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Cancer Research 68, 9413, November 15, 2008. doi: 10.1158/0008-5472.CAN-08-0909
© 2008 American Association for Cancer Research
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Immunology

Involvement of the Leptin Receptor in the Immune Response in Intestinal Cancer

Nijez Aloulou1, Sylvie Bastuji-Garin1,3, Sabine Le Gouvello2, Mohammad Abolhassani1, Marie Thérèse Chaumette4, Antoine Charachon5, Karen Leroy4 and Iradj Sobhani1,5

1 Université Paris XII, INSERM and AP-HP, CIC GIT cancer study team; Departments of 2 Biological Immunology, 3 Public Health, 4 Pathology, and 5 Gastroenterology, Henri Mondor Hospital, Créteil, France

Requests for reprints: Iradj Sobhani, Service d'Hépato-Gastroentérologie, CHU Henri Mondor, 51 Av. Du Mal. Delattre de Tassigny, 94100 Créteil, France. Phone: 33-1-49-81-23-62; Fax: 33-1-49-81-23-52; E-mail: iradj.sobhani{at}hmn.aphp.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
The incidence of colorectal cancers (CRC) may be influenced by environmental factors, including nutrition. The role of peptides regulating food intake in controlling the growth and recurrence of human tumors is controversial. Leptin, a cytokine-like peptide, regulates food intake. We investigated the expression of leptin and its receptor in 171 consecutive patients (78 female and 93 male; 71 years) with CRC. Leptin concentrations in the serum (ELISA) were determined before tumor removal. ObRb was characterized in tumors and normal homologous tissues and culture cells (HT29, HCT116, and HCT116 with a transferred chromosome 3) by using immunocytochemistry, immunohistochemistry, reverse transcription-PCR (RT-PCR), and Western blotting. Microsatellite instability (MSI) phenotype was characterized by immunohistochemistry and pentaplex PCR. mRNAs of cytokines and chemokines were quantified in tumors and in normal homologous tissues (RT-PCR) in 43 patients. Adequate statistical tests, including multivariate analysis adjusted for pathologic tumor-node-metastasis (pTNM), MSI-H, and ObRb phenotypes, were used. Higher expression of ObRb in tumors compared with the homologous normal mucosa, pTNM staging but not leptin serum level, was associated with patients' progression-free survival (PFS). Tumor ObRb phenotype and pTNM were independent predictive factors of PFS. ObRb was more strongly expressed in HCT116 cells than in HCT116-Ch3 cells as well as in MSI-H tumors than in microsatellite stability and potentially associated with efficient cytotoxic antitumoral response as assessed by immunohistochemistry and RT-PCR measurements. We suggest that leptin receptor expression in tumors is involved in adaptive immune response in sporadic colon and rectal tumors likely via MSI-H phenotype orientation. [Cancer Res 2008;68(22):9413–22]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 
Colorectal cancer (CRC) is the second most common cause of cancer mortality in the Western world. Tumor progression is governed both by genetic events, intrinsic to cancer cells, and epigenetic and environmental factors, such as nutrition. Although energy restriction unequivocally reduces tumor development (1, 2), the contribution of nutritional factors to human deaths from cancer remains controversial (1, 3, 4).

Leptin, a 16-kDa product of the ob gene, is mainly produced by fat tissue in human adults (5–7). ObRb is the main isoform of the leptin receptor; it is expressed by colonocytes and is preserved in human colonic adenomas and carcinomas (8). It may activate downstream transcription factors, including signal transducer and activator of transcription 3, and induce the expression of other genes, notably nuclear factor {kappa}B (NF{kappa}B; ref. 9). Leptin targets colonocytes and promotes cell proliferation via NF{kappa}B stimulation (10), and it has been suggested that this could be a mechanism by which nutrition uptake contributes to colon tumor growth (11). However, no tumorigenic effects have been documented in animals in response to leptin (10, 12–14). Activation of NF{kappa}B also leads to the production of proinflammatory and inflammatory cytokines and chemokines. In animals, exogenous leptin promotes experimental colitis by acting on colonocytes (15). These effects could be a consequence of its cytokine-like properties, the involvement of leptin in tumor growth, and/or a microenvironment triggering inflammatory and immune responses. Increased development of colonic, chemical-induced premalignant tumors in mice carrying the leptin receptor db/db mutation compared with wild-type animals (16) and increased susceptibility to spontaneous colonic tumors in mice deficient in one or more components of the immune system (13, 14) should be viewed as experimental evidence supporting this hypothesis.

Antitumoral immunity in CRC includes local control of metastatic invasion in CRC that has been recently shown to be linked to a continuous process from inflammatory cell to cytotoxic cell infiltration in tumors (17, 18). In addition, tumors infiltrated by lymphocytes (19, 20), particularly those with high-level microsatellite instability (known as MSI-H or MSI), have a good prognosis probably due to enhanced specific cytotoxic response (21). MSI is a genome-wide instability of repetitive DNA sequences observed at the nucleotide level; it is caused by the inactivation or loss of expression of mismatch repair (MMR) genes as a result of either mutations or epigenetic silencing (20).

