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(The FASEB Journal. 2000;14:2329-2338.)
© 2000 FASEB

Leptin promotes invasiveness of kidney and colonic epithelial cells via phosphoinositide 3-kinase-, Rho-, and Rac-dependent signaling pathways

SAMIR ATTOUB, VEERLE NOE*, LUCIANO PIROLA{dagger}, ERIK BRUYNEEL*, ERIC CHASTRE, MARC MAREEL*, MATTHIAS P. WYMANN{dagger} and CHRISTIAN GESPACH1

INSERM U482, Signal Transduction and Cellular Functions in Diabetes and Digestive Cancers, and IFR65, Hôpital Saint-Antoine, 75571 Paris Cedex 12, France;
* The Laboratory of Experimental Cancerology, Ghent University, B-9000 Gent, Belgium; and
{dagger} Institute of Biochemistry, CH-1700, Fribourg, Switzerland

1Correspondence: INSERM Unit U482, Hôpital Saint-Antoine, 184 Rue du Faubourg Saint-Antoine, 75571 Paris Cedex 12, France. E-mail: gespach{at}st-antoine.inserm.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Leptin plays a key role regulating food intake, body weight and fat mass. These critical parameters are associated with an increased risk for digestive and mammary gland cancer in the Western population. Here we determined whether leptin contributes to the invasive phenotype of colonic and kidney epithelial cells at various stages of the neoplastic progression. First, leptin potently (EC50 = 10–30 ng/ml) induces invasion of collagen gels by premalignant familial adenomatous colonic cells PC/AA/C1 and nontumorigenic MDCK kidney epithelial cells, their src-transformed counterparts, and the human adenocarcinoma colonic cells LoVo and HCT-8/S11. Leptin and its Ob-Rb receptors were consistently identified by RT-PCR and immunoblotting in these cell lines, as well as in human colonic epithelial crypts, polyps, colonic tumor resections, and adjacent mucosa. Leptin-induced invasion was effectively blocked by pharmacological inhibitors of several downstream signaling pathways involved in cell transformation, namely, JAK2 tyrosine kinase (AG490), phosphoinositide PI3'-kinase (wortmannin and LY294002), mTOR kinase (rapamycin), and protein kinases C (GF109203X, Gö6976). Accordingly, leptin induces transient elevation of the PI3'-kinase lipid products in JAK2 immunoprecipitates prepared from parental MDCK cells. The leptin effect on invasion was potentiated by the activated form of the small GTPase RhoA and was abrogated by dominant negative mutants of RhoA, Rac1, and the p110{alpha} of PI3'-K. Our data indicate that leptin may exert a local and beneficial effect on migration of normal colonic epithelial cells and reparation of the inflamed or wounded digestive mucosa. We also emphasize a new role for leptin, linking the nutritional and body fat status to digestive cancer susceptibility by stimulating the invasive capacity of colonic epithelial cells at early stages of neoplasia. This finding has potential clinical implications for colon cancer progression and management of obesity.—Attoub, S., Noe, V., Pirola, L., Bruyneel, E., Chastre, E., Mareel, M., Wymann, M. P., Gespach, C. Leptin promotes invasiveness of kidney and colonic epithelial cells via phosphoinositide 3-kinase-, Rho-, and Rac-dependent signaling pathways.


Key Words: wortmannin • PI3'-kinase • MDCK cells • leptin signaling • pertussis toxin


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
OVERWEIGHT AND DIET-INDUCED obesity are increasingly important public health problems at the basis of several serious pathologies, including multistep carcinogenesis and susceptibility to diabetes. Some aspects of the Western lifestyle, primarily a high caloric intake, high-fat diets, and little physical activity, result in a positive energy balance, weight gain, and obesity, and are suspected of playing a key role in the etiology of colorectal disease (1 2 3) . A common feature in obesity is an increase of serum leptin, suggesting a loss of the regulation of food intake by this hormone. Most humans with obesity have a resistance to leptin that can be explained, at least in part, by the leptin-induced expression of SOCS-3 mRNA (suppressor of cytokine signaling) in regions of the hypothalamus that express the long form of the leptin receptor (4) . In obese individuals, leptin fails to reduce the appetite or induce energy expenditure (5) . Leptin is predominantly produced by white adipose tissue (6) , and serum levels of leptin correlate directly with body fat mass (7) . However, several other sources of leptin have recently been described in the digestive epithelia, including the liver and gastric mucosa (8 , 9) . Circulating leptin levels are down-regulated by fasting and increased by refeeding and inflammatory mediators (10) . Accordingly, several inflammatory cytokines, such as tumor necrosis factor and interleukin-1, induce anorexia and loss of body mass, two common manifestations of acute or chronic inflammation.

The best-known function of leptin is to exert a pivotal role on the regulation of body weight and food intake, mediated at the level of the central nervous system. Ob-Rb, the functional form of membrane-associated leptin receptor related to the class II cytokine group receptors binding interleukin-2, interferon, and growth hormone and closely related to the gp130 signal-transducing component of the interleukin-6 receptor and the G-CSF receptor (11) , is predominantly expressed in the hypothalamus. One potential mechanism involved in the leptin receptor activation is the reduced secretion of neuropeptide Y (NPY) in the mediobasal hypothalamus (12) , because the neuroendocrine peptide NPY potently stimulates food intake and inhibits thermogenesis (13) .

