Ki16198 on the invasion response to LPA, but not to EGF, were also observed in several pancreatic cancer cell lines, including Panc‐1 (Fig. 1c), CFPAC‐1 (Fig. 1d), and BxPC‐3 (Fig. 1e). Thus, Ki16198 similarly to Ki16425 inhibited LPA‐induced migration and invasion of pancreatic cancer cells.

Figure 1.

Figure 1

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Effects of Ki16425 and Ki16198 on motility responses to lysophosphatidic acid (LPA) and epidermal growth factor in pancreatic cancer cell lines. (a,b) YAPC‐PD cells were treated with the indicated concentrations of Ki16425 (circle) or Ki16198 (triangle) and then assayed to measure migration response to 100 nM LPA (a) and invasion response to 1 μΜ LPA (B). (c to e) Effects of Ki16425 (10 μΜ) and/or Ki16198 (10 μΜ) on invasion responses to 1 μM LPA and 10 ng/mL EGF in Panc‐1 (c), CFPAC‐1 (d), or BxPC‐3 pancreatic cancer cells (e). Data are the means ± SD of the representative result of three separate experiments.

In these motility assays, the change in cell number in the well may affect the activity. It is unlikely, however, that the change in the activity of migration and invasion by LPA and its antagonists is due to the change in cell numbers in the motility assay conditions. Thus, the MTT activity reflecting cell numbers increased at most only 1.2 times by LPA treatment during 24 h incubation in YAPC‐PD and Panc‐1 cells (Suppl. Fig. S4), whereas 10% FBS stimulated about two times the activity (data not shown). On the other hand, motility activity was increased 5–10 times by LPA (Fig. 1).

LPA stimulation of metastasis of YAPC‐PD pancreatic cancer cells in vivo

Although an involvement of LPA in proliferation and migration of a variety origins of cancer cells has been suggested by a great number of in vitro experiments, the role of LPA in tumorigenicity in vivo has not been well characterized. In preliminary experiments, we observed that intraperitoneal inoculation of Panc‐1, BxPC‐3, or CFPAC‐1 into nude mice showed very weak metastatic activity; peritoneal metastasis or ascites formation was visually recognized only in one of five mice, two of five mice, and two of five mice, respectively, after 5 weeks. Since YAPC‐PD cells are highly metastatic and almost 100% of mice showed peritoneal dissemination 2–3 weeks after inoculation,4, 14 we used YAPC‐PD pancreatic cancer cells for the in vivo experiments. On day 7 after the inoculation of YAPC‐PD cells, we detected invasion or metastasis to liver (Fig. 2) as assessed by the expression of human GAPDH mRNA in six of nine control mice and eight of nine LPA‐injected mice, respectively. Thus, we failed to observe a clear difference in the incidence rate of metastasis between the control and LPA treatment under our experimental conditions. However, the degree of the invasion or metastasis of cancer cells to the liver (Fig. 2) was significantly higher in LPA‐treated mice than in control mice.

Figure 2.

Figure 2

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Effect of lysophosphatidic acid (LPA) administration on metastasis or invasion to liver in vivo. Nude mice were inoculated with YAPC‐PD cells. LPA or phosphate‐buffered saline (PBS) was intraperitoneally injected every day from day 0 to day 7 after the inoculation of the cells. On day 7, mice were killed and individual invasive or metastasis activity to liver (left panel) was assessed as the ratio of mRNA expression of human glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) to mouse GAPDH (n = 9). Right panel shows the means ± SE of their activities. *Effect of LPA administration was significant (P < 0.05).

Ki16198 inhibits peritoneal dissemination and metastasis of YAPC‐PD cells in the in vivo xenograft model

