Fibroblast growth factor receptor expression reflects cellular differentiation in human oral squamous carcinoma cell lines

This study examined the expression of fibroblast growth factor receptor 2 (FGFR 2) splice variants, IIIb and IIIc, in normal and malignant human oral keratinocytes and in normal oral fibroblasts by RT-PCR using both exon-specific primers and primers common to both FGFR 2 isoforms. Fibroblasts expressed exclusively FGFR 2/IIIc whilst the normal and malignant keratinocytes co-expressed FGFR 2/IIIb and FGFR 2/IIIc. Well- differentiated keratinocytes expressed proportionally more FGFR 2/IIIb than IIIc whereas the poorly-differentiated cells expressed more FGFR 2/IIIc than IIIb. The normal and malignant keratinocytes, but not fibroblasts, expressed an additional amplification product, which consisted of both IIIb and IIIc of FGFR 2 joined by an extra base pair and with the intronic sequence removed. The results indicate that the expression of FGFR 2 isoforms reflects the degree of cellular differentiation in normal and malignant human oral keratinocytes and that receptor complexes of FGFR 2/IIIb and IIIc may regulate ligand- receptor interactions.      

Nuclear expression of Y‐box binding protein‐1 is associated with poor prognosis in patients with pancreatic cancer and its knockdown inhibits tumor growth and metastasis in mice tumor models

The objective of this study was to examine the implication of Y‐box‐binding protein‐1 (YB‐1) for the aggressive phenotypes, prognosis and therapeutic target in pancreatic ductal adenocarcinoma (PDAC). YB‐1 expression in PDAC, pancreatic intraepithelial neoplasia (PanIN) and normal pancreas specimens was evaluated by immunohistochemistry, and its correlation with clinicopathological features was assessed in patients with PDAC. The effects of YB‐1 on proliferation, invasion and expressions of cell cycle‐related proteins and matrix metalloproteinases (MMPs) were analyzed by WST‐8, cell cycle and Matrigel invasion assays, Western blotting and quantitative RT‐PCR in PDAC cells transfected with YB‐1‐siRNAs. To verify the significance of YB‐1 for tumor progression in vivo, the growth and metastasis were monitored after intrasplenic implantation of ex vivo YB‐1 siRNA‐transfected PDAC cells, and YB‐1‐targeting antisense oligonucleotides were intravenously administered in nude mice harboring subcutaneous tumor. The intensity of YB‐1 expression and positivity of nuclear YB‐1 expression were higher in PDAC than PanIN and normal pancreatic tissues. Nuclear YB‐1 expression was significantly associated with dedifferentiation, lymphatic/venous invasion and unfavorable prognosis. YB‐1 knockdown inhibited cell proliferation via cell cycle arrest by S‐phase kinase‐associated protein 2 downregulation and consequent p27 accumulation, and decreased the invasion due to downregulated membranous‐type 2 MMP expression in PDAC cells. Tumor growth and liver metastasis formation were significantly suppressed in nude mice after implantation of YB‐1‐silenced PDAC cells, and the YB‐1 targeting antisense oligonucleotide significantly inhibited the growth of subcutaneous tumors. In conclusion, YB‐1 may be involved in aggressive natures of PDAC and a promising therapeutic target.     

Silencing circular RNA UVRAG inhibits bladder cancer growth and metastasis by targeting the microRNA‐223/fibroblast growth factor receptor 2 axis

Circular RNA UVRAG (circUVRAG), a type of non‐coding RNA, is derived and cyclized by part of the exon from the UVRAG gene. However, the role of circUVRAG in bladder cancer (BLCA) has not been reported. The purpose of the present study was therefore to characterize the role of circUVRAG in BLCA. Bioinformatics analysis showed interactive relationships among circUVRAG, microRNA‐223 (miR‐223), and fibroblast growth factor receptor 2 (FGFR2). Quantitative real‐time PCR was used to detect the expression of circUVRAG in BLCA cell lines. UM‐UC‐3 cells were stably transfected with siRNA against circUVRAG, and cell proliferation and migration ability were tested using the CCK8 assay, clone formation, and Transwell assays in vitro. Tumor xenograft formation and metastasis were determined using nude mice. Fluorescence in situ hybridization was used to confirm the subcellular localization of circUVRAG, and the luciferase reporter assay was used to confirm the relationships among circUVRAG, miR‐223, and FGFR2. Results showed that circUVRAG was upregulated in BLCA cell lines. Downregulation of circUVRAG expression suppressed proliferation and metastasis both in vitro and in vivo. Downregulation of circUVRAG suppressed FGFR2 expression by “sponging” miR‐223, which was confirmed by rescue experiments and luciferase reporter assay. Overall, the results showed that downregulation of circUVRAG suppressed the aggressive biological phenotype of BLCA. Taken together, silencing circular RNA UVRAG inhibited bladder cancer growth and metastasis by targeting the miR‐223/FGFR2 axis, which may provide a potential biomarker and therapeutic target for the management of BLCA.     

