Expression Of Fibroblast Growth Factor Receptor Research Articles

Abstract 3116: Targeting fibroblast growth factor receptors in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue sarcoma and is classified into two main histopathological subtypes: embryonal RMS (eRMS), characterized by different genomic changes or alveolar RMS (aRMS), driven by the oncogenic fusion protein PAX3-FOXO1. The significant toxicity associated with conventional chemotherapies represents a major complication in pediatric oncology. To improve current therapies, we adopted two different strategies targeting fibroblast growth factor receptors (FGFR) in RMS. FGFR1-4 are a family of transmembrane receptor tyrosine kinases. Their activation upon binding of fibroblast growth factors (FGF) triggers pro-survival and proliferative signals. Our goal is to deliver drugs specifically to the tumor site by taking advantage of FGFR4 overexpression in RMS. To this end, we will covalently link FGFR4 specific nanobodies to the surface of liposomal vincristine in order to actively target RMS cells. We have established the optimal conditions to formulate liposomes loaded with vincristine. Following nanobody phage display selection on recombinant FGFR4 we focused on the top scoring candidates. Flow cytometry analysis on FGFR4-expressing versus FGFR4 knock-out RMS cell lines showed receptor-specific binding of three nanobodies. In activation assays with the FGFR4 specific growth factor FGF19, we demonstrated that the three binding candidates also blocked downstream ERK activation in RMS cells. We will now assess their theranostic potential on drug-loaded and fluorescently labeled nanovesicles on RMS tumor cells in vitro and in xenografts in vivo. Surprisingly, we observed change in cell morphology followed by cell death upon exposure to FGF2 in a subset of cultured cells established from eRMS patient-derived xenografts. Inappropriate expression of FGFRs and FGF signaling is implicated in tumor progression and therefore our findings appear contradictory. Dose-response experiments have shown that FGFR inhibition with small molecule inhibitors completely rescued FGF2 toxicity. In contrast, however, we detected high expression levels of FGFR1, 2 and 4 as well as activating mutations of FGFR4 in FGF2-sensitive eRMS cells. Therefore, our results are of upmost clinical relevance since genetically-based drug selection could lead to an inappropriate treatment inducing tumor promoting conditions. Hence, our second goal is to further unravel the molecular mechanism underlying the toxic effect of FGF-2 in a subset of eRMS tumors to avoid potentially harmful treatments. In summary, we have identified FGFR4 specific nanobodies that bind to the receptor and block downstream signaling in RMS cells. Active drug delivery of liposomal vincristine to the tumor site has the potential to enhance the therapeutic impact and decrease side effects. Moreover, we discovered a toxic effect of FGF2 in a subgroup of eRMS patient derived xenograft cells which might open new avenues for treatment. Citation Format: Nagjie Alijaj, Sandrine Moutel, Maxim Gray, Maurizio Roveri, Gabriele Manzella, Marco Wachtel, Franck Perez, Beat Schäfer, Michele Bernasconi. Targeting fibroblast growth factor receptors in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3116.

Fibroblast growth factor receptor promotes progression of cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is a keratinocyte-derived invasive and metastatic tumor of the skin. It is the second-most commonly diagnosed form of skin cancer striking 200 000 Americans annually. Further, in organ transplant patients, there is a 65- to 100-fold increased incidence of cSCC compared to the general population. Excision of cSCC of the head and neck results in significant facial disfigurement. Therefore, increased understanding of the mechanisms involved in the pathogeneses of cSCC could identify means to prevent, inhibit, and reverse this process. In our previous studies, inhibition of fibroblast growth factor receptor (FGFR) significantly decreased ultraviolet B-induced epidermal hyperplasia and hyperproliferation in SKH-1 mice, suggesting an important role for FGFR signaling in skin cancer development. However, the role of FGFR signaling in the progression of cSCC is not yet elucidated. Analysis of the expression of FGFR in cSCC cells and normal epidermal keratinocytes revealed protein overexpression and increased FGFR2 activation in cSCC cells compared to normal keratinocytes. Further, tumor cell-specific overexpression of FGFR2 was detected in human cSCCs, whereas the expression of FGFR2 was low in premalignant lesions and normal skin. Pretreatment with the pan-FGFR inhibitor; AZD4547 significantly decreased cSCC cell-cycle traverse, proliferation, migration, and motility. Interestingly, AZD4547 also significantly downregulated mammalian target of rapamycin complex 1 and AKT activation in cSCC cells, suggesting an important role of these signaling pathways in FGFR-mediated effects. To further bolster the in vitro studies, NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice with SCC12A tumor xenografts treated with AZD4547 (15 mg/kg/bw, twice weekly oral gavage) exhibited significantly decreased tumor volume compared to the vehicle-only treatment group. The current studies provide mechanistic evidence for the role of FGFR and selectively FGFR2 in the early progression of cSCC and identifies FGFR as a putative therapeutic target in the treatment of skin cancer.