We therefore investigated the involvement of leptin and of the leptin receptor (ObRb) in tumor progression in reference to pathologic tumor-node-metastasis (pTNM) staging, the MMR system, and cytokine and chemokine expression in the tumor microenvironment. Our aim was to determine whether fasting serum leptin concentration and leptin receptor expression in tumor cells were associated with CRC, including patient outcomes in a series of cases of sporadic intestinal cancer. The study was ethically acceptable: the patients were routinely informed about the research, and consent was obtained for genetic investigations whenever required.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 References
 
Patients
Consecutive patients (n = 183) who underwent surgery for primary rectal or colon tumor between 2000 and 2002 were selected for this study. Patients (7%) with known hereditary cancer, inflammatory bowel disease, and those for whom tumor or normal tissues were not available in the tissue collection bank, or who did not give consent for biological research, were excluded. Thus, 171 cases were included in the analysis. Surgical specimens were used for diagnosis and tumor description by routine H&S staining on slides. Tumor extensions to vascular, nervous, and nodular sites were assessed by the same pathologist in all cases. The mean (±1 SD) number of lymph nodes per patient on surgical specimens was 22 (±5); an extra colic (lymph node or other organs) tumor cell infiltration was evidenced in 91 patients (53.2%): lymph nodes in 86, liver in 19, and pulmonary or other synchronous metastases in 10 (Table 1 ). Radiology examinations were used to establish pTNM staging.


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  		Table 1.

 
Methods
Serum leptin level. Fasting serum was obtained from 136 (80%) participants before surgery. Leptin concentrations in the serum were measured by ELISA (DRG International, Inc.) and expressed in ng/mL. The leptin concentrations reported are means of two determinations within the same sample. The sensitivity of the test was 0.10 ng/mL.

Immunohistochemistry. Representative samples (n > 5 per tissue) from tumors and normal homologous mucosa were selected for each case and paraffin-embedded 4-µm sections.

For the MMR proteins, sections were pretreated by boiling in citrate buffer (pH 6.1) in a water bath, and immunohistochemistry was performed by means of an avidin-biotin peroxidase complex technique on an automated immunostaining module (Ventana) using antibodies targeting hMLH1, hMSH2, and hMSH6 proteins as follows: G168-728 (diluted 1:40; PharMingen), FE11 (diluted 1:25; Calbiochem), and 44 (diluted 1:40; Zymed Laboratories, Inc.), respectively. No specific binding sites were blocked before the slides were treated with the appropriate antibodies, and positive and negative immunostained slides were used in each experiment.

For leptin receptor, CD3, interleukin (IL)-17, and perforin staining, the sections were pretreated (boiling in buffer, pH 6.1 or 8, 1:20 dilution of horse serum in PBS for 20 min; Vector Laboratories). The serum was then removed and incubated overnight with C-20 (diluted 1:200; Santa Cruz Biotechnology) for 1 h with A0452 (diluted 1:50; Dako), for 1 h with AF-317-NA (diluted 1:40; R&D Systems), or for 2 h with mouse anti-human clone 5B10 (diluted 1:20; Novocastra) to detect the leptin receptor (ObRb), CD3, IL-17, and perforin, respectively. Staining was undertaken according to the manufacturer's instructions (Vector Laboratories). The chromogen SigmaFast 3,3'-diaminobenzidine (DAB; Sigma-Aldrich) was incubated with the tissue sections in the dark at room temperature for 4 min to visualize the antibody complex. The reaction was terminated by a water wash before being counterstained.

For perforin/CD3 double staining, the rabbit anti-human CD3 (diluted 1:50 in PBS; Dako) antibody was added for 1 h, and then the immunostaining was revealed with Naphthol/Fast Red (Sigma-Aldrich). The mouse anti-human perforin antibody was then added for 2 h. Immunohistochemical staining was undertaken using Envision-PER kit (Dako) and visualization was done with DAB substrate.

For perforin/ObRb double staining, the mouse anti-human perforin (clone 5B10, diluted 1:20) was applied first and incubated for 2 h. Staining was undertaken using Envision-PER kit and revealed by DAB substrate. Subsequently, goat anti-human leptin receptor (ObRb) C-20 (diluted 1:200) was incubated for overnight and revealed with Naphthol/Fast Red.

For IL-17/CD3 double staining, the goat anti-human IL-17 antibody (diluted 1:40) was added for 2 h, and then staining was undertaken using Vectastain AP kit (Vector Laboratories) and revealed by Naphthol/Fast Red. Rabbit anti-human CD3 antibody (diluted 1:50 in PBS) was added for 1 h, staining was carried out using the ImmPRESS system (Vector Laboratories), and visualization was done with DAB substrate.