To further establish the role of LPA, the involvement of endogenous LPA in the tumorigenicity and metastasis of pancreatic cancer cells was examined. We examined the effects of Ki16198 on peritoneal dissemination and metastasis on day 28. In the experiments shown in Figure 3, each of 10 mice was administrated with Ki16198 or its vehicle and then inoculated with YAPC‐PD; however, three control mice and one Ki16198‐administered mouse died within 28 days after cancer cell inoculation. As shown in Figure 3a, the total metastatic node weight in the peritoneal cavity was significantly decreased by Ki16198. Ascites formation was also significantly decreased to roughly 50% by the administration of the LPA antagonist (Fig. 3b). We further examined the effects of Ki16198 on the metastasis activity of YAPC‐PD. On day 28, metastasis to the liver was detected in all control mice and four of nine Ki16198‐treated mice (Suppl. Fig. S5 and Fig. 3c). Furthermore, the degree of metastasis activity in Ki16198‐treated mice was significantly less than that in control mice (Fig. 3c). Similarly to liver, metastasis to lung (Fig. 3d) and brain (Fig. 3e) was observed, although the metastasis activity was roughly one tenth in lung and one thousandth in brain of that in liver. The metastasis to lung and brain was inhibited by the administration of Ki16198 to a similar degree of incidence and activity to those in liver (Fig. 3d,e).

Figure 3.

Figure 3

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Effect of Ki16198 on tumor volume, ascites formation, and metastasis or invasive activity. Twenty nude mice were inoculated with YAPC‐PD cells. Ki16198 or vehicle was orally administered to each 10 mice every day from day 0 to day 28, when mice were killed. Three control mice and one Ki16198‐treated mouse died before day 28. Tumor size (a) and ascites volume (b) were measured. **The effect of Ki16198 was significant (P < 0.01). The invasive or metastasis activities to liver (c), lung (d), and brain (e) were also measured. Results are shown as an individual activity (left panel) and means ± SE of their activities (right panel). The effect of Ki16198 was significant (*P < 0.05; **P < 0.01).

Ki16198 inhibition of peritoneal dissemination and metastasis of YAPC‐PD is associated with the inhibition of accumulation of MMPs in ascites

Matrix metalloproteinases are a multigene family of zinc‐dependent endopetidases and have been shown to be involved in the progression of many types of cancer, including pancreatic cancer.19, 20 Many types of cells, including cancer cells, have been shown to release MMPs.21 We, therefore, measured MMPs, including MMP‐2 and MMP‐9, in ascites of mice inoculated with pancreatic cancer cells. As shown in Figure 4a, both MMP‐9 and MMP‐2 and their active forms are detected in the malignant ascites, as evaluated by gelatin zymography. In the ascites of Ki16198‐treated mice, however, the gelatinase activities were remarkably attenuated (Fig. 4b).

Figure 4.

Figure 4

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Effect of Ki16198 on matrix metalloproteinase (MMP) activity in ascites. The ascites collected in Figure 3 was used. The representative gelatin zymography (a) and the means ± SE of the respective MMP (proMMP‐9, active MMP‐9, proMMP‐2, and active MMP‐2) activity evaluated by densitometry (b) are shown. **The effect of Ki16198 was significant (P < 0.01).

Ki16198 inhibits LPA‐induced MMP‐9 in YAPC‐PD in vitro

We examined the effects of LPA and its antagonist on MMP production in pancreatic cancer cells in vitro in order to clarify the sources of MMPs. Either EGF or LPA significantly increased proMMP‐9 activity (Fig. 5a,b), which was associated with MMP‐9 mRNA expression (Fig. 5c); however, the accumulation of active MMP‐9 was not detected under the conditions. As for MMP‐2, the pro‐form was detected in basal medium and slightly, but not significantly, increased by these agonists. The active‐form was not detected regardless of the agonist stimulation (Fig. 5a,b). Consistent with the protein expression, neither EGF nor LPA showed any significant effect on MMP‐2 mRNA expression (Fig. 5c). Thus, the profile of MMP accumulation is somehow different in ascites in cancer‐bearing mice and the culture medium of pancreatic cancer cells. As expected, however, the LPA‐induced expression of proMMP‐9 protein and mRNA was specifically inhibited by either Ki16425 or Ki16198 (Fig. 5). Thus, proMMP‐9 is, at least in part, produced by LPA‐stimulated cancer cells.

Figure 5.