MicroRNA-889-3p targets FGFR2 to inhibit cervical cancer cell viability and invasion.

MicroRNAs (miRNAs) are frequently dysregulated in cervical cancer, and the aberrant regulation of miRNAs may be involved in the regulation of various cancer-associated biological processes. Therefore, further exploration of the specific roles of dysregulated miRNAs in cervical cancer and their associated mechanism may promote the development of effective therapeutic approaches. miRNA-889-3p (miR-889) serves crucial roles in esophageal squamous cell carcinoma and hepatocellular carcinoma. However, to the best of our knowledge, no studies concerning the relationship between miR-889 and cervical cancer were performed. The aims of this study were to measure miR-889 expression in cervical cancer and to examine the potential effects of miR-889 in cervical cancer development on a molecular level to provide potential clinical insight. The present study revealed that miR-889 was downregulated in cervical cancer tissues and cell lines. Reduced miR-889 expression was significantly associated with International Federation of Gynecology and Obstetrics cancer staging and with lymph node metastasis. In addition, miR-889 overexpression reduced cervical cancer cell viability and invasive ability. Using bioinformatics analysis, fibroblast growth factor receptor 2 (FGFR2) was predicted to be a potential target of miR-889, which was confirmed using luciferase reporter assay. Reverse transcription-quantitative PCR and western blot analysis results suggested that miR-889 overexpression decreased FGFR2 expression in cervical cancer cells at the mRNA and the protein level, respectively. Conversely, FGFR2 silencing using small interfering RNA imitated the tumor suppressive effects of miR-889 overexpression in cervical cancer cells, which was successfully reversed by plasmid-facilitated FGFR2 overexpression. These observations demonstrated that miR-889 may serve tumor suppressive roles in cervical cancer by directly targeting FGFR2, which indicated that this miRNA may be a promising therapeutic target for patients with cervical cancer.

Deleting Suppressor of Cytokine Signaling-3 in chondrocytes reduces bone growth by disrupting mitogen-activated protein kinase signaling

To investigate the impact of deleting Suppressor of Cytokine Signaling (SOCS)-3 (SOCS3) in chondrocytes during murine skeletal development. Mice with a conditional Socs3 allele (Socs3fl/fl) were crossed with a transgenic mouse expressing Cre recombinase under the control of the type II collagen promoter (Col2a1) to generate Socs3Δ/Δcol2 mice. Skeletal growth was analyzed over the lifespan of Socs3Δ/Δcol2 mice and controls by detailed histomorphology. Bone size and cortical bone development was evaluated by micro-computed tomography (micro-CT). Growth plate (GP) zone width, chondrocyte proliferation and apoptosis were assessed by immunofluorescence staining for Ki67 and TUNEL. Fibroblast growth factor receptor-3 (FGFR3) signaling in the GP was assessed by immunohistochemistry, while the effect of SOCS3 overexpression on FGFR3-driven pMAPK signaling in HEK293T cells was evaluated by Western blot. Socs3Δ/Δcol2 mice of both sexes were consistently smaller compared to littermate controls throughout life. This phenotype was due to reduced long bone size, poor cortical bone development, reduced Ki67+ proliferative chondrocytes and decreased proliferative zone (PZ) width in the GP. Expression of pMAPK, but not pSTAT3, was increased in the GPs of Socs3Δ/Δcol2 mice relative to littermate controls. Overexpression of FGFR3 in HEK293T cells increased Fibroblast Growth Factor 18 (FGF18)-dependent Mitogen-activated protein kinase (MAPK) phosphorylation, while concomitant expression of SOCS3 reduced FGFR3 expression and abrogated MAPK signaling. Our results suggest a potential role for SOCS3 in GP chondrocyte proliferation by regulating FGFR3-dependent MAPK signaling in response to FGF18.

Fibroblast Growth Factor Receptor Signaling in Skin Cancers.

