The role of the signaling pathway FGF/FGFR in pancreatic cancer

Fibroblast growth factors (FGFs) are signaling molecules with a wide range of actions that are involved in various processes in the body. Specifically for pancreas, FGFs are important for both organogenesis and carcinogenesis. They belong to the factors involved in the interaction between cancer and stromal cells representing a key component in the development of pancreatic cancer. Pathological changes in the FGF/FGFR signaling pathway is a complex process, which depends on type/isoforms of FGF receptors (FGFR) regulating the remodeling effect and subsequent activation of pancreatic cancer cells by FGF. FGFs and their receptors FGFR are considered as potential specific markers and putative targets for treatment of pancreatic cancer.     

FGFR1 cleavage and nuclear translocation regulates breast cancer cell behavior

FGF-10 and its receptors, FGFR1 and FGFR2, have been implicated in breast cancer susceptibility and progression, suggesting that fibroblast growth factor (FGF) signaling may be co-opted by breast cancer cells. We identify a novel pathway downstream of FGFR1 activation, whereby the receptor is cleaved and traffics to the nucleus, where it can regulate specific target genes. We confirm Granzyme B (GrB) as the protease responsible for cleavage and show that blocking GrB activity stopped FGFR1 trafficking to the nucleus and abrogates the promigratory effect of FGF stimulation. We confirm the in vivo relevance of our findings, showing that FGFR1 localized to the nucleus specifically in invading cells in both clinical material and a three-dimensional model of breast cancer. We identify target genes for FGFR1, which exert significant effects on cell migration and may represent an invasive signature. Our experiments identify a novel mechanism by which FGF signaling can regulate cancer cell behavior and provide a novel therapeutic target for treatment of invasive breast cancer.     

Phosphorylation of RSK2 at Tyr529 by FGFR2-p38 enhances human mammary epithelial cells migration

The members of p90 ribosomal S6 kinase (RSK) family of Ser/Thr kinases are downstream effectors of MAPK/ERK pathway that regulate diverse cellular processes including cell growth, proliferation and survival. In carcinogenesis, RSKs are thought to modulate cell motility, invasion and metastasis. Herein, we have studied an involvement of RSKs in FGF2/FGFR2-driven behaviours of mammary epithelial and breast cancer cells. We found that both silencing and inhibiting of FGFR2 attenuated phosphorylation of RSKs, whereas FGFR2 overexpression and/or its stimulation with FGF2 enhanced RSKs activity. Moreover, treatment with ERK, Src and p38 inhibitors revealed that p38 kinase acts as an upstream RSK2 regulator. We demonstrate for the first time that in FGF2/FGFR2 signalling, p38 but not MEK/ERK, indirectly activated RSK2 at Tyr529, which facilitated phosphorylation of its other residues (Thr359/Ser363, Thr573 and Ser380). In contrast to FGF2-triggered signalling, inhibition of p38 in the EGF pathway affected only RSK2-Tyr529, without any impact on the remaining RSK phosphorylation sites. p38-mediated phosphorylation of RSK2-Tyr529 was crucial for the transactivation of residues located at kinase C-terminal domain and linker-region, specifically, in the FGF2/FGFR2 signalling pathway. Furthermore, we show that FGF2 promoted anchorage-independent cell proliferation, formation of focal adhesions and cell migration, which was effectively abolished by treatment with RSKs inhibitor (FMK). These indicate that RSK2 activity is indispensable for FGF2/FGFR2-mediated cellular effects. Our findings identified a new FGF2/FGFR2-p38-RSK2 pathway, which may play a significant role in the pathogenesis and progression of breast cancer and, hence, may present a novel therapeutic target in the treatment of FGFR2-expressing tumours.     

Targeting FGFR4 inhibits hepatocellular carcinoma in preclinical mouse models

The fibroblast growth factor (FGF)-FGF receptor (FGFR) signaling system plays critical roles in a variety of normal developmental and physiological processes. It is also well documented that dysregulation of FGF-FGFR signaling may have important roles in tumor development and progression. The FGFR4-FGF19 signaling axis has been implicated in the development of hepatocellular carcinomas (HCCs) in mice, and potentially in humans. In this study, we demonstrate that FGFR4 is required for hepatocarcinogenesis; the progeny of FGF19 transgenic mice, which have previously been shown to develop HCCs, bred with FGFR4 knockout mice fail to develop liver tumors. To further test the importance of FGFR4 in HCC, we developed a blocking anti-FGFR4 monoclonal antibody (LD1). LD1 inhibited: 1) FGF1 and FGF19 binding to FGFR4, 2) FGFR4-mediated signaling, colony formation, and proliferation in vitro, and 3) tumor growth in a preclinical model of liver cancer in vivo. Finally, we show that FGFR4 expression is elevated in several types of cancer, including liver cancer, as compared to normal tissues. These findings suggest a modulatory role for FGFR4 in the development and progression of hepatocellular carcinoma and that FGFR4 may be an important and novel therapeutic target in treating this disease.     