Figure 5

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Effects of Ki16198 and Ki16425 on matrix metalloproteinase (MMP) activity and mRNA expression in YAPC‐PD cells. YAPC‐PD cells were incubated for 48 h (a,b) or 12 h (c) with or without of epidermal growth factor (10 ng/mL) or lysophosphatidic acid (LPA) (1 μΜ) in the presence or absence of 10 μM Ki16198 or 10 μM Ki16425 in RPMI1640 medium containing 0.1% bovine serum albumin (BSA) to measure MMP activities (a,b) and MMP mRNA expression (c). The representative gelatin zymography (a) and the means ± SE of proMMP‐9 and proMMP‐2 activities evaluated by densitometry (b) are shown. Note that unidentified band was sometimes but not always detected in the upper position of proMMP‐2 without any constant effect of agonists. In (c), the mRNA expression was expressed as percentages of the control value without test agents: the control value (a relative ratio to glyceraldehyde 3‐phosphate dehydrogenase [GAPDG] × 103) for MMP‐9 mRNA and MMP‐2 mRNA was 0.78 ± 0.14 and 0.54 ± 0.11, respectively. *The effect of EGF or LPA was significant (P < 0.05).

Discussion

Since pancreatic cancer is highly metastatic and has a poor prognosis, novel strategies for its treatment are required.1 Since LPA has been well recognized to be involved in the tumorigenesis and metastasis of a variety of cancers, LPA receptor antagonists and LPA synthesis inhibitors have been proposed to be promising drugs for cancer treatment.22 Recently, LPA‐related drugs have been challenged for the treatment of several cancers, including breast cancer and ovarian cancer23, 24, 25, 26 in vivo. In these previous studies, however, LPA antagonists and LPA synthesis inhibitors were administered in a slightly invasive manner, i.e., subcutaneously or intraperitoneally. Although LPA antagonists have been shown to effectively inhibit the migration and invasion of pancreatic cancer cells in vitro,4, 5 there have been no reports of the therapeutic effects of LPA‐related drugs for pancreatic cancer in vivo. In the present study, in order to ensure the least invasive drug administration for a long‐term therapy, we developed an orally active LPA antagonist, Ki16198, and showed for the first time that the LPA antagonist effectively suppressed pancreatic cancer invasion and metastasis to the liver, lung, and brain in the animal model. Thus, the LPA antagonist is a potential drug for the treatment of highly metastatic pancreatic cancer.

In addition to its oral availability, regarding its specificity, Ki16198 is superior to pan‐LPA antagonist/autotaxin inhibitors, such as α‐bromophosphonate,25, 26 which may inhibit all the LPA‐induced physiological actions as well. Ki16198 specifically inhibits LPA1 and LPA3. In pancreatic cancer cells, LPA1 is abundantly expressed4 and exerts a stimulatory role in the migration and invasion of the cells, whereas LPA2 inhibits migratory activity.5 Thus, Ki16198 was supposed to be useful for the treatment of pancreatic cancer cells. However, ovarian cancer shows a significant reduction in LPA1 expression levels relative to normal tissues,9 and LPA1 seems to be involved in the apoptosis of the cells.27 This raises the possibility that LPA1 antagonists might enhance the metastasis of ovarian cancer cells. In addition, LPA2 and LPA3 appear to play a greater role in determining the aggressiveness of ovarian cancer than LPA1.9, 10 Thus, the specificity of the drugs for LPA receptor subtypes appears to be critical for the treatment of the targeted cancers.

Matrix metalloproteinases induce the degradation of the ECM, the disruption of cell–cell interaction by cleaving E‐cadherin, the liberation of angiogenic factors, including bFGF and VEGF, and the processing of membrane‐associated growth factors and cytokines, eventually facilitating angiogenesis, tumor cell invasion, and metastasis.20, 21 The activity and expression of MMPs have been shown to be elevated in tumor tissues and/or serum in patients with a variety of cancers, including pancreatic cancer.20 In tumor tissues, serum, and/or pancreatic juice of pancreatic cancer patients, the levels of MMP‐919 and MMP‐228 have been shown to be more elevated than those in normal or healthy controls. A strong link of MMP‐9 to vascular angiogenesis and metastasis has been demonstrated in a number of mouse models of cancer, including a pancreatic cancer model.29, 30 Consistently with these studies, we detected a significant amount of MMP‐9 and MMP‐2 in ascites in tumor‐bearing mice. The administration of Ki16198 clearly inhibited the accumulation of MMPs in ascites, suggesting the involvement of these tumorigenic factors in the invasion and metastasis of pancreatic cancer cells. We showed that pancreatic cancer cells significantly release MMP‐9 in response to LPA in a manner sensitive to Ki16198 in vitro. Thus, at least MMP‐9 in malignant ascites of mice bearing pancreatic cancer cells may be partially derived from cancer cells themselves through LPA1 or LPA3 receptors. In contrast to MMPs in ascites, which were partly activated, as evidenced by the co‐existence of an active form of MMPs with proMMPs, those in the culture medium were almost a pro‐form of MMPs and the formation of an active form was not modulated by LPA. These results suggest that the activation or removal of a prodomain of proMMPs by proteolytic cleavage requires additional factors that are not supplied by pancreatic cancer cells.31