Fibroblast growth factor (FGF)/Fibroblast growth factor receptor (FGFR) signaling regulates various cellular processes during the embryonic development and in the adult organism. In the skin, fibroblasts and keratinocytes control proliferation and survival of melanocytes in a paracrine manner via several signaling molecules, including FGFs. FGF/FGFR signaling contributes to the skin surface expansion in childhood or during wound healing, and skin protection from UV light damage. Aberrant FGF/FGFR signaling has been implicated in many disorders, including cancer. In melanoma cells, the FGFR expression is low, probably because of the strong endogenous mutation-driven constitutive activation of the downstream mitogen-activated protein kinase-extracellular signal-regulated kinase (MAPK-ERK) signaling pathway. FGFR1 is exceptional as it is expressed in the majority of melanomas at a high level. Melanoma cells that acquired the capacity to synthesize FGFs can influence the neighboring cells in the tumor niche, such as endothelial cells, fibroblasts, or other melanoma cells. In this way, FGF/FGFR signaling contributes to intratumoral angiogenesis, melanoma cell survival, and development of resistance to therapeutics. Therefore, inhibitors of aberrant FGF/FGFR signaling are considered as drugs in combination treatment. The ongoing LOGIC-2 phase II clinical trial aims to find out whether targeting the FGF/FGFR signaling pathway with BGJ398 may be a good therapeutic strategy in melanoma patients who develop resistance to v-Raf murine sarcoma viral oncogene homolog B (BRAF)/MEK inhibitors.

Research on Mechanism of FGFR1 Inhibitor BAY1163877 against Proliferation of Breast Cancer Cells

To investigate the effect and mechanism of fibroblast growth factor receptor 1 (FGFR1) inhibitor BAY1163877 on proliferation and apoptosis of breast cancer cells. The expression of FGFR1 in human breast cell lines was detected by qRT-PCR and western blot. IC50 of BAY1163877 and cell viability were measured by CCK-8 method. Cell proliferation was observed by colony assay. Cell apoptosis after treatment of BAY1163877 was tested by flow cytometry. The expressions of p-FGFR1/FGFR1 and p-STAT3/STAT3 protein were detected by Western blot. According to the results of qRT-PCR and Western blot, FGFR1 high expression of breast cancer cell line MDA-MB-231 and FGFR1 low expression of breast cancer cell line MCF-7 were selected. BAY1163877 inhibits proliferation of MDA-MB-231 and MCF-7 cells and induces apoptosis of MDA-MB-231 and MCF-7 cells. The results of Western blot showed that the expression of p-FGFR1 and p-STAT3 protein in MDA-MB-231 was reduced after BAY1163877 treatment and the expression in MCF-7 was not significantly changed. BAY1163877 inhibits the proliferation and induces the apoptosis of high FGFR1 expression breast cancer cell line MDA-MB-231, and its mechanism may be related to the decrease of p-FGFR1 and p-STAT3 protein expression. BAY1163877 inhibits the proliferation and induces the apoptosis of MCF-7, but its mechanism still needs the further study.

Design and implementation of quality control plans that integrate moving average and internal quality control: Incorporating the best of both worlds

To explore the inhibitory effects of zoledronate (ZOL) on adipose-derived stem cells (ADSCs) into osteoblasts for repairing jaw necrosis.ADSCs were induced to differentiate into osteoblasts. The differentiation characteristics of osteoblasts was observed under inverted microscope by alizarin red staining. The transwell assay was performed to evaluate the migration of ADSCs co-cultured with osteoblasts and divided into ZOL group treated with ZOL and N-ZOL group without ZOL treatment. The differentiation and proliferation characteristics of ADSCs differentiated osteoblasts were observed respectively. The expression of CTSK (Cathepsin K) and FGFR3 (Fibroblast growth factor receptor 3) in osteoblasts were analyzed by immunofluorescence and western blot.The differentiation degree and proliferation of ADSCs to osteoblasts in N-ZOL group were both higher than those in ZOL group. The migratory cell number in ADSCs differentiation in ZOL group was higher than that of N-ZOL group. The protein expression of CTSK and FGFR3 in ADSCs differentiated to osteoblasts in ZOL group was higher than that in N-ZOL group.The differentiation of ADSCs into osteoblasts is significantly inhibited by ZOL. Due to this reason, it may be difficult to achieve good results by ZOL induced ADSCs into osteoblasts in repairing jaw necrosis.

