Role of FGF Receptors and Their Pathways in Adrenocortical Tumors and Possible Therapeutic Implications.

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy and treatment of advanced disease is challenging. Clinical trials with multi-tyrosine kinase inhibitors in the past have yielded disappointing results. Here, we investigated fibroblast growth factor (FGF) receptors and their pathways in adrenocortical tumors as potential treatment targets. We performed real-time RT-PCR of 93 FGF pathway related genes in a cohort of 39 fresh frozen benign and malignant adrenocortical, 9 non-adrenal tissues and 4 cell lines. The expression of FGF receptors was validated in 166 formalin-fixed paraffin embedded (FFPE) tissues using RNA in situ hybridization (RNAscope) and correlated with clinical data. In malignant compared to benign adrenal tumors, we found significant differences in the expression of 16/94 FGF receptor pathway related genes. Genes involved in tissue differentiation and metastatic spread through epithelial to mesechymal transition were most strongly altered. The therapeutically targetable FGF receptors 1 and 4 were upregulated 4.6- and 6-fold, respectively, in malignant compared to benign adrenocortical tumors, which was confirmed by RNAscope in FFPE samples. High expression of FGFR1 and 4 was significantly associated with worse patient prognosis in univariate analysis. After multivariate adjustment for the known prognostic factors Ki-67 and ENSAT tumor stage, FGFR1 remained significantly associated with recurrence-free survival (HR=6.10, 95%CI: 1.78 - 20.86, p=0.004) and FGFR4 with overall survival (HR=3.23, 95%CI: 1.52 - 6.88, p=0.002). Collectively, our study supports a role of FGF pathways in malignant adrenocortical tumors. Quantification of FGF receptors may enable a stratification of ACC for the use of FGFR inhibitors in future clinical trials.

Expression of FGF8, FGF18, and FGFR4 in Gastroesophageal Adenocarcinomas.

Even though distinctive advances in the field of esophageal cancer therapy have occurred over the last few years, patients' survival rates remain poor. FGF8, FGF18, and FGFR4 have been identified as promising biomarkers in a number of cancers; however no data exist on expression of FGF8, FGF18, and FGFR4 in adenocarcinomas of the esophago-gastric junction (AEG). A preliminary analysis of the Cancer Genome Atlas (TCGA) database on FGF8, FGF18, and FGFR4 mRNA expression data of patients with AEG was performed. Furthermore, protein levels of FGF8, FGF18, and FGFR4 in diagnostic biopsies and post-operative specimens in neoadjuvantly treated and primarily resected patients using immunohistochemistry were investigated. A total of 242 patients was analyzed in this study: 87 patients were investigated in the TCGA data set analysis and 155 patients in the analysis of protein expression using immunohistochemistry. High protein levels of FGF8, FGF18, and FGFR4 were detected in 94 (60.7%), 49 (31.6%) and 84 (54.2%) patients, respectively. Multivariable Cox proportional hazard regression models revealed that high expression of FGF8 was an independent prognostic factor for diminished overall survival for all patients and for neoadjuvantly treated patients. By contrast, FGF18 overexpression was significantly associated with longer survival rates in neoadjuvantly treated patients. In addition, FGF8 protein level correlated with Mandard regression due to neoadjuvant therapy, indicating potential as a predictive marker. In summary, FGF8 and FGF18 are promising candidates for prognostic factors in adenocarcinomas of the esophago-gastric junction and new potential targets for new anti-cancer therapies.

Forkhead box C1 promotes colorectal cancer metastasis through transactivating ITGA7 and FGFR4 expression.

Metastatic colorectal cancer (CRC) is one of the most common causes of cancer death worldwide; however, the molecular mechanism underlying CRC metastasis remains unknown. Using an integrated approach, we identified forkhead box C1 (FOXC1) as a novel regulator of CRC metastasis. Elevated expression of FOXC1 is significantly correlated with metastasis, recurrence and reduced survival. FOXC1 overexpression promotes CRC invasion and lung metastasis, whereas FOXC1 knockdown has the opposite effect. In addition, FOXC1 directly binds its target genes integrin α7 (ITGA7) and fibroblast growth factor receptor 4 (FGFR4) and activates their expression. Genetic epistasis analysis confirmed that ITGA7 and FGFR4 act downstream of FOXC1. Furthermore, pharmaceutical inhibition of FGFR4 can reverse CRC metastasis mediated by FOXC1 overexpression. These results suggest that FOXC1 is a prognostic biomarker in CRC patients and targeting the FGFR4 signaling pathway may provide a promising strategy for the treatment of FOXC1-driven CRC metastasis.