In peripheral tissues, leptin also exerts its control on body weight homeostasis via inhibitory actions on glucose metabolism and insulin secretion (5) . The Ob-Rb receptor is therefore present in various peripheral organs, including inflammatory blood cells, lung, kidney, liver, intestine, and insulin-secreting pancreatic B cells (5 , 11 , 14 , 15) . The leptin receptor oligomerizes with itself and upon leptin interaction activates the Janus kinase JAK-2 via transphosphorylation. Activated JAKs phosphorylate tyrosine residues on signal transducers and activators of transcription STATs (16 , 17) . Phosphorylated STAT proteins then dimerize and translocate to the nucleus to activate the transcription of target genes (16) . Thus, the leptin receptor can activate its associated JAK/STAT elements and mitogen-activated protein kinase MAPK signal transduction pathways (18 , 19) . In agreement, leptin modulates cell proliferation in hematopoietic and embryonic stem cells and CD4+ human T lymphocytes (14 , 19 20 21) .

In view of the critical role of the JAK/STAT and IRS-2/MAPK signaling pathways in cell proliferation, apoptosis, morphogenesis, and transformation (22 23 24 25 26) and because the contribution of leptin in the control of body weight homeostasis, we hypothesized that leptin might be implicated in the modulation of tumor progression and invasion. To determine whether such an effect might be relevant to disease, we used colonic PC/AA/C1 and kidney MDCK epithelial cells at various stages of the neoplastic transformation controlled by the src and Met oncogenes (27 , 28) . The signaling activity of the leptin receptors involved has been examined with reference to a possible connection with several signaling cascades involved in cell transformation and tumor progression, including PI3'-K, Rho-like G-proteins, the Akt/PKB and p70 kDa S6K, protein kinase C (PKC), and phospholipase C (PLC). Our data demonstrate that both systemic and tumor stroma-derived leptin may play a role in local and distant invasiveness of colonic epithelial cells that are already engaged in the neoplastic transformation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
Dulbecco’s modified Eagle’s medium was from GIBCO BRL (Cergy Pontoise, France) and fetal calf serum from Boehringer Mannheim (Meylan, France). Leptin was from R&D Systems Europe Ltd. (Oxon, U.K.). Hepatocyte growth factor (HGF) was a generous gift from Pr. Paolo Comoglio (University of Turin, Turin, Italy). Wortmannin (WORT), the JAK2 inhibitor AG490, Gö6976 (a selective inhibitor of PKC{alpha} and PCKß I isoenzymes that has no effect on the atypical Ca2+-independent PKCs), GF109203X (abbreviated as GF109, a PKC inhibitor with high selectivity for PKC{alpha}, ßI, ßII, {gamma}, and {varepsilon} isoenzymes), and the phosphatidylinositol-phospholipase inhibitor U-73122 were from Calbiochem (Meudon, France). Forskolin, rapamycin, pertussis toxin (PTx), LY294002, L-{alpha}-phosphatidylinositol, phosphatidylserine, and phenylmethysulfonyl fluoride (PMSF) were from Sigma (Saint Quentin Fallavier, France). Collagen type I was from Upstate Biotechnology (Lake Placid, N.Y.).

Antibodies
The anti-leptin receptor polyclonal antibody (pAb K-20) was from Santa Cruz Biotechnologies (Santa Cruz, California), the rabbit pAb against JAK2 from Upstate Biotechnology, and Sepharose-protein A beads from Amersham Pharmacia Biotech AB (Uppsala, Sweden). Enhanced chemiluminescence (ECL) immunodetection system and Hybond ECL nitrocellulose membranes were from Pharmacia Biotech (Buckinghamshire, England) and [{gamma}-32P]ATP was from Amersham Pharmacia Biotech AB (Les Ullis, France).

Cell lines and human tissue samples
Parental MDCK canine kidney epithelial cells and MDCKp110* cells stably transfected with a constitutively activated form of bovine p110*{alpha} by addition of the carboxyl-terminal farnesylation signal from Ha-Ras were a generous gift from Dr. J. Downward (29) . MDCKts.src transformed by a temperature-sensitive mutant of v-src (MDCKts.src, Cl2) and the MDCKts.src-p110DN cell line (transfected with the dominant negative mutant p110 {alpha} EcoS of PI3'-K) were previously described (27 , 30) . The MDCKT23 cells expressing the mutant G-proteins RhoAV14, RhoAN19, Rac1V12, or Rac1N17 under the tetracycline-repressible transactivator were a generous gift from Dr. J. Nelson (31) . Expression of V14RhoA, V12Rac1, and N17Rac1 was induced by removing doxycycline (DOX, Sigma) for 16–18 h or for 40 h (N19RhoA) from the culture media, as described previously (31) . Human colorectal cell lines LoVo, HCT-8/S11, PC/AA/C1, and PCmsrc were routinely grown in 6 cm diameter Petri dishes, as described previously (28 , 30 , 32) .