The efficacy of cisplatin on nasopharyngeal carcinoma cells may be increased via the downregulation of fibroblast growth factor receptor 2.

Cisplatin is one of the primary compounds used in the treatment of nasopharyngeal carcinoma (NPC), and fibroblast growth factor receptor 2 (FGFR2) has emerged to be a promising target for treatment in various tumors. Therefore, the present study aimed to explore whether the expression levels of FGFR2 in NPC tissues and cell lines were altered, and whether the efficiency of cisplatin was increased following the downregulation of FGFR2. The downregulation of FGFR2 was achieved by transfection with a small interfering RNA against FGFR2. Tissues of patients with NPC were analyzed by immunohistochemistry. Cell viability was examined using a Cell Counting Kit-8 assay. Cell cycle analysis was performed using flow cytometry. mRNA and protein levels were measured by reverse transcription quantitative polymerase chain reaction and western blot analysis, respectively. FGFR2 was observed to be overexpressed in cancer tissues of patients with NPC and in the NPC SUNE1, C666-1, 6-10B and HNE-3 cell lines, and resulted in an unfavorable prognosis. Cisplatin treatment decreased cell viability and increased FGFR2 expression. The silencing of FGFR2 was demonstrated to augment the effects of cisplatin treatment, including decreasing the cell viability and inducing cell cycle arrest, which involved the increase and decrease of the durations of G1 and S phases, respectively, and a decrease in the expression levels of cyclin D1 and CDC25A, and increasing the rate of apoptosis via the intrinsic apoptosis pathway, as demonstrated by the upregulation of cleaved caspase-3 and B-cell lymphoma 2 (Bcl-2)-associated X protein and downregulation of Bcl-2, in SUNE1 and C666-1 cell lines. FGFR2 was overexpressed in the cancer tissues of patients with NPC and in NPC cell lines, resulting in an unfavorable prognosis. The downregulation of FGFR2 decreased cell viability via cell cycle arrest at G1 phase, and increased the efficacy of the cisplatin-based induction of apoptosis through the intrinsic apoptosis pathway.

Fibroblast Growth Factor Receptor 1 (FGFR1) Amplification Detected by Droplet Digital Polymerase Chain Reaction (ddPCR) Is a Prognostic Factor in Colorectal Cancers.

PurposeThe purpose of this study was to reveal the clinicopathological characteristics and prognostic implications associated with fibroblast growth factor receptor 1 (FGFR1) amplification in colorectal cancers (CRCs).Materials and MethodsWe measured the copy number of FGFR1 by droplet digital polymerase chain reaction (ddPCR), and analyzed the FGFR1 expression by immunohistochemistry, in 764 surgically resected CRCs (SNUH2007 dataset, 384 CRCs; SNUH Folfox dataset, 380 CRCs).ResultsCRCs with ≥ 3.3 copies of the FGFR1 gene were classified as FGFR1 amplified. FGFR1 amplification was found in 10 of the 384 CRCs (2.6%) in the SNUH2007 dataset, and in 28 of the 380 CRCs (7.4%) in the SNUH Folfox dataset. In the SNUH2007 dataset, there was no association between the FGFR1 copy number status and sex, gross appearance, stage, or differentiation. High FGFR1 expression was associated with female sex and KRAS mutation. At the molecular level, FGFR1 amplification was mutually exclusive with BRAF mutation, microsatellite instability, and MLH1 methylation, in both SNUH2007 and SNUH Folfox datasets. Survival analysis revealed that FGFR1 amplification was associated with significantly worse clinical outcome compared with no FGFR1 amplification, in both SNUH2007 and SNUH Folfox datasets. Within the SNUH2007 dataset, CRC patients with high FGFR1 expression had an inferior progression-free survival compared with those with low FGFR1 expression. The FGFR inhibitor, PD173074, repressed the proliferation of a CRC cell line overexpressing FGFR1, but not of cells with FGFR1 amplification.ConclusionFGFR1 amplification measured by ddPCR can be a prognostic indicator of poor clinical outcome in patients with CRCs.