Positive controls included slides from normal human fundic mucosa for ObRb sections of tumors with a known mutation in the MMR system, leading to undetectable hMLH1, hMSH2, and hMSH6 proteins; primary antibodies were omitted for the negative controls. CRC tumors were recorded to have lost hMLH1, hMSH2, and hMSH6 expression when nuclear staining was absent from all the malignant cells but were detected in normal epithelial and stroma cells. Two observers, blind to the PCR results, assessed all cases independently, and any cases in which they did not agree were further considered until the observers reached agreement.

ObRb morphometric analysis. Interindividual and intraindividual variability was first checked by using at least 8 to 10 slides per patient. Second, a semiquantitative method was used to evaluate ObRb immunoreactivity in tissues as "I x E" (I = intensity and E = extent of immunostaining; each being scored from 1 to 4). Accordingly, the I x E score varied from 6 to 9 (median, 6) in normal tissue and from 8 to 16 (median, 8) in tumor tissues, with colonocytes being the main immunoreactive cells (Fig. 1A ). Third, the procedure was routinely validated by comparing reverse transcription-PCR (RT-PCR) and immunohistochemical analysis in normal and tumor colonic tissues, with confirmation by histopathology of H&S-stained slides (see Fig. 1 in Results). For quantifying the immunostained cells, the "I x E score" was considered to be a qualitative variable by calculating (five slide samples per patient) the ratio between the scores for tumor over homologous normal tissues: a ratio of >1 was considered to indicate ObRb overexpression in the tumor, noted herein as ObRb+ (Fig. 1). The pathologists were unaware of patient characteristics and MMR status when assessing the ObRb phenotype by immunohistochemistry. However, ObRb can be expressed either in the colonocytes or in various inflammatory and immune cells (9), suggesting that the mRNA extracted from tumor tissue samples might come from normal, tumoral, or inflammatory cells. This is why semiquantitative evaluation by immunohistochemistry was preferred to RT-PCR for comparing tumor tissues with the normal colonic mucosa. In addition, inflammatory and lymphocyte cell infiltrations within tumors were evaluated semiquantitatively, and their association with ObRb overexpression and/or MSI (20) was investigated.


Figure 1
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  		Figure 1. Leptin receptor expression in colon and rectal mucosa. A, paraffin-embedded sections of normal (x60 magnification) and tumoral (T; x40 magnification) formalin-fixed tissues obtained by surgery were processed with an automated immunostainer using a specific leptin receptor antibody (C-20 diluted 1:200). A semiquantitative method was used to assess ObRb immunoreactivity in tissues as I x E (where I = intensity and E = extent of immunostaining; both scored from 1 to 4; see Materials and Methods). This score was then expressed as qualitative variable by calculating the ratio between the scores in tumor and normal tissue: a ratio of >1 was considered to indicate ObRb overexpression and designated the ObRb+ phenotype. B, RT-PCR analysis of leptin receptor expression and leptin immunohistochemistry in tissues: This procedure was used for internal validation by comparing RT-PCR and immunohistochemical results. RNA was extracted from 90 samples after histopathology examination on H&S-stained slides: 45 were from normal mucosa and 45 from homologous tumors. RT-PCR (40 cycles) was used to amplify a region of 237 bp of the Ob-R gene mRNA with the primers 5'-GCCAACAACTGTGGTCTCTC-3' and 5'-AGAGAAGCACTTGGTGACTG-3'. The amount of amplified ObRb fragment was normalized to that of GAPDH. The value for ObRb mRNA abundance is expressed according to the immunoreactivity score (see above). The I x E score ranged from 6 to 12 in normal tissues and from 8 to 16 in tumor tissues. C, Western blotting of ObRb: Samples of 20 to 40 µg of total protein from normal colonic tissue, cancers, HT29, and Caco-2 cells (1, 2, 3, and 4, respectively) were homogenized and resolved by 7.5% SDS-PAGE gel electrophoresis. The proteins were transferred to a membrane and probed with polyclonal anti-leptin receptor C-20 antibodies. Immunoreactivity was revealed by a chemiluminescence detection system. The specificity of the immunoreactive bands was checked by preincubating the antibodies overnight with the homologous peptide.

 
ObRb RT-PCR analysis. A 237-bp region flanking the Ob-Rb gene was amplified by RT-PCR from mRNA using appropriate primers (Supplementary Data, Annex A) to detect the expression of a gene in colonocytes as previously described (8, 15). RT-PCR was performed on total RNA extracted with Trizol reagent (Life Technologies). First-strand cDNA was prepared from 4 µg of total RNA according to the Pharmacia Biotech procedure and amplified by PCR [glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the control housekeeping gene (HKG)]. The sample was denatured (95°C; 3 min), and PCR was carried out using 40 cycles. The amplification was terminated by a final 10-min extension step at 72°C. PCR products were separated on 1% agarose gels and visualized under UV light after ethidium bromide staining. The fragments detected were isolated and sequenced to confirm that they corresponded to the ObR gene. In controls, the reverse transcriptase was omitted. HT29 cells were used to calibrate RT-PCR quantification, and data are expressed as the amount of the ObRb fragment, normalized versus GAPDH expression.