Cancer-associated fibroblasts secrete FGF-1 to promote ovarian proliferation, migration, and invasion through the activation of FGF-1/FGFR4 signaling.

Ovarian cancer is the most lethal gynecologic malignancy, due to its high propensity for metastasis. Cancer-associated fibroblasts, as the dominant component of tumor microenvironment, are crucial for tumor progression. However, the mechanisms underlying the regulation of ovarian cancer cells by cancer-associated fibroblasts remain little known. Here, we first isolated cancer-associated fibroblasts from patients' ovarian tissues and found that cancer-associated fibroblasts promoted SKOV3 cells' proliferation, migration, and invasion. Fibroblast growth factor-1 was identified as a highly increased factor in cancer-associated fibroblasts compared with normal fibroblasts by quantitative reverse transcription polymerase chain reaction (~4.6-fold, p < 0.01) and ELISA assays (~4-fold, p < 0.01). High expression of fibroblast growth factor-1 in cancer-associated fibroblasts either naturally or through gene recombination led to phosphorylation of fibroblast growth factor receptor 4 in SKOV3 cells, which is followed by the activation of mitogen-activated protein kinase/extracellular signal-regulated protein kinase pathway and epithelial-to-mesenchymal transition-associated gene Snail1 and MMP3 expression. Moreover, treatment of SKOV3 cell with fibroblast growth factor receptor inhibitor PD173074 terminated cellular proliferation, migration, and invasion, reduced the phosphorylation level of fibroblast growth factor receptor 4, and suppressed the activation of mitogen-activated protein kinase/extracellular signal-regulated protein kinase pathway. In addition, the expression level of Snail1 and MMP3 was reduced, while the expression level of E-cadherin increased. These observations suggest a crucial role for cancer-associated fibroblasts and fibroblast growth factor-1/fibroblast growth factor receptor 4 signaling in the progression of ovarian cancer. Therefore, this fibroblast growth factor-1/fibroblast growth factor receptor 4 axis may become a potential target for the treatment of ovarian cancer.

Multiplexed gene expression profiling identifies the FGFR4 pathway as a novel biomarker in intrahepatic cholangiocarcinoma.

BACKGROUND: The fibroblast growth factor receptor 4 (FGFR4) pathway is an essential regulatory component of bile acid synthesis, and its relationship with hepatocellular carcinoma (HCC) has been reported. We investigated the gene expression and clinical significance of FGFR4 and related pathways in intrahepatic cholangiocarcinoma (iCCA). RESULTS: The median age was 56 years (range 30-78) and 34 patients (74%) were male. Six patients (13%) had hepatitis B virus infection, with or without liver cirrhosis. Overall survival was significantly associated with FGFR4 (p = 0.004), FGF19 (p = 0.047), FGF21 (p = 0.04), and KLB (p = 0.03) expression. In the multivariate analysis with potential prognostic factors, high expression of FGF19, FGF21, and FGFR4 was significantly associated with better survival. In the analysis using the TCGA iCCA dataset, mRNA overexpression of at least 1 of the FGFR4-related genes was significantly associated with better disease-free survival (p = 0.02). MATERIALS AND METHODS: We assessed the expression of 98 genes in formalin-fixed paraffin embedded tumor tissue specimens from 46 patients with surgically resected iCCA using a NanoString platform. This included 10 FGF pathway genes (e.g. FGFR1-4, KLB, FGF3, 4, 19, 21, and 23), 19 distal marker genes (e.g. CYP7A1 and CYP17A1), 31 genes relevant to HCC and iCCA (e.g. AFP, TS), 18 copy number variation matched genes, and 20 control genes. Log-transformation of gene expression was performed for normalization and statistical analysis. Overall survival was correlated with gene expression (< median vs. ≥ median) using a log-rank test. The prognostic impact of FGFR4-related genes was validated using the public TCGA dataset for iCCA. CONCLUSIONS: Our results indicate that mRNA expression of FGFR4-related genes may be a biomarker to define the distinctive molecular phenotype of iCCA. Future preclinical and clinical validation is required to define the role of the FGFR4 pathway in iCCA.

Prognostic roles for fibroblast growth factor receptor family members in malignant peripheral nerve sheath tumor.

