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, 42 (3), 334-343

Adipocytes Sustain Pancreatic Cancer Progression Through a Non-Canonical WNT Paracrine Network Inducing ROR2 Nuclear Shuttling

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Adipocytes Sustain Pancreatic Cancer Progression Through a Non-Canonical WNT Paracrine Network Inducing ROR2 Nuclear Shuttling

C Carbone et al. Int J Obes (Lond).

Abstract

Background: Solid epidemiological evidences connect obesity with incidence, stage and survival in pancreatic cancer. However, the underlying mechanistic basis linking adipocytes to pancreatic cancer progression remain largely elusive. We hypothesized that factors secreted by adipocytes could be responsible for epithelial-to-mesenchymal transition (EMT) induction and, in turn, a more aggressive phenotype in models of pancreatic preneoplastic lesions.

Methods: We studied the role of factors secreted by two adipogenic model systems from primary human bone marrow stromal cells (hBMSCs) in an in vitro experimental cell transformation model system of human pancreatic ductal epithelial (HPDE) cell stably expressing activated KRAS (HPDE/KRAS),Results:We measured a significant induction of EMT and aggressiveness in HPDE and HPDE/KRAS cell lines when cultured with medium conditioned by fully differentiated adipocytes (ADIPOCM) if compared with the same cells cultured with medium conditioned by hBMSC (hBMSCCM) from two different healthy donors. Several genes coding for soluble modulators of the non-canonical WNT signaling pathway, including FRZB, SFRP2, RSPO1, WNT5A and 5B were significantly overexpressed in fully differentiated adipocytes than in their respective in hBMSC. ADIPOCM induced the overexpression and the nuclear translocation of the Frizzled family member receptor tyrosine kinase-like orphan receptor (Ror) 2 in HPDE and HPDE/KRAS cells. Vantictumab, an anti-Frizzled monoclonal antibody, reduced ROR2 nuclear translocation and in turn the EMT and aggressiveness in HPDE and HPDE/KRAS cells.