Specimens from patients who had undergone surgery for colonic cancer were obtained from the Center de Chirurgie Digestive (Prof. R. Parc, Hôpital Saint-Antoine, Paris, France). Tissue samples were immediately frozen in liquid nitrogen and stored at -80°C until use. For each tumor, a frozen section was subjected to histological analysis to confirm the neoplastic origin of the sample. The relative amount of stromal tissue in tumor specimens ranged from 15 to 20% of the sample. Individual colon carcinomas were staged according to Dukes’ classification, as modified by Astler and Coller (33) .

For preparation of human colonic epithelial crypts, fresh samples of nontumorous colonic mucosa were washed and incubated for 90 min in ice-cold solution containing 2.5 mM EDTA and 250 mM NaCl (pH 7.5). Colonic crypts were obtained by serial shaking of the samples in the same solution (28) .

Western blots, RNA isolation, and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis
For immunoblotting, cells were homogenized at 4°C in RIPA buffer containing 0.1 mg/ml PMSF, 100 µM benzamidine, and 100 mM Na3VO4 as protease inhibitors. A Polytron apparatus was used, with three bursts of 15 s. Insoluble material was removed by centrifugation for 15 min at 4°C and 12,000 g. Proteins were resolved in reducing conditions and 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, then transferred to Hybond-C Extra membranes (Pharmacia Biotech). Membranes were blocked in Tris-buffered saline (TBS: 20 mM Tris-HCl, pH 8, 150 mM NaCl) containing 5% dried skimmed milk. The blots were incubated in blocking solution for 1 h at 25°C with goat polyclonal antibodies (pAb) that recognize the synthetic peptides corresponding to the carboxyl-terminal sequence 32–51 of the mouse leptin receptor Ob-R (Santa Cruz Biotechnologies). Membranes were washed in TBS containing 0.1% Tween 20 and probed with a goat anti-mouse immunoglobulin G pAb (Santa Cruz Biotechnologies), then revealed by chemiluminescence Western detection (ECL, Amersham).

For RNA isolation, frozen tissue samples were homogenized in 4.7 M guanidinium isothiocyanate lysis buffer (10% w/v) with a Polytron apparatus and centrifuged for 20 h at 20°C through a 5.7 M cesium-chloride solution at 180,000 g. RNA samples (4 µg) were reverse-transcribed for 50 min at 42°C, using 200 U of Super Script II reverse transcriptase (Life Technologies, Cergy Pontoise, France). The cDNAs (1 µg) underwent PCR amplification in the presence of 25 pmol of each primer, 100 mM deoxyribonucleotide triphosphates, and 1.25 U of Goldstar polymerase (Eurogentec, Seraing, Belgium). For leptin and leptin receptor, amplification consisted of 40 cycles of denaturation for 60 s at 95°C, annealing for 30 s at 62°C for leptin and the leptin receptor, and 2 min extension at 72°C in an automated thermal cycler (Robocycler Gradient 96, Stratagene, La Jolla, Calif.). The reaction was initiated by 3 min of incubation at 95°C and ended after 10 min of extension at 72°C. PCR products were run on 1% agarose gels stained with ethidium bromide. To identify the leptin and leptin receptor transcripts, we used the following sense and antisense oligonucleotides (OligoExpress, Paris, France), spanning exon 3: 5'-CCTGACCTTATCCAAGATGG-3' and 5'-GAGTAGCCTGAAGCTTCCAG-3' (ob leptin gene), and spanning exon 20: 5'-GCCAACAACTGTGGTCTCTC-3' and 5'-AGAGAAGCACTTGGTGACTG-3' (Ob-Rb leptin receptor). The expected sizes of the PCR products were 224 and 246 bp for leptin and leptin receptors, respectively.

Collagen invasion assay
For invasion of collagen gels by renal and intestinal epithelial cells, petri dishes were filled with 1.35 ml of neutralized type I collagen (UBI) and incubated overnight at 37°C to allow gelling. Cells were harvested using Moscona buffer and trypsin/EDTA, and seeded on top of the collagen gels. Cultures were incubated for 24 h at the indicated temperature, in the presence or absence of leptin alone or combined with appropriate inhibitors of signal transduction pathways. The depth of cell migration inside the gels was measured, using an inverted microscope (34) . Invasive and superficial cells were counted in 12 fields of 0.157 mm2. The invasion index is the percentage of cells invading the gel over the total number of cells.