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNST) are rare, highly malignant, and poorly understood sarcomas. The often poor outcome of MPNST highlights the necessity of identifying prognostic predictors for this aggressive sarcoma. Here, we investigate the role of fibroblast growth factor receptor (FGFR) family members in human MPNSTs. RESULTS: aCGH and bioinformatics analysis identified frequent amplification of the FGFR1 gene. FISH analysis revealed that 26.9% MPNST samples had amplification of FGFR1, with both focal and polysomy patterns observed. IHC identified that FGFR1 protein expression was positively correlated with FGFR1 gene amplification. High expression of FGFR1 protein was associated with better overall survival (OS) and was an independent prognostic predictor for OS of MPNST patients. Additionally, combined expression of FGFR1 and FGFR2 protein characterized a subtype of MPNST with better OS. FGFR4 protein was expressed 82.3% of MPNST samples, and was associated with poor disease-free survival. MATERIALS AND METHODS: We performed microarray-based comparative genomic hybridization (aCGH) profiling of two cohorts of primary MPNST tissue samples including 25 patients treated at The University of Texas MD Anderson Cancer Center and 26 patients from Tianjin Medical University Cancer Institute and Hospital. Fluorescence in situ hybridization (FISH) was used to validate the gene amplification detected by aCGH analysis. Another cohort of 63 formalin-fixed paraffin-embedded MPNST samples (including 52 samples for FISH assay) was obtained to explore FGFR1, 2, 3, and 4 protein expression by immunohistochemical (IHC) analysis. CONCLUSIONS: Our integrated genomic and molecular studies provide evidence that FGFRs play different prognostic roles in MPNST.

Effects of FGFR Signaling on Cell Proliferation and Differentiation of Apert Dental Cells.

The Apert syndrome is a rare congenital disorder most often arising from S252W or P253R mutations in fibroblast growth factor receptor (FGFR2). Numerous studies have focused on the regulatory role of Apert FGFR2 signaling in bone formation, whereas its functional role in tooth development is largely unknown. To investigate the role of FGFR signaling in cell proliferation and odontogenic differentiation of human dental cells in vitro, we isolated dental pulp and enamel organ epithelia (EOE) tissues from an Apert patient carrying the S252W FGFR2 mutation. Apert primary pulp and EOE cells were established and shown to exhibit normal morphology and express alkaline phosphatase under differentiation conditions. Similar to control cells, Apert dental pulp and EOE cells expressed all FGFRs, with highest levels of FGFR1 followed by FGFR2 and low levels of FGFR3 and FGFR4. However, Apert cells had increased cell growth compared with control cells. Distinct from previous findings in osteoblast cells, gain-of-function S252W FGFR2 mutation did not upregulate the expression of epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFRα), but elevated extracellular signal-regulated kinase (ERK) signaling in cells after EGF stimulation. Unexpectedly, there was little effect of the S252W mutation on odontogenic gene expression in dental pulp and EOE cells. However, after inhibition of total FGFR signaling or ERK signaling, the expression of odontogenic genes was upregulated in both dental cell types, indicating the negative effect of whole FGFR signaling on odontogenic differentiation. This study provides novel insights on FGFR signaling and a common Apert FGFR2 mutation in the regulation of odontogenic differentiation of dental mesenchymal and epithelial cells.

Frequent methylation of the KLOTHO gene and overexpression of the FGFR4 receptor in invasive ductal carcinoma of the breast.

Invasive ductal carcinoma of the breast is the most common cancer affecting women worldwide. The marked heterogeneity of breast cancer is matched only with the heterogeneity in its associated or causative factors. Breast cancer in Saudi Arabia is apparently an early onset with many of the affected females diagnosed before they reach the age of 50 years. One possible rationale underlying this observation is that consanguinity, which is widely spread in the Saudi community, is causing the accumulation of yet undetermined cancer susceptibility mutations. Another factor could be the accumulation of epigenetic aberrations caused by the shift toward a Western-like lifestyle in the past two decades. In order to shed some light into the molecular mechanisms underlying breast cancer in the Saudi community, we identified KLOTHO (KL) as a tumor-specific methylated gene using genome-wide methylation analysis of primary breast tumors utilizing the MBD-seq approach. KL methylation was frequent as it was detected in 55.3 % of breast cancer cases from Saudi Arabia (n = 179) using MethyLight assay. Furthermore, KL is downregulated in breast tumors with its expression induced following treatment with 5-azacytidine. The involvement of KL in breast cancer led us to investigate its relationship in the context of breast cancer, with one of the protagonists of its function, fibroblast growth factor receptor 4 (FGFR4). Overexpression of FGFR4 in breast cancer is frequent in our cohort and this overexpression is associated with poor overall survival. Interestingly, FGFR4 expression is higher in the absence of KL methylation and lower when KL is methylated and presumably silenced, which is suggestive of an intricate relationship between the two factors. In conclusion, our findings further implicate "metabolic" genes or pathways in breast cancer that are disrupted by epigenetic mechanisms and could provide new avenues for understanding this disease in a new context.