Upregulation of FGFR3 and HIF-1α expression in muscle invasive bladder cancer

BACKGROUND The major risks in patients diagnosed with non-muscle invasive bladder cancer (NMIBC)are recurrence, progression of muscle invasive bladder cancer (MIBC), and metastasis. Biological markers such as fibroblast growth factor receptor-3 (FGFR3) and hypoxia-inducible factor-1α (HIF-1α) are related to muscle invasiveness of bladder cancer. This study was aimed to analyze the expression of FGFR3 and HIF-1α to predict muscle invasiveness in bladder cancer patients.METHODS This was an observational study with a case-control design. Sixty patients with bladder cancer, who underwent histopathology examinations at the Department of Pathology, Faculty of Medicine, Universitas Sumatera Utara/H. Adam Malik Hospital from January 2012 to December 2015, were included in this study. Samples were then classified into 30 NMIBC and 30 MIBC groups. All samples were analyzed with an immunohistochemistry assay for FGFR3 and HIF-1α. H-scores were used to determine the relationships between each group.RESULTS FGFR3 was expressed in 29 (96.7%) patients of the NMIBC group, and 23 (76.7%) patients of the MIBC group (p=0.026, OR=8.8; 95% CI=1.01–76.96). HIF-1α was expressed in only 1 (3.33%) patient of the NMIBC group, and 15 (50%) patients of the MIBC group (p<0.001, OR=29; 95% CI=3.49–241.13).CONCLUSIONS There was a difference in upregulation of FGFR3 and HIF-1α expression in both the NMIBC and MIBC groups.

Significance of FGFR2 – RSK2 interdependence for breast cancer progression. In vitro studies and clinical material analyses

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. Elevated level of RSK1 and RSK2 was found in about 50% of breast tumours and is associated with a poor survival outcome. The project aims to investigate the role of RSK2 kinase in breast cancer progression out at two complementary levels: in vitro studies and analyses of samples from breast cancer patients. We demonstrated that in breast epithelial and cancer cell lines stimulation of FGFR2 (fibroblast growth factor receptor 2) with FGF2 induced activation of RSK2-Tyr529 and that this new signalling pathway promoted anchorage-independent cell proliferation, formation of focal adhesions and cell migration. In clinical analyses FGFR2 showed a positive correlation with RSK2 at both protein (p = 0.003) and mRNA (p = 0.001) levels. The values of correlation coefficients were the highest in triple- -negative breast cancer (TNBC), which is more aggressive and has a poorer prognosis than other breast cancer subtypes. Moreover, expression of FGFR2 and/or activated RSK (RSK-P) significantly correlated with poor disease-free survival (DFS) of patients (p = 0.01). Taken together, our results suggest that FGFR2-RSK2 signalling pathway is associated with breast cancer progression and further studies of FGFR2-RSK2 interdependence may contribute to the development of new therapeutic strategies in the management of patients with breast cancer.

FGFR2-mediated phosphorylation of PTEN at tyrosine 240 contributes to the radioresistance of glioma.

Ionizing radiation (IR) is a standard-of-care treatment for glioma patients; however, the clinical efficacy is limited due to therapeutic resistance. A recent study published by Ma et al. (Cancer Cell 35:504-518, 2019) reported that the phosphorylation of phosphatase and tensin homolog (PTEN) at tyrosine 240 (pY240-PTEN) promotes the radioresistance of human glioma cells. After treatment with IR, the fibroblast growth factor receptor 2 (FGFR2)-mediated phosphorylation that generated pY240-PTEN could effectively promote the decondensation of chromatin through an interaction with Ki-67, leading to DNA damage repair and radioresistance. However, such promising findings need to be addressed in detail after considering the following points. (1) The authors should take into consideration whether or not the nuclear-cytoplasmic translocalization of PTEN occurs. (2) The roles of FGFR2-PTEN downregulation should be validated using both genetic and pharmacological inhibition models. (3) Some of the data shown by the authors are confusing and did not support the conclusion that patients with higher PTEN and FGFR2 expression were relatively IR resistant.

Proliferation of poorly differentiated endometrial cancer cells through autocrine activation of FGF receptor and HES1 expression.