PI3'-kinase assay
Cultured parental MDCK cells were serum-starved overnight. At different times after treatment with 100 ng/ml recombinant leptin, cells were washed with ice-cold phosphate-buffered saline and lysed with lysis buffer containing 20 mM Tris-HCL (pH 7.5), 138 mM NaCl, 2.7 mM KCl, 5% glycerol, 1% Nonidet P-40, 20 mM NaF, and 1 mM Na3VO4 supplemented with 1 mM PMSF, 1 µg/ml aprotinin, and leupeptin. The lysate was centrifuged at 15,000 g for 10 min at 4°C and the supernatant was incubated with anti-JAK-2 pAb for 1 h at 4°C under constant agitation. The immunocomplex was then precipitated with protein A-Sepharose for 2 h at 4°C. The precipitate was washed once with lysis buffer, twice with LiCl buffer (100 mM Tris-HCl (pH, 7.5), 0.5 M LiCl), and once with the PI3'-kinase buffer containing 20 mM HEPES, 5 mM MgCl2 (pH 7.5).

For measurement of PI3'-Kinase activity, the beads were incubated for 10 min at 30°C in 50 µl of kinase buffer containing 10 µg of sonicated L-{alpha}-phosphatidylinositol, 10 µg phosphatidylserine, 50 µM ATP, and 10 µCi [{gamma}-32P]ATP. The reaction was stopped by the addition of 50 µl of 1M HCl; lipids were extracted with 200 µl of chloroform/methanol (v/v), centrifuged at 13,000 g for 5 min, and the organic phase containing the phospholipids was dried in a speed vac. The samples were resuspended in 15 µl chloroform/methanol (2:1) and spotted onto silica gel 60 thin-layer chromatography (TLC) plates (Aldrich, Steinheim, Germany). Chromatography of the TLC plates was in chloroform/methanol/ammonium hydroxide/water (45/38/1.5/8.5) for ~2 h and then the plates were dried and autoradiographed. The radioactivity incorporated into PtdIns-3-P was quantitated using a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.).

Lipid kinase activity
Parental MDCK cells were serum- and phosphate-starved for 24 h. Each plate was labeled with 360 µCi [32P]-Pi (200 µCi/ml) for 3 h. At various times after treatment with recombinant 100 ng/ml leptin, the radioactive medium was aspirated; the plate was scrapped once with 1 ml of 2.4 M HCl containing 5 mM tetra-butyl ammonium-hydrogen sulfate (TBHS) and 25 mM EDTA, and twice with 650 µl methanol. The extract was transferred in a tube containing 2.66 ml chloroform with carrier lipids (5 µg/ml) and 0.33 mg/ml of butyl-hydroxytoluene. The sample was vortexed vigorously for 1 min, centrifuged, and the organic phase was put to a new tube containing 2.3 ml of synthetic upper phase (chloroform/methanol/HCl: 8/4/3). The 1 M HCl phase was supplemented with 25 mM EDTA and 5 mM TBHS. Each tube was vigorously agitated with a Vortex and the organic phase was dried, deacylated, and analyzed by high-performance liquid chromatography.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of leptin and leptin receptors
Western blot analysis of crude protein lysates (40 µg) prepared from the human colorectal cancer cell lines (LoVo, PCmsrc, HCT-8/511, and Caco-2) and canine kidney epithelial cells in culture (MDCK and MDCKts-src) was performed using the K20 pAb raised against the extracellular domain of the leptin receptor Ob-R. We observe in Fig. 1A that the intestinal and renal leptin receptor exhibited a broad band on SDS-PAGE, corresponding to ~125,000 daltons and a minor band at 90 kDa. This estimated molecular mass of 120 kDa corresponds to the full-length Ob-Rb isoform reported in other tissues (35) and is the only isoform for which biological activity has been clearly established.



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  		Figure 1. Expression of the leptin receptor and leptin by Western blot and RT-PCR in colonic and kidney epithelial cell lines and human colorectal tumors at various stages of neoplastic progression. A) Western blot of the human leptin receptor. The long isoform of the leptin receptor Ob-Rb (Ob-Rb: 120 kDa) is expressed in cultured human colonic epithelial cells LoVo, PCmsrc, HCT-8/S11, and Caco-2, parental and src-transformed kidney epithelial cells MDCK and MDCKts.src. Immunoblotting of total cell lysates was performed using the K-20 pAb raised against the leptin receptor C-amino terminus that is common to all Ob-R isoforms. B) RT-PCR analysis of the human leptin receptor was performed in epithelial crypts isolated from normal colonic mucosa, in adenomatous polyps, resections from Dukes’ stages adenocarcinomas (ADK) B2, C2, D, and their adjacent nontumorous mucosa (Mc) and a liver metastasis (Meta) with its adjacent liver tissue (Liv), using human Ob-Rb specific primers (see Materials and Methods). The transcript of the Ob-Rb receptor was clearly detected as the 246 bp expected product. C) RT-PCR analysis of leptin expression in the corresponding human colonic cancer resections. The expected amplicon of 224 bp was identified in all samples examined.

Using oligonucleotide primers that specifically amplify the functional form of the human leptin receptor, we examined the expression of the gene by RT-PCR in human colonic epithelia and mucosa at various stages of cancer progression. Using total RNA prepared from normal human colonic epithelial crypts and polyps, we detected the leptin receptor Ob-Rb gene transcripts as a single 246 bp product (Fig. 1B ). The same PCR product was clearly identified in samples of colonic tumors (Dukes’ stages B2, C2, D, and a liver metastasis) as well as in their adjacent paired control mucosa. The specificity and identity of the Ob-Rb amplicons have been verified by sequence analysis. Using the same cDNAs prepared from colonic crypts, polyps, and ADK (B2, C2, D, liver metastasis), leptin gene expression was also detected as a single predicted 224 bp amplicon (Fig. 1C ).