Nestin is a novel target for suppressing pancreatic cancer cell migration, invasion and metastasis

Nestin, is a class VI intermediate filament (IF) that is expressed in 30% of pancreatic ductal adenocarcinoma (PDAC) cases, and its expression in PDAC positively correlates with peripancreatic invasion. An expression vector carrying a short hairpin RNA (shRNA) targeting nestin was stably transfected into PANC-1 and PK-45H human pancreatic cancer cells, which express high nestin levels. Alterations in morphology and alignment of actin filaments and α-tubulin were examined by phase-contrast and immunocytochemistry. Effects on cell growth, migration in scratch and Boyden chamber assays, invasion, cell adhesion, and in vivo growth were determined. Differences in mRNA levels were examined by arrays. Nestin shRNA-transfected cells exhibited decreased nestin expression, a sheet-like appearance with tight cell-cell adhesion, increased expression of filamentous F-actin and E-cadherin, and attenuated migration and invasion, both of which were enhanced following nestin re-expression. Expression of α-tubulin, and in vitro cell growth and adhesion were not altered by nestin down-regulation, whereas hepatic metastases were decreased. Thus, nestin plays important roles in pancreatic cancer cell migration, invasion and metastasis by selectively modulating the expression of actin and cell adhesion molecules, and may therefore be a novel therapeutic target in PDAC.     

Establishment and characterization of CRISPR/Cas9‐mediated NF2−/− human mesothelial cell line: Molecular insight into fibroblast growth factor receptor 2 in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM), a highly refractory tumor, is currently incurable due to the lack of an early diagnosis method and medication, both of which are urgently needed to improve the survival and/or quality of life of patients. NF2 is a tumor suppressor gene and is frequently mutated in MPM. Using a CRISPR/Cas9 system, we generated an NF2‐knockout human mesothelial cell line, MeT‐5A (NF2‐KO). In NF2‐KO cell clones, cell growth, clonogenic activity, migration activity, and invasion activity significantly increased compared with those in NF2‐WT cell clones. Complementary DNA microarray analysis clearly revealed the differences in global gene expression profile between NF2‐WT and NF2‐KO cell clones. Quantitative PCR analysis and western blot analysis showed that the upregulation of fibroblast growth factor receptor 2 (FGFR2) was concomitant with the increases in phosphorylation levels of JNK, c‐Jun, and retinoblastoma (Rb) in NF2‐KO cell clones. These increases were all abrogated by the exogenous expression of NF2 in the NF2‐KO clone. In addition, the disruption of FGFR2 in the NF2‐KO cell clone suppressed cell proliferation as well as the phosphorylation levels of JNK, c‐Jun, and Rb. Notably, FGFR2 was found to be highly expressed in NF2‐negative human mesothelioma tissues (11/12 cases, 91.7%) but less expressed in NF2‐positive tissues. Collectively, these findings suggest that NF2 deficiency might play a role in the tumorigenesis of human mesothelium through mediating FGFR2 expression; FGFR2 would be a candidate molecule to develop therapeutic and diagnostic strategies for targeting MPM with NF2 loss.     

Inhibition of endogenous SPARC enhances pancreatic cancer cell growth: modulation by FGFR1-III isoform expression

Background:

Secreted protein acidic and rich in cysteine (SPARC) is a multi-faceted protein-modulating cell–cell and cell–matrix interactions. In cancer, SPARC can be not only associated with a highly aggressive phenotype, but also acts as a tumour suppressor. The aim of this study was to characterise the function of SPARC and its modulation by fibroblast growth factor receptor (FGFR) 1 isoforms in pancreatic ductal adenocarcinoma (PDAC).