Tissue and serum microRNAs in the Kras(G12D) transgenic animal model and in patients with pancreatic cancer

microRNAs (miRs) modulate the expression levels of mRNAs and proteins and can thus contribute to cancer initiation and progression. In addition to their intracelluar function, miRs are released from cells and shed into the circulation. We postulated that circulating miRs could provide insight into pathways altered during cancer progression and may indicate responses to treatment. Here we focus on pancreatic cancer malignant progression. We report that changes in miR expression patterns during progression of normal tissues to invasive pancreatic adenocarcinoma in the p48-Cre/LSL-Kras(G12D) mouse model mirrors the miR changes observed in human pancreatic cancer tissues. miR-148a/b and miR-375 expression were found decreased whereas miR-10, miR-21, miR-100 and miR-155 were increased when comparing normal tissues, premalignant lesions and invasive carcinoma in the mouse model. Predicted target mRNAs FGFR1 (miR-10) and MLH1 (miR-155) were found downregulated. Quantitation of nine microRNAs in plasma samples from patients distinguished pancreatic cancers from other cancers as well as non-cancerous pancreatic disease. Finally, gemcitabine treatment of control animals and p48-Cre/LSL-Kras(G12D) animals with pancreatic cancer caused distinct and up to 60-fold changes in circulating miRs that indicate differential drug effects on normal and cancer tissues. These findings support the significance of detecting miRs in the circulation and suggests that circulating miRs could serve as indicators of drug response.     

Mechanisms of FGFR-mediated carcinogenesis

In this review, the evidence for a role of fibroblast growth factor receptor (FGFR) mediated signalling in carcinogenesis are considered and relevant underlying mechanisms highlighted. FGF signalling mediated by FGFR follows a classic receptor tyrosine kinase signalling pathway and its deregulation at various points of its cascade could result in malignancy. Here we review the accumulating reports that revealed the association of FGF/FGFRs to various types of cancer at a genetic level, along with in vitro and in vivo evidences available so far, which indicates the functional involvement of FGF signalling in tumour formation and progression. An increasing number of drugs against the FGF pathways is currently in clinical testing. We will discuss the strategies for future FGF research in cancer and translational approaches.     

Neutrophil extracellular DNA traps promote pancreatic cancer cells migration and invasion by activating EGFR/ERK pathway

Neutrophil extracellular DNA traps (NETs) are newly discovered forms of activated neutrophils. Increasing researches have shown that NETs play important roles in cancer progression. Our previous study has proved that tumour-infiltrating NETs could predict postsurgical survival in patients with pancreatic ductal adenocarcinoma (PDAC). However, the roles of NETs on the progression of pancreatic cancer are unknown. Here, we investigated the effects of NETs on pancreatic cancer cells. Results showed that both PDAC patients’ and normal individuals’ neutrophils-derived NETs could promote migration and invasion of pancreatic cancer cells with epithelial-mesenchymal transition. Further, study confirmed that EGFR/ERK pathway played an important role in this progression. The addition of neutralizing antibodies for IL-1β could effectively block the activation of EGFR/ERK companied with reduction of EMT, migration and invasion. Taken together, NETs facilitated EMT, migration and invasion via IL-1β/EGFR/ERK pathway in pancreatic cancer cells. Our study suggests that NETs may provide promising therapeutic targets for pancreatic cancer.     