Analysis of microsatellite stability by PCR pentaplex. Formalin-fixed, paraffin-embedded (FFPE) sections from tumors were stained with hematoxylin and erythrosin B and, if necessary, macrodissected to obtain a tissue fragment comprising >40% tumor cells. DNAs were extracted from 50-µm-thick FFPE or frozen tumor and normal autologous tissue sections with QIAamp DNA Mini kits (Qiagen) according to the manufacturer's instructions. The genetic instability (MSI) status of the tumors was established using pentaplex PCR for markers as described by Suraweera and colleagues (22). Briefly, microsatellites were coamplified in a single 20 µL pentaplex fluorescent PCR reaction, containing 0.1 µmol/L of Bat-25, 0.25 µmol/L of Bat-26 and NR-22, 0.5 µmol/L of NR-24 and NR-21 primers, 0.15 mmol/L deoxynucleotide triphosphate (dNTP), 1.5 mmol/L MgCl2, 1x GeneAmp PCR Buffer II, 50 ng genomic DNA, and 0.5 unit AmpliTaq Gold DNA polymerase (Applied Biosystems). PCR was performed after denaturing (95°C; 10 min) and involved 35 amplification cycles [denaturing (95°C; 30 s), annealing (55°C; 45 s), and extension (72°C; 30 s)] and a final elongation step (72°C, 7 min). An ABI PRISM 3100 Genetic Analyzer was used to separate and detect the fluorescent PCR products, and the data were analyzed using GeneScan Analysis Software (Applied Biosystems). The MSI-H phenotype (designated MSI) was defined as the detection of length differences for at least three markers between tumoral and normal DNAs from the same individual (22). Thirty-one of the 45 tumors were phenotyped as microsatellite stability (MSS), and 14 as MSI. All remaining specimens (with two or fewer markers) were considered to be MSS.

Quantitative RT-PCR analysis of chemokines and cytokines. Total mRNA was prepared from a subset of 43 CRCs and homologous normal specimens using Trizol reagent and following the manufacturer's protocol. This procedure was originally optimized for extracting total RNA from frozen tissue samples measuring approximately 5 mm x 5 mm x 5 mm (30 mg). Serial sections (50 µ) of selection tissue were ground using stainless steel beads (5 mm) after adding cold Trizol. Tumors were selected to have a high (> 60%) percentage of tumoral cells.

First-strand cDNA was synthesized in reverse transcriptase samples, each containing 2 µg total RNA isolated from the colorectal biopsy, 16 units/µL Moloney murine leukemia virus reverse transcriptase (Life Technologies), 4 µmol/L oligo(dT)12-18, and 0.8 mmol/L mixed dNTP (Amersham-Pharmacia Biotech). Quantitative PCR was performed in a LightCycler 2.0 System (Roche Diagnostics) using a SYBR Green PCR kit or a Hybridization Probe PCR kit from Roche Diagnostics. The sequences of the primers and probes are indicated in Annex A (Supplementary Materials and Methods). The β2-microglobulin gene was used as the control HKG for normalizing the result because of the three HKGs tested it displayed the most stable expression in both tumoral and nontumoral specimens (data not shown). All PCR conditions were adjusted to obtain equivalent optimal amplification efficiency in the different assays. Real-time PCR was used for relative quantification of CXCR1, CD3, IL-8, IL-17, granzyme A, perforin, and FoxP3 mRNAs according to Gibson and colleagues (23) using the SYBR Green PCR kit, and peripheral blood mononuclear cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin for 1 h as calibrator samples. Real-time PCR was used to provide absolute quantification of CD3, IL-1b, tumor necrosis factor {alpha} (TNF{alpha}), IL-6, IFN{gamma}, IL-4, transforming growth factor β (TGFβ), IL-10, granzyme B, and FasL mRNAs using a Hybridization Probe PCR kit. The copy number of the mRNA for all target genes and the HKG was determined by plotting the sample Ct values against the standard curve obtained with the corresponding "quantitative DNA standard" (24) dilution series using LightCycler software 4.0. The abundance of the target gene mRNA was calculated as the copy number of the target gene per 106 copies of β2-microglobulin. All PCR experiments were done in duplicate.