Methods and results:

Exogenous SPARC inhibited growth, movement, and migration. ShRNA inhibition of endogenous SPARC in ASPC-1 and PANC-1 cells resulted in increased anchorage-dependent and -independent growth, transwell migration, and xenograft growth as well as increased mitogenic efficacy of fibroblast growth factor (FGF) 1 and FGF2. Endogenous SPARC expression in PANC-1 cells was increased in FGFR1-IIIb over-expressing cells, but decreased in FGFR1-IIIc over-expressing cells. The up-regulation of endogenous SPARC was abrogated by the p38-mitogen-activated protein kinase inhibitor SB203580. SPARC was detectable in conditioned medium of pancreatic stellate cells (PSCs), but not PDAC cells. Conditioned medium of PDAC cells reduced endogenous SPARC expression of PSCs.

Conclusion:

Endogenous SPARC inhibits the malignant phenotype of PDAC cells and may, therefore, act as a tumour suppressor in PDAC. Endogenous SPARC expression can be modulated by FGFR1-III isoform expression. In addition, PDAC cells may inhibit endogenous SPARC expression in surrounding PSCs by paracrine actions.     

The novel driver gene ASAP2 is a potential druggable target in pancreatic cancer

Targeting mutated oncogenes is an effective approach for treating cancer. The 4 main driver genes of pancreatic ductal adenocarcinoma (PDAC) are KRAS, TP53, CDKN2A, and SMAD4, collectively called the “big 4” of PDAC, however they remain challenging therapeutic targets. In this study, ArfGAP with SH3 domain, ankyrin repeat and PH domain 2 (ASAP2), one of the ArfGAP family, was identified as a novel driver gene in PDAC. Clinical analysis with PDAC datasets showed that ASAP2 was overexpressed in PDAC cells based on increased DNA copy numbers, and high ASAP2 expression contributed to a poor prognosis in PDAC. The biological roles of ASAP2 were investigated using ASAP2-knockout PDAC cells generated with CRISPR-Cas9 technology or transfected PDAC cells. In vitro and in vivo analyses showed that ASAP2 promoted tumor growth by facilitating cell cycle progression through phosphorylation of epidermal growth factor receptor (EGFR). A repositioned drug targeting the ASAP2 pathway was identified using a bioinformatics approach. The gene perturbation correlation method showed that niclosamide, an antiparasitic drug, suppressed PDAC growth by inhibition of ASAP2 expression. These data show that ASAP2 is a novel druggable driver gene that activates the EGFR signaling pathway. Furthermore, niclosamide was identified as a repositioned therapeutic agent for PDAC possibly targeting ASAP2.     

Ras inhibits endoplasmic reticulum stress in human cancer cells with amplified Myc

In neuroblastoma LAN‐1 cells harboring an amplified MycN gene, disruption of cooperation between Ras and MycN proteins by the Ras inhibitor farnesylthiosalicylic acid (FTS, Salirasib) reportedly arrests cell growth. Our aim was to establish whether this is a general phenomenon. We examined the effects of FTS on gene‐expression profiles, growth and death of NCIH929 myeloma cells and K562 leukemia cells, which—like LAN‐1 cells—exhibit Myc gene amplification and harbor active Ras. Under specified conditions, FTS reduced Ras and Myc and induced cell growth arrest and death in all Myc‐amplified cell lines but not in SHEP, a neuroblastoma cell line without Myc gene amplification. Gene‐expression analysis revealed a common pattern of FTS‐induced endoplasmic reticulum (ER) stress, known as the unfolded protein response (UPR), in Myc‐amplified cells, but not in SHEP. Thus, Ras negatively regulates ER stress in cells with amplified Myc. ER stress was also inducible by dominant‐negative (DN)‐Ras or shRNA to Ras isoforms, all of which induced an increase in BIP (the master regulator of ER stress) and its downstream targets Nrf2 and eIF2α, both regulated by active p‐PERK. FTS also induced an increase in p‐PERK, while small interfering RNA to PERK reduced Nrf2 and ATF4 and rescued cells from FTS‐induced death. BIP and its downstream targets were also increased by inhibitors of MAPK p38 and MEK. Ras, acting through MAPK p38 and MEK, negatively regulates the ER stress cascades BIP/PERK/Nrf2 and eIF2α/ATF4/ATF3. These findings can explain the Ras‐dependent protection of Myc‐amplified cells from ER stress‐associated death.     