Combination of the FGFR4 inhibitor PD173074 and 5-fluorouracil reduces proliferation and promotes apoptosis in gastric cancer.

Our previous findings revealed that FGFR4 may be a novel therapeutic target for gastric cancer. The aim of the present study was to explore the effects of a combination of PD173074 (PD) and 5-fluorouracil (5-Fu) on the biological behavior of gastric cancer cell lines and the relevant mechanisms involved. MKN45, a gastric cancer cell line, was treated with each single agent alone or a combination of FGF19, PD and 5-Fu. Then, a series of functional assays were performed using CCK-8 assay and flow cytometry. Western blot analysis was used to determine the expression of signaling pathway and downstream-related molecules in the MKN45 cells following the different treatments. As the concentration of PD and 5-Fu increased, the cell viability gradually decreased; the viability of the combination group was less than the viability following single administration. Western blot analysis showed that FGFR4 expression was weak in the 5-Fu-treated groups when compared with the control. PD markedly increased the apoptosis rate of MKN45 cells when compared to the control; the apoptosis rate in the cells treated with the combination of PD and 5-Fu was higher than that in the cells following single treatment. Furthermore, PD reduced the expression of p-ERK and Bcl-xl and increased caspase-3 expression. Inhibition of the activity of FGFR4 may be the main mechanisms of PD effect while 5-Fu reduced FGFR4 expression. Furthermore, the effects of the combination of 5-Fu and PD in inhibiting proliferation, increasing apoptosis and arresting cell cycle were superior to these effects following the single agent treatments, suggesting that the two drugs applied in combination may contribute to the effective treatment of gastric cancer.

Outlier kinase expression by RNA sequencing as targets for precision therapy.

Protein kinases represent the most effective class of therapeutic targets in cancer; therefore, determination of kinase aberrations is a major focus of cancer genomic studies. Here, we analyzed transcriptome sequencing data from a compendium of 482 cancer and benign samples from 25 different tissue types, and defined distinct "outlier kinases" in individual breast and pancreatic cancer samples, based on highest levels of absolute and differential expression. Frequent outlier kinases in breast cancer included therapeutic targets like ERBB2 and FGFR4, distinct from MET, AKT2, and PLK2 in pancreatic cancer. Outlier kinases imparted sample-specific dependencies in various cell lines, as tested by siRNA knockdown and/or pharmacologic inhibition. Outlier expression of polo-like kinases was observed in a subset of KRAS-dependent pancreatic cancer cell lines, and conferred increased sensitivity to the pan-PLK inhibitor BI-6727. Our results suggest that outlier kinases represent effective precision therapeutic targets that are readily identifiable through RNA sequencing of tumors.

Differential effects of polymorphic alleles of FGF receptor 4 on colon cancer growth and metastasis.

A gly(388)arg polymorphism (rs351855) in the transmembrane domain of the fibroblast growth factor receptor (FGFR4) is associated with increased risk, staging, and metastasis in several different types of cancer. To specifically assess the impact of the polymorphic FGFR4 in colorectal cancer (CRC), we engineered CRC cell lines with distinct endogenous expression patterns to overexpress either the FGFR4(gly) or FGFR4(arg) alleles. The biologic analyses revealed an oncogenic importance for both polymorphic alleles, but FGFR4(gly) was the stronger inducer of tumor growth, whereas FGFR4(arg) was the stronger inducer of migration. An evaluation of clinical specimens revealed that FGFR4 was upregulated in 20/71 patients independent of gly(388)arg status. There was no correlation between the presence of an FGFR4(arg) allele and CRC or polyp risk in 3,471 participants of the CORSA study. However, among 182 patients with CRC, FGFR4(arg)-carriers had a fivefold higher risk of tumors that were stage II or greater. Together, our results established that both allelic forms of FGFR4 exert an oncogenic impact and may serve equally well as therapeutic targets in CRC. One important implication of our findings is that FGFR4(arg)-carriers are at a higher risk for more aggressive tumors and therefore may profit from early detection measures.

Overexpression of fibroblast growth factor receptor 4 in high-grade pancreatic intraepithelial neoplasia and pancreatic ductal adenocarcinoma.