FGFR signalling in women's cancers

FGFs, in a complex with their receptors (FGFRs) and heparan sulfate (HS), are responsible for a range of cellular functions, from embryogenesis to metabolism. Both germ line and somatic FGFR mutations are known to play a role in a range of diseases, most notably craniosynestosis dysplasias, dwarfism and cancer. Because of the ability of FGFR signalling to induce cell proliferation, migration and survival, FGFRs are readily co-opted by cancer cells. Mutations in, and amplifications of, these receptors are found in a range of cancers with some of the most striking clinical findings relating to their contribution to pathogenesis and progression of female cancers. Here, we outline the molecular mechanisms of FGFR signalling and discuss the role of this pathway in women's cancers, focusing on breast, endometrial, ovarian and cervical carcinomas, and their associated preclinical and clinical data. We also address the rationale for therapeutic intervention and the need for FGFR-targeted therapy to selectively target cancer cells in view of the fundamental roles of FGF signalling in normal physiology.     

FGFR4 GLY388 isotype suppresses motility of MDA-MB-231 breast cancer cells by EDG-2 gene repression

Clinical investigations of an FGFR4 germline polymorphism, resulting in substitution of glycine by arginine at codon 388 (G388 to R388), have shown a correlation between FGFR4 R388 and aggressive disease progression in cancer patients. Here, we studied the differential effects of the two FGFR4 isotypes on cellular signalling and motility in the MDA-MB-231 human breast cancer cell model. cDNA array analysis showed the ability of FGFR4 G388 to suppress expression of specific genes involved in invasiveness and motility. Further investigations concentrating on cell signalling and motility revealed an abrogation of phosphatidylinositol-3-kinase-dependent LPA-induced Akt activation and cell migration due to downregulation of the LPA receptor Edg-2 in FGFR4 G388-expressing MDA-MB-231 cells. Moreover, FGFR4 G388 expression attenuated the invasivity of the breast cancer cell line and decreased small Rho GTPase activity. We conclude that FGFR4 G388 suppresses cell motility of invasive breast cancer cells by altering signalling pathways and the expression of genes that are required for metastasis. Therefore, the positive effect of FGFR4 R388 on disease progression appears to result from a loss of the tumour suppressor activity displayed by FGFR4 G388 rather than the acquisition or enhancement of oncogenic potential.     

Reactivation of Mitogen-activated Protein Kinase (MAPK) Pathway by FGF Receptor 3 (FGFR3)/Ras Mediates Resistance to Vemurafenib in Human B-RAF V600E Mutant Melanoma

Oncogenic B-RAF V600E mutation is found in 50% of melanomas and drives MEK/ERK pathway and cancer progression. Recently, a selective B-RAF inhibitor, vemurafenib (PLX4032), received clinical approval for treatment of melanoma with B-RAF V600E mutation. However, patients on vemurafenib eventually develop resistance to the drug and demonstrate tumor progression within an average of 7 months. Recent reports indicated that multiple complex and context-dependent mechanisms may confer resistance to B-RAF inhibition. In the study described herein, we generated B-RAF V600E melanoma cell lines of acquired-resistance to vemurafenib, and investigated the underlying mechanism(s) of resistance. Biochemical analysis revealed that MEK/ERK reactivation through Ras is the key resistance mechanism in these cells. Further analysis of total gene expression by microarray confirmed a significant increase of Ras and RTK gene signatures in the vemurafenib-resistant cells. Mechanistically, we found that the enhanced activation of fibroblast growth factor receptor 3 (FGFR3) is linked to Ras and MAPK activation, therefore conferring vemurafenib resistance. Pharmacological or genetic inhibition of the FGFR3/Ras axis restored the sensitivity of vemurafenib-resistant cells to vemurafenib. Additionally, activation of FGFR3 sufficiently reactivated Ras/MAPK signaling and conferred resistance to vemurafenib in the parental B-RAF V600E melanoma cells. Finally, we demonstrated that vemurafenib-resistant cells maintain their addiction to the MAPK pathway, and inhibition of MEK or pan-RAF activities is an effective therapeutic strategy to overcome acquired-resistance to vemurafenib. Together, we describe a novel FGFR3/Ras mediated mechanism for acquired-resistance to B-RAF inhibition. Our results have implications for the development of new therapeutic strategies to improve the outcome of patients with B-RAF V600E melanoma.     