ZFP36L2 promotes cancer cell aggressiveness and is regulated by antitumor microRNA‐375 in pancreatic ductal adenocarcinoma

Due to its aggressive nature, pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal and hard‐to‐treat malignancies. Recently developed targeted molecular strategies have contributed to remarkable improvements in the treatment of several cancers. However, such therapies have not been applied to PDAC. Therefore, new treatment options are needed for PDAC based on current genomic approaches. Expression of microRNA‐375 (miR‐375) was significantly reduced in miRNA expression signatures of several types of cancers, including PDAC. The aim of the present study was to investigate the functional roles of miR‐375 in PDAC cells and to identify miR‐375‐regulated molecular networks involved in PDAC aggressiveness. The expression levels of miR‐375 were markedly downregulated in PDAC clinical specimens and cell lines (PANC‐1 and SW1990). Ectopic expression of miR‐375 significantly suppressed cancer cell proliferation, migration and invasion. Our in silico and gene expression analyses and luciferase reporter assay showed that zinc finger protein 36 ring finger protein‐like 2 (ZFP36L2) was a direct target of miR‐375 in PDAC cells. Silencing ZFP36L2 inhibited cancer cell aggressiveness in PDAC cell lines, and overexpression of ZFP36L2 was confirmed in PDAC clinical specimens. Interestingly, Kaplan–Meier survival curves showed that high expression of ZFP36L2 predicted shorter survival in patients with PDAC. Moreover, we investigated the downstream molecular networks of the miR‐375/ZFP36L2 axis in PDAC cells. Elucidation of tumor‐suppressive miR‐375‐mediated PDAC molecular networks may provide new insights into the potential mechanisms of PDAC pathogenesis.     

Enhanced FGFR signalling predisposes pancreatic cancer to the effect of a potent FGFR inhibitor in preclinical models

Background:

Fibroblast growth factor receptor (FGFR) signalling has been implicated in pancreas carcinogenesis. We investigated the effect of FGFR inhibition in pancreatic cancer in complementary cancer models derived from cell lines and patient-derived primary tumour explants.

Methods:

The effects of FGFR signalling inhibition in pancreatic cancer were evaluated using anti-FRS2 shRNA and dovitinib. Pancreatic cancers with varying sensitivity to dovitinib were evaluated to determine potential predictive biomarkers of efficacy. Primary pancreatic explants with opposite extreme of biomarker expression were selected from 13 tumours for in vivo dovitinib treatment.

Results:

Treatment with anti-FRS2 shRNA induced significant in vitro cell kill in pancreatic cancer cells. Dovitinib treatment achieved similar effects and was mediated by Akt/Mcl-1 signalling in sensitive cells. Dovitinib efficacy correlated with FRS2 phosphorylation status, FGFR2 mRNA level and FGFR2 IIIb expression but not phosphorylation status of VEGFR2 and PDGFRβ. Using FGFR2 mRNA level, a proof-of-concept study using primary pancreatic cancer explants correctly identified the tumours'' sensitivity to dovitinib.

Conclusion:

Inhibiting FGFR signalling using shRNA and dovitinib achieved significant anti-cancer cancer effects in pancreatic cancer. The effect was more pronounced in FGFR2 IIIb overexpressing pancreatic cancer that may be dependent on aberrant stimulation by stromal-derived FGF ligands.     

Circulating extracellular vesicle‐encapsulated HULC is a potential biomarker for human pancreatic cancer