In vitro human colonic cell and cancer line studies. Human cancer cell lines, HCT116+Ch3 (HCT116 with homozygote mutation of hMLH1 with transferred cDNA of hMLH1) and HCT116 parent cells and Caco-2 and HT-29 cells from the American Type Culture Collection, were cultured in Eagle's MEM (Life Technologies) supplemented with 10% decomplemented fetal bovine serum (EuroBio) in a humid atmosphere with 5% CO2 at 37°C and used for experiments once the cultures reached confluence. For RT-PCR, Western blotting, and immunocytochemistry, confluent cells were collected (13,000 in 300 µL). mRNA and proteins were extracted. For Western blotting, samples from human normal mucosa (n = 3), colon cancer tissue (n = 3), and confluent cancer cell lines were homogenized at 4°C in radioimmunoprecipitation assay buffer (Upstate Biotechnology) containing the protease inhibitors (Roche Diagnostics), 0.1 mg/mL phenylmethylsulfonyl fluoride, 100 µmol/L benzamidine, and 100 mmol/L Na3VO4. These crude protein extracts were resolved by 7.5% SDS-PAGE (20–40 µg of total protein in each lane). Proteins were transferred to nitrocellulose sheets and probed with 1:500 polyclonal anti-leptin receptor antibody (C-20) and 1:1,000 GAPDH antibody (Santa Cruz Biotechnology). The specificity of the immunoreactive bands was checked by preincubating the antibodies overnight with 40 µg of their homologous peptide. Reaction was revealed using the enhanced chemiluminescence detection system (Amersham), and for immunocytochemistry, cells were collected on microscope slides (Superfrost Plus, Fisher Scientific) and fixed in acetone (hMLH1, hMSH2 and hMSH6) or in methanol (ObRb) solution. For RT-PCR, mRNA was extracted and the amplification procedure was performed as described.

Statistical analysis. Quantitative variables are expressed as mean (±1 SD) or medians with interquartile range (IQR), as appropriate; categorical variables are expressed as numbers (%). All tests were two tailed.

Baseline characteristics of patients with an ObRb+ tumor were compared with those of patients with an ObRb– tumor by using {chi}2, Fisher's exact, or Mann-Whitney U test as appropriate. mRNA expressions in tumoral and nontumoral tissues were compared according to the leptin receptor and MMR status in tumor cells. Overall differences in the expression of immune markers between tumor tissues and autologous nontumor specimens were analyzed by the paired, nonparametric Wilcoxon signed-rank test. When significant differences were identified, the gene expression levels were also compared in tumoral and nontumoral specimens for each phenotypic subgroup, combining leptin receptor expression and phenotype (MSS&ObRb–, MSS&ObRb+, and MSI&ObRb+); two patients with the ObRb–&MSI phenotype were excluded from these analyses because of the small size of the subgroup. mRNA abundance was also compared between these phenotypes by using the Kruskall-Wallis test. For mRNA analyses, the Bonferroni correction was applied to adjust for multiple testing, and P < 0.004 (0.05/12 = 0.0042) was considered to be significant.

Factors associated with progression-free survival. The end point was the progression-free survival (PFS) measured from surgery to progression of the cancer or death and estimated by Kaplan-Meier survival analysis. Factors that may influence cancer progression, including leptin receptor expression and MSI in tumors, were first tested in univariate analysis by comparing survival curves with the log-rank test or Cox proportional hazards model, as appropriate. Hazard ratios (HR) with their two-sided 95% confidence interval (95% CI) were estimated separately for each variable. Variables with a P value of <0.15 were then considered for multivariate analysis. A systematic search for statistical association between these variables was performed using {chi}2, Fisher's exact, or Mann-Whitney U test, as appropriate. Multiple 2 x 2 analyses were used to assess first-order interaction and confounding. Finally, a Cox proportional hazards model was used to identify the independent variables associated with PFS. Stata Statistical software (release 8.0; StataCorp.) was used for data analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 References
 
Patient characteristics are summarized in Table 1. The mean follow-up was 18.9 (±18.0) months (median, 15.4; range, 0.4–62.1).

Leptin receptor and MMR status. Most colonocytes were immunostained by the ObRb-specific antibody in both normal colonic mucosa and tumors. Immunostaining was strong in the apical membrane of the colonocytes lining the colonic lumen (Fig. 1A). There were no obvious differences in the staining patterns in "normal tissue" from different segments of the large intestine using immunohistochemistry, Western blotting, or RT-PCR. Staining within the cytoplasm was stronger in tumor cells than in normal cells, possibly indicating the presence of sites of intracellular leptin receptor synthesis and metabolism. ObRb expression, as assessed by semiquantitative immunohistochemical estimation, was similar to the quantitative results obtained by RT-PCR (Fig. 1B). A tumor was classified as ObRb+ when the score was higher in the tumor than in the normal homologous mucosa and as ObRb– in all other cases. Accordingly, 124 (72.5%) patients were classified as ObRb+. Thirty-one (18%) patients had tumors with the MSI phenotype (Table 1) as assessed by pentaplex PCR technique and lacked expression of hMLH1 (n = 29; 80% right sided) or hMSH2 (n = 2) on immunohistochemistry. All other tumors with the MSS phenotype showed normal nuclear hMLH1, hMSH2, and hMSH6 protein expression on immunohistochemistry. There was no discrepancy between the immunohistochemical and pentaplex PCR results.