Promotion of invasiveness by leptin
Because Ob-Rb receptors are connected with several signaling pathways involved in cell proliferation, apoptosis, and cancer progression, we addressed the question of whether leptin receptors may participate in the regulation of invasion in normal and transformed kidney and colonic epithelial cells. As shown in Fig. 2A , human colonic epithelial cells PC/AAC1 (PC) exhibited a remarkable invasiveness of collagen gels in response to 100 ng/ml leptin. Thus, leptin can promote invasion in premalignant, nontumorigenic epithelial cells derived from a familial adenomatous polyposis (FAP) patient. Subsequent transformation of the PC adenoma cells by ectopic expression of the c-src oncogene induced tumorigenicity of PCmsrc cells in nude mice (28) and retention of the leptin-induced invasiveness in this model (Fig. 2A ). Similar results were observed in immortalized, nontumorigenic kidney epithelial cells MDCK and in MDCKts.src cells incubated at the restrictive temperature of 40°C for src activation. No cooperativity was noticed between leptin and the src oncogene in MDCKts.src cells incubated at the permissive temperature of 35°C. Leptin also promoted invasiveness of two human colonic epithelial cell lines established from sporadic tumors (LoVo and HCT-8/S11).



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  		Figure 2. Induction of invasion of collagen type I gels by leptin in colonic and kidney epithelial cells at various stages of the neoplastic progression. A) Effect of 100 ng/ml leptin in human colonic cancer cell lines LoVo, HCT-8/S11, the noncancerous cells PC/AA/C1 (colonic epithelial cells established from a patient with familial adenomatosis polyposis), and MDCK cell lines (parental kidney epithelial cells) and their src-transformed counterparts, the PCmsrc and MDCKts.src cells. B) Effect of various concentrations of leptin on parental MDCK cells and src-transformed colonic and kidney epithelial cells PCmsrc and MDCKts.src. After 24 h incubation at 37°C in the presence or absence of leptin, the number of MDCK and PCmsrc cells that invaded the collagen gel as well as the depth of the invasive cells was monitored under a microscope with a computer-controlled step motor (34) . The effect of leptin was compared in MDCKts.src cells incubated at the restrictive temperature for src activation, i.e., 40°C. Data are means ± SE from four to five separate experiments.

We next examined the concentration dependence of the leptin effect on invasion. As shown in Fig. 2B , src-transformed PCmsrc and MDCKts.src cells incubated at 40°C exhibited significant invasiveness in response to 1 ng/ml leptin (P<0.001). An apparent half-maximal effect on invasiveness was induced by similar concentrations of leptin (10–30 ng/ml) in both parental and src-transformed MDCK cells. Normal and src-transformed MDCK cells appear to be equally sensitive to leptin.

Leptin signaling and invasion
In view of the critical role of PI3'-K in tumor invasion induced by HGF/scatter factor and {alpha}6ß4 integrins (30 , 36 , 37) , we first examined the contribution of this lipid/protein kinase in the functional regulation of invasiveness by leptin. For this purpose, we used two PI3'-K inhibitors (wortmannin and LY294002), MDCK cells transfected by constitutively activated p110*{alpha} of PI3'-K, and MDCKts.src cells stably transfected by the dominant negative mutant p110{alpha}EcoS of PI3'-K (29 , 30) .

As shown in Fig. 3A , the PI3'-K inhibitors wortmannin (10 nM) and LY294002 (10 µM) abolished almost completely invasiveness induced by 100 ng/ml leptin and 10 U/ml HGF in MDCKts.src cells incubated at the restrictive temperature of 40°C. Wortmannin also abolished invasion induced by the activated form of PI3'-K in stably transfected MDCK-p110* cells. In this assay, p110* showed constitutive activation after plasma membrane targeting by a carboxy-terminal farnesylation signal from H-Ras. Since the dominant negative form of PI3'-K completely blocked the leptin and HGF effects in stably transfected MDCKts.src-p110DN cells (Fig. 3A ), these results confirm the contribution of PI3'-K in the leptin-induced invasiveness in kidney epithelial cells.



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  		Figure 3. Leptin signaling and induction of invasion in MDCKts.src kidney epithelial cells. Invasion index in collagen type I gels was measured using MDCKts.src cells incubated at 40°C in the presence and absence of 100 ng/ml leptin or 10 units/ml HGF, alone or combined with one of the following inhibitors or activators of signal transduction pathways: A) PI3'-kinase-dependent signaling: WORT (10 nM) and LY294002 (LY: 10 µM) as PI3'-K inhibitors. Collagen gel invasion was measured at 37°C in MDCK cells stably transfected by the activated form of p110*{alpha} and in MDCKts.src cells (40°C) transfected by the dominant negative mutant p110-EcoS (p110{alpha}DN) of PI3'-K (29 , 30) . B) Other signaling pathways: AG490 (50 µM as a JAK2 tyrosine kinase inhibitor), GF109, and Gö6976 (1 µM) as PKC inhibitors, the G{alpha}i/0 protein subunits inhibitor PTx (200 ng/ml), and the PLC inhibitor U-73122 (1 µM). The latter was added 30 min before leptin in the invasion assay. Data are means ±SE from four experiments.