TOP2A induces malignant character of pancreatic cancer through activating β-catenin signaling pathway

It has been reported that Topoisomerase II alpha (TOP2A) could induce tumor development and progression in many cancer types. Herein, through analysis of different independent cohorts, we found TOP2A was up-regulated in pancreatic cancer as compared with non-tumor tissues. Moreover, the up-regulation of TOP2A was significantly correlated with tumor metastasis and shorter survival in patients with pancreatic cancer. Knockdown of TOP2A in pancreatic cancer cell lines inhibited cell proliferation and migration. Furthermore, bioinformatics analysis revealed TOP2A activatesβ-catenin pathway in pancreatic cancer. Mechanistically, we demonstrated TOP2A acts as a co-activator ofβ-catenin and activates EMT process. Further investigation showed TOP2A was a direct target of mir-139, which was validated by dual-luciferase reporter gene assay. The effects of mir-139 on pancreatic cancer were also mechanistically, functionally and clinically investigated. Taken together, our research identified a novel miR-139\TOP2A\β-catenin axis driving the malignant progression of pancreatic cancer.     

A super-enhancer controls TGF- β signaling in pancreatic cancer through downregulation of TGFBR2

Pancreatic cancer is one of the most lethal malignant tumors due to a late diagnosis and highly invasion and metastasis. Transforming growth factor-β (TGF-β) signaling plays a vital role in the progression of pancreatic cancer. The delicate activity of TGF-β signaling is particular important for the development of aggression and metastasis which must be fine-tuned. Here, we investigated the role of super-enhancers in regulating the expression of TGF-β signaling pathway in pancreatic cancer. TGFBR2 owns the modification of H3K27Ac around the gene in pancreatic cancer cells. Inhibition of BRD4 by JQ1 robustly blocked the expression of TGFBR2 in a dose dependent manner. We successfully mapped a super-enhancer in TGFBR2 by sgRNA. Deletion of the super-enhancer in TGFBR2 (sgTGFBR2-SEΔ) significantly reduced the expression of TGFBR2 in pancreatic cancer cells. TGF-β-induced p-SMAD2/3 was greatly impaired in TGFBR2 super-enhancer deleted cells. Both migration and EMT induced by TGF-β in pancreatic cancer cells were impaired after deleting the super-enhancer of TGFBR2. Our data suggest a novel molecular mechanism by which a super-enhancer regulates TGFBR2, affecting the activity of TGF-β as well as its function in pancreatic cancer progression.     

Aberrant hypermethylation of the FGFR2 gene in human gastric cancer cell lines

We have previously shown that fibroblast growth factor receptor 2 (FGFR2) plays an important role in gastric carcinogenesis. In this study, we assessed DNA methylation status in the promoter region of FGFR2 gene in gastric cancer cell lines, and indicated that this region was highly methylated, compared with FGFR2-expressing gastric cancer cell lines. Moreover, the restoration of FGFR2 expression by treating methylated cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine strongly suggests that the loss of FGFR2 expression may be due to the aberrant hypermethylation in the promoter region of the FGFR2 gene. Thus, our results suggest that the epigenetic silencing of FGFR2 through DNA methylation in gastric cancer may contribute to tumor progression.     

Tumor‐suppressor Fbxw7 targets SIK2 for degradation to interfere with TORC2‐AKT signaling in pancreatic cancer

The tumor suppressor F‐box/WD repeat‐containing protein 7 (Fbxw7) is a substrate‐recognition subunit of a ubiquitin ligase complex. We have previously proposed that Fbxw7 inhibited pancreatic cancer cell proliferation and invasion by targeting β‐catenin. To identify other targets of Fbxw7 involved in pancreatic carcinogenesis, we screened the human protein database for Fbxw7 target candidates using the conserved Fbxw7‐recognizing sequences. Twenty‐three candidates are identified, including five known Fbxw7 targets and two cancer‐related genes (salt inducible kinase 2 [SIK2] and ZMIZ1). We identified SIK2 as an Fbxw7 target for degradation by binding to the “TPPPS” motif of SIK2 in pancreatic cancer cells. We also demonstrated that SIK2 promoted proliferation and mitotic progression of pancreatic cancer cells. Moreover, endogenous Fbxw7 downregulates SIK2 protein level for controlling cell cycle progression, possibly by interfering the SIK2/TORC2/AKT signaling pathway to modulate p21 expression. Collectively, these data demonstrate that Fbxw7 targets the cell cycle controller, SIK2, for degradation, thereby leading to the disruption of downstream TORC2/AKT signaling to inhibit pancreatic cancer cell proliferation and cell cycle progression.     