Although the LPA antagonist effectively suppressed invasion and metastasis, the inhibitory action of Ki16198 on tumor volume was smaller than that on invasion or metastasis. In fact, in vitro experiments using pancreatic cancer cells showed that LPA is a strong agonist for motility but not so much for proliferation (Suppl. Fig. S4).4 Therefore, combined use of drugs to inhibit proliferation with the LPA antagonist may be effective for the practical treatment of pancreatic cancer. Alternatively, the LPA antagonist might be used as a drug to suppress metastasis after removal of the main tumor in patients for whom surgical resection is applicable.

In conclusion, the orally active LPA receptor antagonist, Ki16198, effectively suppressed the invasion and metastasis of peritoneally inoculated pancreatic cancer cells, which were associated with the inhibition of MMP accumulation in ascites. LPA stimulates MMP accumulation and migration in pancreatic cancer cells through Ki16198‐sensitive LPA receptors, which may in part account for the inhibitory Ki16198 effects on invasion and metastasis in vivo. Thus, Ki16198 may be a novel drug for unresectable pancreatic cancer therapy.

Disclosure Statement

The authors have no conflict of interest.

Supporting information

Fig. S1. Chemical structure of Ki16425 and Ki16198 
 Fig. S2. LPA receptor subtype selectivity of Ki16425 and Ki16198. 
 Fig. S3. Effect of Ki16425 or Ki16198 on LPA‐induced cell rounding in LPA6‐expressing B103 cells. 
 Fig. S4. Effect of Ki16198 or Ki16425 on EGF‐ or LPA‐induced proliferation in pancreatic cancer cells. 
 Fig. S5. Typical picture of invasion or metastasis of YAPC‐PD pancreatic cancer cells to liver and its inhibition by Ki16198.

Click here for additional data file. (457.7KB, pdf)

Data S1. Inositolphosphate response; Neurite retraction assay of B103‐LPA6 cells; Proliferation assay; Cell migration assay; Matrigel invasion assay; Quantitative real‐time PCR (RT‐qPCR).

Click here for additional data file. (52.5KB, doc)

Acknowledgments

We are grateful to Dr Takao Shimizu of Tokyo University for encouraging our study and Ms Mutsumi Takano for technical assistance. This work was funded by Grants‐in‐Aid for scientific research from the Japan Society for the Promotion of Science (21390016, 23112503, and 23659029 to F.O. and 22791267 to M.K.). This work was also supported by Global COE Program from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to M.K.), by the joint research program of the Institute for Molecular and Cellular Regulation, Gunma University (10004) (to F.O. and T.K.), and by 2010 Japan–Korean joint research project.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1. Chemical structure of Ki16425 and Ki16198 
 Fig. S2. LPA receptor subtype selectivity of Ki16425 and Ki16198. 
 Fig. S3. Effect of Ki16425 or Ki16198 on LPA‐induced cell rounding in LPA6‐expressing B103 cells. 
 Fig. S4. Effect of Ki16198 or Ki16425 on EGF‐ or LPA‐induced proliferation in pancreatic cancer cells. 
 Fig. S5. Typical picture of invasion or metastasis of YAPC‐PD pancreatic cancer cells to liver and its inhibition by Ki16198.

Click here for additional data file. (457.7KB, pdf)

Data S1. Inositolphosphate response; Neurite retraction assay of B103‐LPA6 cells; Proliferation assay; Cell migration assay; Matrigel invasion assay; Quantitative real‐time PCR (RT‐qPCR).

Click here for additional data file. (52.5KB, doc)

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