Patients' characteristics according to ObRb status. The ObRb+ phenotype was significantly associated with an older age, a right-sided localization of tumors, and MSI-H (Table 1). No significant association with pTNM staging was observed. The lymphocyte infiltration was found more frequently in tumors with ObRb+ phenotype (76% versus 24% in ObRb– tumors; P < 0.01).

Factors associated with progression-free survival. In univariate analysis, the leptin receptor and classic prognostic factors such as age and pTNM staging were significantly associated with PFS (Table 1B; Fig. 2 ). A trend toward an association was also observed for the absence of chemotherapy and for the MSI phenotype (Table 1B). Considering the strong relationship between ObRb expression and MSI (Table 1A), patients were classified in the following groups according to tumor phenotypes: MSS&ObRb–, MSS&ObRb+, and MSI&ObRb+. The two patients classified as having a MSI&ObRb– tumor phenotype were not included in the subsequent analyses. No significant interaction was observed between variables associated with progression-free survival (PFS). The pTNM staging was strongly associated with age (P = 0.01) and with chemotherapy (P = 0.0001). Therefore, when analyses were adjusted for pTNM staging, age and chemotherapy were no longer associated with PFS. In the multivariate analysis adjusted for pTNM classification, the MSI&ObRb+ phenotype was shown to be independently associated with progression-free disease. The relative risk was nearly significant for MSS&ObRb+ phenotype [0.53 (95% CI, 0.28–1.00)], and a trend toward an association was observed for MSI&ObRb+ [0.37 (95% CI, 0.12–1.12)].


Figure 2
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  		Figure 2. Event-free survival as estimated by Kaplan-Meier construction according to MMR and leptin receptor status: At baseline, 72.5% of patients' tumors displayed ObRb overexpression (ObRb+) in tumors compared with that in homologous normal mucosa. During the follow-up of 171 patients with rectal (19.3%) or colon cancer (80.7%), 45 patients were diagnosed with events including death, recurrence, and disease progression (30% of them with ObRb+ and 66% with ObRb– tumors at baseline). A, having a tumor with ObRb– phenotype carried a higher risk for event occurrence. B, having a MSS tumor phenotype did not constitute a significantly higher risk for event occurrence. C, the combination of both MSI and ObRb+ phenotypes together was associated with a lower risk of event occurrence. Two patients with MSI and ObRb– phenotype tumors were excluded from these analyses. P is the P value of the log-rank test comparing survival curves according to the tumor phenotypes.

 
Possible ObRb and MSI dependence in vitro. The colon HCT116 cell line is a cancer model with high MSI due to a mutation in the hMLH1 gene. HCT116-Ch3 is HCT116 carrying chromosome 3, which corrects this hMLH1 abnormality and shifts the phenotype from a high level to a low level of MSS. Both immunohistochemistry and RT-PCR showed that ObRb was expressed thrice more strongly in HCT116 cells than in HCT116-Ch3 cells (Fig. 3 ).


Figure 3
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  		Figure 3. ObRb expression in colon cancer cells according to the level of MSI level. A, hMLH1 and ObRb proteins in HCT116 and HCT116-Ch3 cells were immunostained on air-dried cytospin cultured cells using specific antibodies against the leptin receptor ObRb (top) and against the MMR protein hMLH1 (bottom). HCT116 cells (a and c) do not produce normal hMLH1 protein, whereas HCT116-Ch3 cells (b and d) do, as they carry the corresponding cDNA. B and C, ObRb is less abundant in HCT116-Ch3 than in the parental HCT116 cells, as shown by Western blotting (1 = HT29 cells as control; 2 = HCT116; 3 = HCT116-Ch3), immunocytochemistry, and RT-PCR (2 = HCT116; 3 = HCT116-Ch3).

 
Chemokine and cytokine expression according to the ObRb+ and MSI phenotypes. Univariate analysis revealed that the mRNAs for IL-8, CXCR1, IL-6, TGFβ, IL-17, FoxP3, FasL, granzyme B, and perforin were significantly more abundant in tumors than in homologous normal tissues (Table 2 ); no difference was observed between tumors and homologous normal tissues for IL-4, IL-10, and TNF{alpha} mRNAs. In tumor tissues, relationships were observed between perforin, FasL, and granzyme B on the one hand and between IL-8 and CXCR1 on the other hand (25). The expression of the CXCR1, FasL, granzyme B, and perforin genes was more strongly expressed in ObRb+ tumors than in ObRb– tumors (Table 2). Expressions of IL-8 and its receptor, CXCR1, were 3- to 6-fold higher in tumor tissues than in homologous normal tissues (Table 2). IL-17 and perforin immunoreactive cells were rarely detected in normal tissues in the lamina propria. However, their numbers were higher in sections of tumor tissues than in those of homologous normal tissues. Expressions of IL-17 and perforin on immunohistochemistry were consistent with those of RT-PCR, with higher IL-17 and lower perforin immunoreactive cells in MSS&ObRb– tumors contrasting with lower IL-17 and higher perforin immunoreactive cells in MSI&ObRb+ tumors (Fig. 4 ).