Conclusions: We demonstrated that adipocytes could induce EMT and aggressiveness in models of pancreatic preneoplastic lesions by orchestrating a complex paracrine signaling of soluble modulators of the non-canonical WNT signaling pathway that determine, in turn, the activation and nuclear translocation of ROR2. This signaling pathway could represent a novel target for pancreatic cancer chemoprevention. Most importantly, these factors could serve as novel biomarkers to select a risk population among obese subjects for screening and, thus, early diagnosis of pancreatic cancer.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Adipocytes secreted factors induce EMT and aggressiveness in in vitro models of pancreatic preneoplastic lesions. (a) Isolation and adipogenic differentiation of hBMSCs. The morphology of adherent hBMSCs and adipogenic differentiation. The accumulation of cytoplasmic triglycerides was detected by Oil Red O staining and visualized under a microscope. (b) Quantitative real-time PCR analysis of leptin (Lep) and adiponectin (ADIPOQ) along adipogenic differentiation. The values represent the fold change in RNA expression between the gene of interest and b-actin. (c) Western blot analysis of EMT marker vimentin in HPDE models cultured with conditional medium (CM) from hBMSC (hBMSCCM) and adipocytes (ADIPOCM). γ-Tubulin was used as loading control. (d) Cell proliferation assay of HPDE and HPDE/KRAS cell lines cultured with the conditional medium from hMBSC and adipocytes. The mean values and 95% confidence interval (CI) from three independent experiments done in quadruplicate are shown. (e) Levels of cell migration in HPDE and HPDE/KRAS cell lines co-cultured with hBMSC and adipocytes. The mean values and 95% CI from three independent experiments done in quadruplicate are shown. ***P<0.001 by two-tailed unpaired Student’s t-tests. Photographs of the wound area were taken by using phase-contrast microscopy immediately at indicated time point after the incision. All these experiments were performed by using hBMSC isolated from the healthy donor 1 (#1) and its relative adipocytes.
Figure 2
Figure 2
Adipocytes sustain pancreatic preneoplastic lesions aggressiveness through a WNT paracrine network. (a) Interaction network derived from secreted genes upregulated in adipocytes versus the hBMSC cells from two healthy donors. Each interaction is supported by at least one literature reference identified in the Ingenuity Pathway Knowledge Base, with solid lines representing direct interactions and dashed lines representing indirect interactions. In the heat map shown in this figure, the logarithms of the gene expression levels are shown in colors (green=decreased expression, red=increased expression). (b) Quantitative real-time PCR gene expression analysis of WNT5A, WNT5B, RSPO1, DKK1, DKK2, DKK3, SFRP2 and FRZB in hBMSC and relative adipocytes from two healthy donor (#1 and #2). The result values represent the values of the fold change in RNA expression between the gene of interest and b-actin. The mean values and 95% CI from three independent experiments done in quadruplicate are shown. ***P<0.001, **P<0.005 and *P<0.05, by two-tailed unpaired Student’s t-tests. (c, d) Western blot analysis for GSK3β and phospho-GSK3 β in HPDE and HPDE/KRAS cells cultured with conditional medium (CM) from hBMSC (hBMSCCM) or adipocytes (ADIPOCM) from two healthy donor. γ-Tubulin was used as loading control. The relative protein quantification, using ImageJ software (https://imagej.nih.gov/ij/), was performed and relative GSK3 and γ-tubulin ratio are indicated.
Figure 3
Figure 3
Adipocytes sustain pancreatic preneoplastic lesions aggressiveness through a WNT paracrine network. (a) Western blot analysis for Vimentin, β-catenin, phpspoGSK3 and total GSK3 of HPDE and HPDE/KRAS cultured with hBMSCCM or adipocytes (ADIPOCM) treated or untreated with vantictumab. γ-Tubulin was used as loading control. The relative protein quantification, using ImageJ software, was performed and relative GSK3 and γ-tubulin ratio are indicated. (b) Cell proliferation assay of HPDE and HPDE/KRAS cell lines cultured with the conditional medium from hMBSC and adipocytes treated or untreated with vantictumab. The mean values and 95% confidence interval (CI) from three independent experiments done in quadruplicate are shown. (c) Bar histograms of cell migration levels of HPDE and HPDE/KRAS cell lines cultured with the conditioned medium from hMBSC and adipocytes treated or untreated with vantictumab. The mean values and 95% CI from three independent experiments done in quadruplicate are shown. ***P<0.001 by two-tailed unpaired Student’s t-tests. Photographs of the wound area were taken by using phase-contrast microscopy immediately at indicated time point after the incision. All these experiments were performed using hBMSC isolated from the healthy donor 1 (#1) and its relative adipocytes. (d) Transwelll invasion assay of HPDE and HPDE/KRAS cell lines cultured with the conditioned medium from adipocytes and hBMSC control cells with or without vantictumab. The invasion chambers were used for treated and untreated group. The values obtained were calculated by averaging the total number of the cells from three filters. All experiments were performed in triplicate. The mean values and 95% CI from three independent experiments done in quadruplicate are shown. ***P< 0.001, **P<0.005, *P<0.05 and NS (Not Significative), by one-way analysis of variance and post Dunnett's mutiple comparison post test using hBMSC as control.
Figure 4
Figure 4
Adipocytes-derived WNT secreted factors induced expression and cellular rearrangement of ROR2 in pancreatic preneoplastic lesions. (a) Western blot analysis for the expression of ROR2 in HPDE and HPDE/KRAS cultured with ADIPOCM and hBMSCCM treated or untreated with vantictumab. GAPDH was used as loading control. (b) Immunofluorescence colocalization analysis for ROR2 (red) and FZD (green) receptors in HPDE and HPDE/KRAS cells cultured with ADIPOCM or with hBMSCCM treated or untreated with vantictumab. Fluorescence intensity analysis was performed by ImageJ software. Fluorescence intensity analysis was performed by ImageJ software and bars represent mean±s.d. from analysis of four separate high-power field images. ***P<0.001, by two-tailed unpaired Student’s t-test.
Figure 5
Figure 5
ROR2 nuclear shuttling mediates pancreatic preneoplastic lesions aggressiveness induced by adipocytes-derived WNT paracrine factors. (a) Western blot analysis for the expression of both 105 and 50 kDA protein isoforms of ROR2 in cytosolic and nuclear compartment from HPDE and HPDE/KRAS cultured with ADIPOCM and hBMSCCM treated or untreated with vantictumab. GAPDH and Histone H3 were used as loading and cellular fraction compartment controls. (b) Immunofluorescence colocalization analysis for ROR2 (green) and nucleus (blue) in HPDE and HPDE/KRAS cells cultured with ADIPOCM or with hBMSCCM treated or untreated with vantictumab. Fluorescence intensity analysis was performed by ImageJ software.

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