We next examined the possible contribution of the JAK2 tyrosine kinase as a signaling component of the Ob-Rb receptor, in the leptin-induced invasiveness of MDCKts.src incubated at 40°C (Fig. 3B ). The JAK2 kinase inhibitor AG490 (38) at 10 µM reduced by 50% the leptin effect (data not shown) and 50 µM of this drug completely abolished the leptin-induced invasiveness. Likewise, the two PKC inhibitors GF109203X (GF109, 1 µM), Gö6976 (1 µM) and PTx (200 ng/ml) prevented the leptin effect. In parallel experiments, the same concentration of PTx was ineffective against src- and p110*-induced invasion in MDCKts.src cells (data not shown). As shown in Fig. 3B , leptin-induced invasiveness was not blocked by 1 µM of the PLC inhibitor U-73122.

Leptin and PI3'-K activation
We next examined the effect of leptin on PI3'-K activity measured in JAK2 immunoprecipitates prepared from parental MDCK cells. As shown in Fig. 4 , leptin increased PI3'-K activity in anti-JAK2 precipitates in a time-dependent manner. This activation was detectable at 1 min (2.65±0.61) and reached a maximum 3 min (threefold) after addition of 100 ng/ml of leptin (Fig. 4A ). Cells were serum-starved, pretreated or not with 200 nM wortmannin for 30 min at 37°C, and stimulated with 100 ng/ml leptin. Leptin increased formation of PtdIns (3 , 4 , 5) P3 lipids in a dose-dependent manner (Fig. 4B ). The physiologically important product of PI3'-K is thought to be phosphatidylinositol (3 , 4 , 5) -triphosphate (PIP3), which can also act by regulating specific isoforms of protein kinase C (39) .



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  		Figure 4. Effects of leptin on PI3'-K activity and PIP3 phospholipid production in MDCK kidney epithelial cells. A) Activation of JAK2-associated PI3'-K by leptin was determined in MDCK cells serum starved for 24 h and stimulated with leptin (100 ng/ml) for different time intervals. Cell lysates were immunoprecipitated with the JAK-2 antibody. Immune complexes were then adsorbed using protein A-Sepharose and subjected to a PI3'-kinase assay. The products of the reaction were analyzed by thin-layer chromatography, visualized by autoradiography, and quantified by a PhosphorImager. Data are means ± SE from three experiments. A representative autoradiogram of the PI3'-kinase assay is shown and the position of phosphatidylinositol phosphate (PIP) is indicated (arrow). B) Leptin-induced PIP3 accumulation was measured as described in Materials and Methods. Data are means ± SE from four experiments.

Contribution of the small GTPases Rho and Rac in leptin-induced invasiveness
The Rho small G-protein family are signaling molecules that regulate cell adhesion and migration through reorganization of the actin cytoskeleton. It is now well established that Rho activates the formation of actin stress fiber bundles and their associated focal adhesion. On the other hand, Rac regulates the formation of ruffling lamellipodia, filopodia in various cell types, and cadherin-dependent adherens junctions at cell–cell contacts. Furthermore, Rac activation was recently associated with the induction of cell migration and tumor invasion (40 , 41) .

To determine whether Rho and Rac contributed to the promotion of tumor invasion by leptin, dominant negative forms of Rho (N19 Rho) or Rac (N17 Rac) and the constitutively active mutants V14 Rho or V12 Rac were investigated in MDCKT23 cells. The ectopic expression of these dominant forms of the Rho/Rac GTPases are controlled by the tetracycline-repressible transactivator tTA, according to Jou (31) . At DOX concentrations below 20 ng/ml, Rac1V12 and RhoAV14 accumulated gradually to levels similar to endogenous Rac1 or RhoA.

As shown in Fig. 5 , MDCKT23 cells were induced to invade collagen gels by 100 ng/ml leptin, in agreement with the data presented in the parental MDCK cell line (Fig. 2) . This effect was blocked by rapamycin (20 nM) which binds to FKBP12. This complex inactivates mTOR/FRAP and thus interferes with the activation of the Akt/PKB-p70 S6 k cascade that controls translation, cell growth, and transcription in response to nutrients (42) . The p70 S6 kinase has been shown to complex with and be activated by the Rho family of GTPases (43) , which contribute to the organization and remodeling of the actin cytoskeleton and its associated sites of cell adhesion. In this context, we observed that the dominant negative mutants Rho N19 and Rac N17 completely abrogated the leptin-induced invasion (Fig. 5) . An inverse situation is observed with the constitutively active Rho mutant, which cooperates with leptin, but not with HGF, in inducing cellular invasion. Such cooperativity was not observed in V12RacMDCK cells because this activated form of Rac already induce a submaximal activation of invasiveness in our system, which is wortmannin-sensitive. In contrast, MDCKT23 cells are not invasive in the presence of 10 U/ml HGF (Fig. 5) , and the same data were obtained in the parental MDCK cell line (not shown).