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  		Table 2. mRNA expression in tumor microenvironment according to leptin receptor and MMR status in tumor cells

 

Figure 4
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  		Figure 4. Leptin receptor, perforin, IL-17, and CD3 expression. A, sample from a colon cancer with ObRb+&MSI phenotype showing double staining with both perforin and CD3 antibodies, with high magnification on the right; double-stained cells are indicated in the window in down control. B, perforin immunoreactive cells (brown) were identified in a subset of cells (A) that were also immunoreactive to leptin receptor antibody (red) in this tumor; high magnification is shown in the windows. For double immunostaining, a rabbit anti-human CD3 antibody was added, immunostaining was revealed with Naphthol/Fast Red, and then a mouse anti-human perforin antibody was added before carrying out immunohistochemical staining and visualization with DAB substrate (brown); high magnification in the window. Immunohistochemistry with double staining clearly revealed the presence of perforin in most CD3 cells. C, in colon cancer with ObRb+&MSS phenotype, IL-17–stained cells were identified. D, double staining of IL-17 and CD3 antibodies showed that CD3 was not the only cell producing IL-17; the goat anti-human IL-17 antibody was added first before staining with Naphthol/Fast (red) followed by the rabbit anti-human CD3 antibody that was revealed with DAB substrate (brown).

 
Immunohistochemical double staining clearly showed that perforin was found in most CD3 cells, whereas IL-17 was found in only a few of them (Fig. 4).

Perforin immunoreactive cells were found more in tumors than in homologous normal mucosa. Among immunoreactive cells to ObRb antibody, some were double immunostained by antibodies targeting both ObRb and perforin proteins (Fig. 4).

Leptin serum level and leptin receptor expression in CRC. No relationship between serum leptin and either pTNM staging or survival was found (Table 1), although leptin levels were related to body mass index (BMI; R2 = 0.392; P < 0.001).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 References
 
We investigated leptin and ObRb, its receptor, in a large series of CRCs. We found that ObRb was overexpressed in a subgroup of sporadic human CRCs that seemed to be associated with a better prognosis than that in the other patients. This new phenotype was also associated with a lymphocytic immune response, with predominant cytotoxic markers that we showed were also associated with the level of MSI in tumors. We found a possible link between ObRb expression and MSI levels in tumors compared with homologous normal mucosa. Thus, ObRb seemed to be a new combined marker for immune response and survival.

Like those of various other peptides, in vivo, the effects of leptin depend on the serum level and on the number of receptors expressed in target cells. Although controversial data have been reported for fasting serum leptin concentrations and CRC (26, 27), we did not observe any significant differences for the fasting serum leptin levels in patients according to pTNM staging at baseline or to the event-free survival in the current study. In contrast, the overexpression of ObRb in tumor cells compared with homologous normal cells was an independent prognostic marker in addition to pTNM staging and age. This new marker was characterized using various methods (Western blotting, RT-PCR, and immunohistochemistry), with immunohistochemistry showing that ObRb was preferentially located in the colonocytes. Although immunohistochemistry has been used in several other studies (27–30), this is the first time that it is entirely consistent with Western blotting and RT-PCR findings (Fig. 1B and C). ObRb is expressed particularly by tumor cells in CRC (current study), in breast cancer, and in hepatocarcinoma (28, 31–33), although some inflammatory cells and lymphocytes (18, 34) also express this receptor. This has led to speculation about the role of leptin in tumor growth and in inflammatory and immune responses occurring in the tumor microenvironment. This is substantially supported by the following observations. ObRb is expressed in normal and tumor colonocytes, and leptin has been shown to stimulate NF{kappa}B activation (35) in these cells (25). Exogenous leptin stimulates the innate immune system via ObRb in the colonic mucosa in vivo (8, 15), whereas colonic tumors are increased in mice deficient in one or more components of the immune system (13, 14) and in db/db mice that carry a mutated leptin receptor (16). Whether these effects are due solely to the infiltration by polymorphonuclear cells and lymphocytes of tumors or colonocytes, or both, is still a matter of investigation. Interestingly, in the current study, lymphocyte infiltration was higher in tumors with ObRb+ than in those with ObRb– phenotype. However, colonocytes seems to be the first trigger of the proinflammatory response in tumors with a possible antitumoral effect in vivo (Supplementary Data).