The overexpression of fibroblast growth factor receptor (FGFR) 4 has been reported in various human cancers, but it has not been studied in pancreatic ductal adenocarcinoma (PDAC) or its precursor lesion, pancreatic intraepithelial neoplasia (PanIN). Moreover, there is controversy as to whether FGFR4 has a mitogenic role in carcinogenesis or other functions. Therefore, the expression and roles of FGFR4 in pancreatic cancer were investigated. Immunohistochemical staining was performed using an anti-FGFR4 antibody in PDAC and PanIN cases. The expression levels of FGFR4 mRNA and protein were investigated in PDAC cell lines by qRT-PCR and Western blot, respectively. Changes were analyzed in cell morphology, proliferation, migration, invasion and attachment in PDAC cell lines with or without the stimulation of FGFR4 by FGF19, as a known specific ligand. The changes in mRNA levels associated with transformation and tumorigenesis as a result of FGF19 administration were also evaluated. FGFR4 was expressed in 39 of 53 PDAC cases (73.6%) and its expression tended to be related to longer overall survival (P=0.068). Moreover, it was frequently expressed in high-grade PanIN lesions [10 of 11 lesions (90.9%)], whereas it was hardly expressed in low-grade PanIN lesions [1 of 10 lesions (10.0%)] (P=0.0003). FGFR4 stimulation of PDAC cells resulted in significantly increased cell adhesion to laminin and fibronectin (P<0.05) and decreased cell migration (P<0.05). The results of PCR array analysis indicated that this was a result of up-regulation of the integrin α4 family. In contrast, cell morphology or proliferation in PDAC cells was not affected. We showed that FGFR4 expression is markedly increased in high-grade PanIN and PDAC compared with that in normal and low-grade PanIN, and that FGFR4 stimulation by FGF19 of PDAC cells contributes to tumor suppression by increasing cell adhesion to extracellular matrix.

Targeting FGF19 inhibits tumor growth in colon cancer xenograft and FGF19 transgenic hepatocellular carcinoma models.

Although fibroblast growth factor 19 (FGF19) can promote liver carcinogenesis in mice its involvement in human cancer is not well characterized. Here we report that FGF19 and its cognate receptor FGF receptor 4 (FGFR4) are coexpressed in primary human liver, lung and colon tumors and in a subset of human colon cancer cell lines. To test the importance of FGF19 for tumor growth, we developed an anti-FGF19 monoclonal antibody that selectively blocks the interaction of FGF19 with FGFR4. This antibody abolished FGF19-mediated activity in vitro and inhibited growth of colon tumor xenografts in vivo and effectively prevented hepatocellular carcinomas in FGF19 transgenic mice. The efficacy of the antibody in these models was linked to inhibition of FGF19-dependent activation of FGFR4, FRS2, ERK and beta-catenin. These findings suggest that the inactivation of FGF19 could be beneficial for the treatment of colon cancer, liver cancer and other malignancies involving interaction of FGF19 and FGFR4.

Genetics and proteomics of pituitary tumors.

Genetics and proteomics determine structure and function of normal tissues, and the molecular alterations that underlie tumorigenesis result in changes in these aspects of tissue biology in neoplasms. We review the known genetic alterations in pituitary tumors. These include the oncogenic Gsalpha protein (GSP)-activating mutations, and pituitary tumor-derived fibroblast growth factor receptor-4 (ptd-FGFR4), as well as tumor suppressor gene mutations associated with multiple endocrine neoplasia type 1 (MEN1). Other candidates identified from expression profiling include pituitary tumor-transforming gene (PTTG), GADD45, and bone morphogenic protein (BMP)4. Proteomic changes in pituitary tumors include classical alterations identified by immunohistochemistry as well as epigenetic reductions in p27. The underlying mechanisms for dysregulated cell adhesive molecules including cadherins and FGFRs are reviewed. The combined use of genetic and proteomic approaches will enhance novel drug therapeutic development.

Expression of basic fibroblast growth factor and its receptors in human ovarian follicles from adults and fetuses.

OBJECTIVE: To investigate the immunocytochemical expression and presence of mRNA transcripts for basic fibroblast growth factor (bFGF) and its four receptors (FGFR-1, -2, -3, and -4) in ovaries from human adults and fetuses. DESIGN: Immunocytochemical and reverse transcription polymerase chain reaction (RT-PCR) study. SETTING: Major tertiary care and referral academic centers. PATIENT(S): Nine women and girls undergoing laparoscopic ovarian biopsy and 26 women undergoing second- and third-trimester pregnancy terminations. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Microscopic morphometric analysis, immunocytochemistry for bFGF and its receptors, and RT-PCR analyses. RESULT(S): The proteins for bFGF, FGFR-2, FGFR-3, and FGFR-4 were identified in oocytes of all follicular classes. Immunocytochemical expression of bFGF and its receptors was detected in granulosa cells of follicles from adolescents/women but not from fetuses. There was no immunocytochemical expression of FGFR-1. Transcripts of bFGF and its four receptors were identified by RT-PCR in all samples. CONCLUSION(S): The expression of bFGF and its receptors in human ovaries suggests that bFGF might have a role in early folliculogenesis.