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  		Figure 5. Effects of dominant-negative or -active forms of the Rho and Rac small GTPases on leptin-induced invasiveness of MDCK epithelial cells incubated in the presence or absence of rapamycin or wortmannin. Invasion index in collagen type I gels was measured using parental and MDCK cells expressing regulated forms of the Rac1 and RhoA mutants under the control of a tetracycline repressible transactivator, as described in Materials and Methods. After induction of the transgenic small G-protein mutants, parental and transfected MDCK were incubated for 24 h in collagen gels in the presence or absence of leptin (100 ng/ml), HGF (10 U/ml) alone, or combined with the PI3'-K inhibitor wortmannin (10 nM), or rapamycin (20 nM, added 30 min before leptin). Data are means ± SE from three experiments.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
As leptin and its Ob-Rb receptors were identified by Western blot and RT-PCR in intestinal and kidney cell lines and resections from human colonic tumors, we decided to extend these data by testing the hypothesis that leptin may influence the invasive potential of normal, immortalized, and cancerous epithelial cells.

Leptin promotes invasiveness of colonic and kidney epithelial cells at various stages on the neoplastic transformation
In the present study it became clear that leptin can trigger invasion of collagen gels by colonic and kidney epithelial cells that are in a premalignant stage of cancer development. Indeed, the FAP cell line PC/AA/C1 and the parental MDCK cells are not tumorigenic in the nude mice and do not invade collagen gels even in presence of the scatter factor HGF. Leptin is therefore the only agent able to induce invasion of collagen gels by these two cell lines. Genetic alterations representative of the early stages of human colorectal carcinogenesis, including truncated mutant APC and activated Ki-ras gene were previously identified in the parental PC/AA/C1 cells (28) . We also observed that leptin promotes invasion in src-transformed epithelial cells PCmsrc and MDCKts.src cells and two other human colonic cancer cell lines established from sporadic primary tumors (LoVo, HCT-8/S11).

Leptin signaling, tumor growth, and invasiveness
Leptin directly stimulates the PI3'-K in JAK2 immunoprecipitates, a tyrosine kinase present in the molecular cluster associated with the leptin receptor. The leptin receptor–JAK2 complex was reported to be involved in the regulation of mitogenic signals in BaF3 cells, a growth factor-dependent hematopoietic cell line (18) . Thus, activation of JAK kinases seems to be a prerequisite for the activation of signaling cascades emerging from the cytokine receptor superfamily, including the Ob-Rb receptor (18) . The JAK2 inhibitor AG490 abolished the leptin-induced invasiveness of MDCKts.src in our experiments, providing further support to this theory. Recently, IRS-2 has been shown to be phosphorylated by JAK2 and thereby associated with the p85 regulatory PI3'-K subunit in response to cytokine receptor signaling (25) , providing a potential signaling cascade between leptin receptors, PI3'-K activation, and endogenous formation of the PIP3 in our study. Our demonstration that PI3'-K inhibitors (wortmannin and LY294002) and the kinase inactive form of PI3'-K in stably transfected MDCKts.src-p110DN cells abrogate the leptin-induced invasion of collagen gels confirms that the activity of PI3'-K is indeed a crucial component of the signaling machinery used by the leptin receptor in this assay. Such a conclusion further emphasizes the key role of PI3-K in tumor invasion induced by growth factors, cytokines, adhesion molecules, and the Rho-like GTPase Rac (36 , 67 , 40 , 44 , 45) . We have shown here that leptin strongly synergizes with activated Rho in MDCKT23-V14Rho cells, but did not synergize with activated V12Rac, presumably because leptin acts, at least in part, on this Rac-dependent signaling pathway. It appears therefore that leptin promotes invasiveness of colonic and kidney epithelial cells by direct activation of multiple signaling pathways in parallel, including its associated signaling components JAK2/p85 and/or indirectly via the IRS-2 bound p85/PI3'-K complex and subsequent activation of downstream elements of the PI3'-K cascades: PKB, p70 S6 k, PKC, and Rho-like G-proteins. Our data using the PKC inhibitors and rapamycin confirm this interpretation, because in mammalian cells mTOR contributes to the activation of transcription via STAT3 (46) . In contrast, the phospholipase C inhibitor U-73122 displayed no effect against leptin- and HGF-induced invasion. It has been suggested that PLC is tyrosine-phosphorylated by the KDR/Flk-1 receptor in response to VEGF, a component of the angiogenic response in human colonic tumors (33 , 47) . Leptin-induced invasion in MDCKts.src was reversed by pertussis toxin, directly implicating substrate G-protein {alpha}-subunits for toxin-catalyzed ADP-ribosylation and inactivation. Leptin Ob-Rb receptors can synergize with a latent signaling pathways connected with G-protein subunits, including G{alpha}i, G{alpha}o, and Gß{gamma}, as proposed by us for the HGF receptor tyrosine kinase (30) and previously for the cross-talk between trimeric G-proteins and EGF receptors (48) . Treatment of MDCKts.src cells with 10 µM forskolin, a drug that induces cAMP formation by activating adenylate cyclase, also abolished the leptin effect on invasion (not shown), suggesting additional cross-talk between leptin signals and protein kinase A-dependent or independent pathways, as recently reported for the guanine-nucleotide-exchange factor for Rap1 directly regulated by cAMP (49) . Thus, cAMP may act as a negative regulator of leptin signaling in invasion, as suggested by our data using the cAMP-inducing agents pertussis toxin and forskolin.