DKK3 blocked translocation of β‐catenin/EMT induced by hypoxia and improved gemcitabine therapeutic effect in pancreatic cancer Bxpc‐3 cell

The Wnt/β‐catenin signalling pathway is activated in pancreatic cancer initiation and progression. Dickkopf‐related protein 3 (DKK3) is a member of the human Dickkopf family and an antagonist of Wnt ligand activity. However, the function of DKK3 in this pathway in pancreatic cancer is rarely known. We examined the expression of DKK3 in six human pancreatic cancer cell lines, 75 pancreatic cancer and 75 adjacent non‐cancerous tissues. Dickkopf‐related protein 3 was frequently silenced and methylation in pancreatic cancer cell lines (3/6). The expression of DKK3 was significantly lower in pancreatic cancer tissues than in adjacent normal pancreas tissues. Further, ectopic expression of DKK3 inhibits nuclear translocation of β‐catenin induced by hypoxia in pancreatic cancer Bxpc‐3 cell. The forced expression of DKK3 markedly suppressed migration and the stem cell‐like phenotype of pancreatic cancer Bxpc‐3 cell in hypoxic conditions through reversing epithelial–mesenchymal transition (EMT). The stable expression of DKK3 sensitizes pancreatic cancer Bxpc‐3 cell to gemcitabine, delays tumour growth and augments gemcitabine therapeutic effect in pancreatic cancer xenotransplantation model. Thus, we conclude from our finding that DKK3 is a tumour suppressor and improved gemcitabine therapeutic effect through inducing apoptosis and regulating β‐catenin/EMT signalling in pancreatic cancer Bxpc‐3 cell.     

VRK2 activates TNFα/NF-κB signaling by phosphorylating IKKβ in pancreatic cancer

NF-κB signaling is active in more than 50% of patients with pancreatic cancer and plays an important role in promoting the progression of pancreatic cancer. Revealing the activation mechanism of NF-κB signaling is important for the treatment of pancreatic cancer. In this study, the regulation of TNFα/NF-κB signaling by VRK2 (vaccinia-related kinase 2) was investigated. The levels of VRK2 protein were examined by immunohistochemistry (IHC). The functions of VRK2 in the progression of pancreatic cancer were examined using CCK8 assay, anchorage-independent assay, EdU assay and tumorigenesis assay. The regulation of VRK2 on the NF-κB signaling was investigated by immunoprecipitation and invitro kinase assay. It was discovered in this study that the expression of VRK2 was upregulated in pancreatic cancer and that the VRK2 expression level was significantly correlated with the pathological characteristics and the survival time of patients. VRK2 promoted the growth, sphere formation and subcutaneous tumorigenesis of pancreatic carcinoma cells as well as the organoid growth derived from the pancreatic cancer mouse model. Investigation of the molecular mechanism indicated that VRK2 interacts with IKKβ, phosphorylating its Ser177 and Ser181 residues and thus activating the TNFα/NF-κB signaling pathway. An IKKβ inhibitors abolished the promotive effect of VRK2 on the growth of organoids. The findings of this study indicate that VRK2 promotes the progression of pancreatic cancer by activating the TNFα/NF-κB signaling pathway, suggesting that VRK2 is a potential therapeutic target for pancreatic cancer.     

FGFR2 upregulates PAI-1 via JAK2/STAT3 signaling to induce M2 polarization of macrophages in colorectal cancer

Fibroblast growth factor receptor 2 (FGFR2) is frequently activated by overexpression or mutation, and an abnormal fibroblast growth factor (FGF)/FGFR signaling pathway is associated with the occurrence, development, and poor prognosis of colorectal cancer (CRC). Our preliminary analysis found that plasminogen activator inhibitor-1 (PAI-1) expression may be related to FGF/FGFR signaling, however, their role in the tumor immune microenvironment remains unclear. In this study, we observed markedly higher PAI-1 expression in CRC patients with poor survival rates. PAI-1 is regulated by FGF/FGFR2 in colon cancer cells and is involved in M2 macrophage polarization. Mechanistically, inhibiting the JAK2/STAT3 signaling pathway could cause PAI-1 downregulation. Furthermore, the activation of phosphorylated STAT3 upregulated PAI-1. In vivo, FGFR2 overexpression in tumor-bearing mouse models suggested that a PAI-1 inhibitor could rescue FGFR2/PAI-1 axis-induced M2 macrophage polarization, which leads to effective immune activity and tumor suppression. Moreover, the combination of a PAI-1 inhibitor and anti-PD-1 therapy exhibited superior antitumor activity in mice. These findings offer novel insights into the molecular mechanisms underlying tumor deterioration and provide potential therapeutic targets for CRC treatment.