The immunosurveillance system for tumor growth has been reported in several carcinomas (36) and involves inflammatory cells and lymphocytes, with regulatory T lymphocytes (LyTreg) and cytotoxic T cells being predictive of ineffective and effective antitumoral effects, respectively (37). In CRC, although higher numbers of inflammatory cells in tumors have been shown to favor tumor progression (38), the presence of markers for Th1 and cytotoxic polarization, the memory T-cell type, a high density, and the location of immune cells within the tumor samples are associated with improved survival (17, 39, 40). However, the fact that lymphocytes are recruited in only a subset of CRC tumors remains to be explained. Lymphocyte infiltration in CRCs is strongly associated with the MSI-H phenotype (20) but was not characterized in these studies. However, it is unlikely that all the tumors (88 of 104) with higher densities of CD3+ plus CD45+ cells, and with more prolonged PFS in related patients (39, 40), had the MSI-H phenotype. This would suggest that lymphocyte infiltration might be observed in MSS tumors too. We have recently shown that the MSI phenotype is significantly associated with cytotoxic T-cell polarization of lymphocytes in the microenvironment of tumors, whereas LyTreg and TH17 cells best characterized proficient MSS CRC (21). In the current study, we report evidence of a hormonal pathway corresponding to polarization of this type. Higher expression of the leptin receptor is shown to be associated with an improved prognosis in the current series of CRC-like hepatocarcinomas; in contrast, it is a marker of worse prognosis in breast cancer (28, 31–33). However, although the leptin receptor was characterized in the latter report, the inflammatory and/or immune responses have not been documented. The present study is the only one in which all well-established classic as well as new markers of prognosis (i.e., pTNM staging, molecular phenotype in MMR status, and the pattern of the immune response) have been taken into consideration. ObRb overexpression in tumors is shown to be a stronger marker than the MSI phenotype, which we found in <20% of CRCs. ObRb overexpression is associated with improved prognosis independently of pTNM staging. Expression of the cytotoxic markers (FasL, granzyme B, and perforin), inflammatory cytokines, and Foxp3, a marker of regulatory T cells, was significantly higher in tumors than in homologous normal tissues to an extent that depends on ObRb expression and MMR status. ObRb expression was accompanied by both proinflammatory cytokine and LyTreg expression, whereas MSI-H status was indicative of polarization to CTL (21). It is of interest that markers of this polarization were more highly expressed in tumors with ObRb+&MSI than in the others. This could account for the longer PFS in these patients. These findings suggest that MMR status and leptin receptor profiling in CRC may be potential biomarkers for a new prognostic "test set" in sporadic CRCs.

We do not know whether the level of MSI is the cause or the consequence of leptin overexpression in colonocytes or whether both phenotypes are due to an epigenetic phenomenon. Our results suggest that ObRb+ and MSI phenotypes are not independent markers because MSI status was associated with the ObRb+ phenotype at baseline and the MSI phenotype per se seemed to be less strongly associated with PFS. In addition, the number of leptin receptor copies and the level of IL-8 cytokine (Supplementary Data) in the HCT116 cell line, which is used as a high-level MSI cancer model, were higher than in neighboring cells (HCT116-Ch3) with a low MSI level. Because leptin has been shown to inhibit Treg lymphocyte (34), we would suggest that leptin receptor expression and MSI level have cumulative effects in regulating the immunosurveillance system. Leptin might stimulate colonocytes to trigger a proinflammatory response when a higher level of MSI leads to the production of tumor-specific neoantigens (41) and thus elicit potentially effective antitumor cytotoxic responses (38). Briefly, although the MSI phenotype seems to be a cytotoxic pathway marker (42, 43), and the leptin receptor a marker of a proinflammatory response, the interaction between the leptin receptor and the level of MSI should be considered in investigations of the tumor immune response.

Our study has some limitations. It is a retrospective study and did not include an analysis of the luminal leptin or genetic polymorphism analysis of ObRb in patients with cancer or of adaptive phenomena due to metabolic pathway (44). These studies clearly need confirmation from biological specimens taken in a prospective trial. Although systemic leptin has not been shown to influence tumor growth (16, 45), larger studies are required to elucidate the role of colonocytes as the main cells bearing the leptin receptor and to characterize the immune response in CRC.


   Disclosure of Potential Conflicts of Interest
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
References
 
No potential conflicts of interest were disclosed.


   Acknowledgments
 
Grant support: Ligue Nationale Contre le Cancer and Association Charles Debray.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Prof. Richard J.M. Ross (Endocrinology and Reproduction Section, University of Sheffield), Dr. John M. Carethers (Division of Gastroenterology, Department of Medicine, University of California, San Diego, CA), and Drs. Mohammad Yaghoubi, Mehdi Karoui, Daniel Cherqui, Catherine Delbaldo, and Philippe Gaullard for their scientific advice and Nadine Martin-Garcia, François Berrehar, Amal Seikour, and Feriel Bouabbas for their technical assistance.


   Footnotes
 
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 3/10/08. Revised 7/10/08. Accepted 8/15/08.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 References
 

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