Leptin, obesity, and cancer progression
The incidence of fatal cancers in the digestive tract and mammary gland is tightly related to dietary habits, increased fat intake, high caloric consumption and body weight (1 2 3) . The Western-style diet led to epithelial cell hyperproliferation in these tissues (50) . Leptin was previously shown to regulate food intake and energy expenditure (5) . Circadian variations of serum leptin between 2 and 3 ng/ml were observed in freely feeding mice, increasing rapidly to 6 ng/ml after short-term fasting and refeeding (10) and from 7.5 to 31.3 ng/ml in normal weight and obese humans (7) . In other pathophysiological situations, the range of serum leptin levels was increased from 2 to 8–10 ng/ml in acute inflammation induced by cytokines or LPS in mice (10) and to 23 ng/ml after efficient recombinant leptin therapy in a patient with congenital leptin deficiency, owing to an inactivating mutation of the leptin gene (51) . After 12 months of treatment with this antiobesity peptide, the amount of body fat decreased by 15.6 kg in a 10-year-old girl, and a maximal peak serum leptin of 108 ng/ml was measured at 8 months (lower values were 20–23 ng/ml before subcutaneous injections of 28 µg leptin once daily). Administration of leptin ameliorated hyperphagia and promoted weight loss while preserving lean mass, suggesting that leptin treatment may have a clinical value with a hypocaloric diet in the treatment of obesity and/or diabetes (51 , 52) . Our study clearly illustrates that significant invasiveness of PCmsrc and MDCKts.src cells was induced by 1 ng/ml leptin, half-maximal effect being observed at 10–30 ng/ml leptin. This Ob-Rb receptor involved in the regulation of invasiveness by kidney and colonic epithelial cells exhibited high affinity for leptin, consistent with the dissociation constant KD of 10 ng/ml measured in COS cells transfected with the Ob-Rb cDNA (11) . Thus, the affinity of the Ob-Rb receptor mediating invasion of collagen gels in our study is consistent with a possible activation of this signal transduction system by luminal (9) or serum leptin levels under pathophysiological situations. Furthermore, our data related to the expression of leptin in normal colonic epithelial crypts, adenomatous polyps, and human colonic tumor resections argue for a local, paracrine role of leptin in the normal and neoplastic growth of the digestive mucosa.

We can therefore propose that leptin might exert a beneficial role on the proliferation, migration, and renewal of normal intestinal epithelial cells along the crypt-villus axis and during the reparation of the transiently wounded, inflamed colonic mucosa. However, an adverse effect during chronic inflammation and multistep carcinogenesis is not excluded. The same situation was recently presented for leptin and endothelial cells (53) , which have an intrinsic ability to ‘invade’ surrounding tissues during the (neo) vascularization process, normal development, and cicatrization. A pejorative effect during angiogenesis of solid tumors is plausible, because leptin has angiogenic activity in cornea from normal rats in vivo (53) , but also induces a directional migration of cultured human umbilical vascular endothelial cells (IC50=60 ng/ml). Thus, leptin may exert cumulative adverse effects during tumor progression by convergent actions on the transformed compartment and vascular endothelium in response to hypoxia and requirements on energy expenditure that are inherent to the development of growing primary tumors and metastases. Calorie and increasing dietary fat intake can further increase the apparent risk on tumor incidence, growth, and patient survival in familial and sporadic cancers, as suggested in the multiple intestinal neoplasia MIN mouse model (54) .

In conclusion, our data reveal that leptin might be, at least in part, a potential link between dietary habits, obesity, and colon cancer. Thus, leptin administration in the treatment of obese patients or congenital leptin deficiency (50) should be considered with caution in the adult population at risk for digestive cancers in relation to the emergence of preneoplastic or inflammatory lesions in colonic mucosal cells that are already engaged in the multistep progression of ulcerative colitis and carcinogenesis.


   ACKNOWLEDGMENTS
 
This work was supported by INSERM, a postdoctoral fellowship, and a research grant from l’Association de la Recherche sur le Cancer, France (to S.A. and C.G.), the Swiss Cancer League 780–2-1999, and Fortis Bank, Verzekeringen (Brussels, Belgique). The authors are grateful to Dr. James Nelson for providing the MDCKT23-derived cell lines transfected with RhoA and Rac1 mutants under the control of the tetracycline repressible transactivator.

Received for publication March 17, 2000. Revision received May 15, 2000.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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