Next-Generation Sequencing Analysis of Gastric Cancer Identifies the Leukemia Inhibitory Factor Receptor as a Driving Factor in Gastric Cancer Progression and as a Predictor of Poor Prognosis

Abstract

Gastric cancer (GC) is the third cause of cancer-related mortality worldwide. Nevertheless, because GC screening programs are not cost-effective, most patients receive diagnosis in the advanced stages, when surgical options are limited. Peritoneal dissemination occurs in approximately one-third of patients with GC at the diagnosis and is a strong predictor of poor outcome. Despite the clinical relevance, biological and molecular mechanisms underlying the development of peritoneal metastasis in GC remain poorly defined. Here, we report results of a high-throughput sequencing of transcriptome expression in paired samples of non-neoplastic and neoplastic gastric samples from 31 patients with GC with or without peritoneal carcinomatosis. The RNA-seq analysis led to the discovery of a group of highly upregulated or downregulated genes, including the leukemia inhibitory factor receptor (LIFR) and one cut domain family member 2 (ONECUT2) that were differentially modulated in patients with peritoneal disease in comparison with patients without peritoneal involvement. Both LIFR and ONECUT2 predicted survival at univariate statistical analysis. LIFR and its major ligand LIF belong to the interleukin-6 (IL-6) cytokine family and have a central role in immune system regulation, carcinogenesis, and dissemination in several human cancers. To confirm the mechanistic role of the LIF/LIFR pathway in promoting GC progression, GC cell lines were challenged in vitro with LIF and a LIFR inhibitor. Among several GC cell lines, MKN45 cells displayed the higher expression of the receptor, and their exposure to LIF promotes a concentration-dependent proliferation and epithelial–mesenchymal transition (EMT), as shown by modulation of relative expression of E-cadherin/vimentin along with JAK and STAT3 phosphorylation and acquisition of a migratory phenotype. Furthermore, exposure to LIF promoted the adhesion of MKN45 cells to the peritoneum in an ex vivo assay. These effects were reversed by the pharmacological blockade of LIFR signaling. Together, these data suggest that LIFR might have a major role in promoting disease progression and peritoneal dissemination in patients with GC and that development of LIF/LIFR inhibitors might have a role in the treatment of GC.

Repositioning Mifepristone as a Leukaemia Inhibitory Factor Receptor Antagonist for the Treatment of Pancreatic Adenocarcinoma

“…However, despite this difference, both cell lines exhibit a concentration-dependent proliferation when challenged with LIF (5, 10, 25, 50, and 100 ng/mL); however, while the MIA PaCa-2 cells showed a biphasic response ( Figure 2 D), PANC1 cells exhibited a linear progression of cell proliferation (measured by the MTS assay) in response to increasing concentrations of LIF ( Figure 2 E). The fact that exposure of MIA PaCa-2 cells to higher concentrations of LIF (25, 50, and 100 ng/mL) promoted a reduction of cell vitality is consistent with the finding that, in some systems, this cytokine exerts direct cytotoxic effects ( Figure 2 D) as reported by us and others in gastric cancer cell lines [ 16 , 42 ]. Because the effects exerted by low concentrations of LIF (5–10 ng/mL) were similar in the two cell lines, we used MIA PaCA-2 for the following experiments.…”

“…The cell viability assay was done using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Milano, Italy), a colorimetric method for accessing the number of viable cells in proliferation, as described previously [ 16 ]. MIA-PaCa 2 and PANC-1 cells were seeded in DMEM complete medium at 36 × 10 3 cells/100 μLl well into a 96-well tissue culture plate.…”

“…Leukaemia Inhibitory Factor (LIF) has been identified as one of the potential biomarkers of poor prognosis in patients with PDAC and is also a potential target for pharmacological intervention. LIF, a pleiotropic cytokine that belongs to the Interleukin (IL)-6 family [ 14 ], is overexpressed in a variety of solid tumours, including pancreas [ 15 ], stomach [ 16 ], liver [ 17 ], colon [ 18 ] and breast [ 19 ], and promotes cancer cell proliferation, remodelling, invasiveness and epithelial-to-mesenchymal transition (EMT) [ 20 ]. In target tissues, LIF signalling is mediated by its binding to an heterodimeric receptor complex formed by the LIF-receptor (LIFR) and the glycoprotein (gp) 130 subunit of the IL-6 receptor [ 21 ].…”

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“…The cell viability assay was done using the CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega, Milano, Italy), a colorimetric method for accessing the number of viable cells in proliferation as described previously ( 13 ). MIA-PaCa 2 cells were seeded in DMEM complete medium at 36 *10 3 cells/100 uL well into 96-well tissue culture plate.…”

“…Upon binding to its ligands, LIFR undergoes a series of conformational rearrangements that promote the phosphorylation of the Jak-Tyk, two proteins that are constitutively associated to cytoplasmic domain of the gp130/LIFR complex ( 11 ), activating the downstream signalling pathways which include JAK1/STAT3, MAPK and AKT. The LIF/LIFR axis and JAK/STAT3 signalling pathway is over-regulated in several type of solid tumours, including PDAC ( 12 ), gastric cancer (GC) ( 13 ), hepatocellular carcinoma (HCC) ( 14 ), colon-rectal cancer (CRC) ( 15 ) and breast cancer ( 16 ), and promotes cancer cell proliferation, epithelial-to-mesenchymal transition (EMT) ( 17 ) and regulates aberrantly the self-renewal of cancer cell-initiating tumors ( 18 ), as well as promoting radio ( 19 ) and chemo-resistance ( 15 ). Several studies support the suppression of LIFR signalling as potential target in inhibiting cell growth and tumour progression ( 9 ), and we have shown recently, that LIFR-mediated antagonism supports the anti-oncogenic effect of mifepristone in pancreatic cancer and chemoresistance ( 20 ).…”