The role of long noncoding RNAs (lncRNAs) in the epithelial‐mesenchymal transition (EMT) in pancreatic ductal adenocarcinoma (PDAC) is unclear. Some lncRNAs can be transferred by extracellular vesicles (EVs) and have potential as biomarkers. Here, we identify an lncRNA that could serve as a biomarker for PDAC and show the functional roles of the lncRNA. Expression profiling of lncRNAs revealed that highly upregulated in liver cancer (HULC) was highly expressed, and induced, by transforming growth factor‐β in PDAC cells and their EVs. Knockdown of HULC decreased PDAC cell invasion and migration by inhibiting the EMT. Thus, HULC could be transferred by EVs, and promote EMT, invasion, and migration in recipient PDAC cells. To assess the roles of HULC, PDAC cell xenografts in nude mice were established. Knockdown of HULC in PDAC cells implanted in mice inhibited tumor growth. Moreover, microRNA‐133b suppressed PDAC cell invasion and migration by inhibiting the EMT through targeting HULC. Furthermore, serum samples were obtained from 20 PDAC and 22 intraductal papillary mucinous neoplasm (IPMN) patients, as well as 21 healthy individuals. Analysis of serum EV HULC expression by digital PCR showed that HULC expression was significantly increased in PDAC patients compared to healthy individuals or IPMN patients. Additionally, HULC showed good predictive performance for discriminating PDAC, suggesting that the analysis of EV‐encapsulated HULC would contribute to the diagnosis for human PDAC. Extracellular vesicle‐transported HULC promotes cell invasion and migration by inducing the EMT, and microRNA‐133b suppresses the EMT by targeting HULC. Extracellular vesicle‐encapsulated HULC could be a potential circulating biomarker for human PDAC.     

Molecular subclasses of hepatocellular carcinoma predict sensitivity to fibroblast growth factor receptor inhibition

A recent gene expression classification of hepatocellular carcinoma (HCC) includes a poor survival subclass termed S2 representing about one‐third of all HCC in clinical series. S2 cells express E‐cadherin and c‐myc and secrete AFP. As the expression of fibroblast growth factor receptors (FGFRs) differs between S2 and non‐S2 HCC, this study investigated whether molecular subclasses of HCC predict sensitivity to FGFR inhibition. S2 cell lines were significantly more sensitive (p < 0.001) to the FGFR inhibitors BGJ398 and AZD4547. BGJ398 decreased MAPK signaling in S2 but not in non‐S2 cell lines. All cell lines expressed FGFR1 and FGFR2, but only S2 cell lines expressed FGFR3 and FGFR4. FGFR4 siRNA decreased proliferation by 44% or more in all five S2 cell lines (p < 0.05 for each cell line), a significantly greater decrease than seen with knockdown of FGFR1‐3 with siRNA transfection. FGFR4 knockdown decreased MAPK signaling in S2 cell lines, but little effect was seen with knockdown of FGFR1‐3. In conclusion, the S2 molecular subclass of HCC is sensitive to FGFR inhibition. FGFR4‐MAPK signaling plays an important role in driving proliferation of a molecular subclass of HCC. This classification system may help to identify those patients who are most likely to benefit from inhibition of this pathway.     

Directionally specific paracrine communication mediated by epithelial FGF9 to stromal FGFR3 in two-compartment premalignant prostate tumors

Tissue homeostasis in normal prostate and two-compartment nonmalignant prostate tumors depends on harmonious two-way communications between epithelial and stromal compartments. Within the fibroblast growth factor (FGF) family, signaling to an epithelial cell-specific FGF receptor (FGFR) 2IIIb-heparan sulfate complex from stromal-specific FGF7 and FGF10 delivers directionally specific instruction from stroma to epithelium without autocrine interference. Using a two-compartment transplantable prostate tumor model in which survival of stromal cells in vivo depends on epithelial cells, we show that signaling from epithelial FGF9 to stromal FGFR3 potentially mediates epithelial-to-stromal communication that also is directionally specific. FGF9 mRNA was expressed exclusively in the epithelial cells derived from well-differentiated, two-compartment Dunning R3327 rat prostate tumors. In contrast, FGFR3 was expressed at functionally significant levels only in the derived stromal cells. Competition binding and immunoprecipitation assays revealed that FGF9 only bound to an FGFR on the stromal cells. FGF9 also failed to covalently cross-link to clonal lines of stromal cells devoid of FGFR3 that expressed FGFR1 and FGFR2IIIc. Furthermore, FGF9 specifically stimulated DNA synthesis in stromal cells expressing FGFR3. These results demonstrate a directionally specific paracrine signaling from epithelial FGF9 and stromal FGFR3. Similar to the FGF7/FGF10 to FGFR2IIIb signaling from the stroma to the epithelium, the directional specificity from epithelium to stroma appears set by a combination of cell-specific expression of isoforms and cell-context specificity of FGFR isotypes for FGF.