Patients with poorly differentiated endometrial cancer show poor prognosis, and effective molecular target-based therapies are needed. Endometrial cancer cells proliferate depending on the activation of HES1 (hairy and enhancer of split-1), which is induced by several pathways, such as the Notch and fibroblast growth factor receptor (FGFR) signaling pathways. In addition, aberrant, ligand-free activation of the FGFR signaling pathway resulting from mutations in FGFR2 was also reported in endometrial cancer. However, a clinical trial showed that there was no difference in the effectiveness of FGFR inhibitors between patients with and without the FGFR2 mutation, suggesting a presence of another signaling pathway for the FGFR activation. Here, we investigated the signaling pathway regulating the expression of HES1 and proliferation of poorly and well-differentiated endometrial cancer cell lines Ishikawa and HEC-50B, respectively. Whereas Ishikawa cells proliferated and expressed HES1 in a Notch signaling-dependent manner, Notch signaling was not involved in HES1 and proliferation of HEC-50B cells. The FGFR inhibitor, NVP-BGJ398, decreased HES1 expression and proliferation of HEC-50B cells; however, HEC50B cells had no mutations in the FGFR2 gene. Instead, HEC-50B cells highly expressed ligands for FGFR2, suggesting that FGFR2 is activated by an autocrine manner, not by ligand-free activation. This autocrine pathway activated Akt downstream of FGFR for cell proliferation. Our findings suggest the usefulness of HES1 as a marker for the proliferation signaling and that FGFR inhibitor may be effective for poorly differentiated endometrial cancers that harbor wild-type FGFR.

MiR‑802 inhibits the aggressive behaviors of non‑small cell lung cancer cells by directly targeting FGFR1.

Emerging reports have revealed that several microRNAs (miRNAs) are abnormally expressed in non‑small cell lung cancer (NSCLC). miRNAs have been identified as oncogenes or tumor suppressors, and regulate various biological processes including oncogenesis and development. miR‑802 is dysregulated in multiple types of human cancer, and exerts tumor‑suppressive or promoting roles. However, the expression levels and functional roles of miR‑802 in NSCLC remain largely unknown. In the present study, miR‑802 expression was demonstrated to be decreased in NSCLC tissues and cell lines. A low miR‑802 expression was significantly correlated with the tumor stage, lymph node metastasis and brain metastasis in NSCLC patients. Restoring miR‑802 expression inhibited NSCLC cell proliferation and colony formation, induced cell apoptosis, decreased cell migration and invasion invitro, and hindered invivo tumor growth. Mechanistically, fibroblast growth factor receptor1 (FGFR1) was confirmed as the target gene of miR‑802 in NSCLC cells. In addition, FGFR1 silencing mimicked the tumor‑suppressing roles of miR‑802 upregulation in NSCLC cells. Furthermore, rescue experiments revealed that FGFR1 reintroduction rescued the miR‑802‑induced inhibition of the malignant phenotypes in NSCLC cells. Notably, miR‑802 was able to deactivate the phosphoinositide3‑kinase (PI3K)/AKT serine/threonine kinase (Akt)/mammalian target of rapamycin (mTOR) pathway in NSCLC cells invitro and invivo. Overall, these results demonstrated that miR‑802 could downregulate FGFR1 expression, thereby deactivating the PI3K/Akt/mTOR pathway and inhibiting the malignant development of NSCLC. Thus, miR‑802 may be a therapeutic candidate for patients with NSCLC.

Benzyl isothiocyanate suppresses IGF1R, FGFR3 and mTOR expression by upregulation of miR-99a-5p in human bladder cancer cells.

Benzyl isothiocyanate (BITC) is known for its pharmacological properties against malignant neoplasm, including bladder cancer(BC). The current study investigated microRNAs (miRNA or miR) expression profiles with an emphasis on the role of miR‑99a‑5p in BITC‑treated BC cells. A quantitative polymerase chain reaction (qPCR) microarray containing 79aberrantly expressed miRNAs in BC was used to detect miRNA expression in BITC‑treated cells. Several dysregulated miRNAs were identified and further confirmed using miRNA stem‑loop reverse transcription(RT)‑qPCR in 5637cells. Insulin‑like growth factor1 receptor(IGF1R), fibroblast growth factor receptor3(FGFR3) and mammalian target of rapamycin(mTOR) expression were determined by RT‑qPCR and western blotting. Cell viability was evaluated using WST‑1 reagent and apoptosis was monitored by determining the levels of cleaved‑poly ADP‑ribose polymerase and cleaved‑caspase‑3. BITC treatment significantly upregulated miR‑99a‑5p levels in a dose‑dependent manner. miR‑99a‑5p overexpression decreased IGF1R, mTOR and FGFR3 expression, predicted targets of miR‑99a‑5p. In addition, antisense miR‑99a‑5p sequences inhibited BITC‑induced miR‑99a‑5p overexpression, resulting in the restoration of protein expression and decreased cell viability. The current study identified multiple miRNAs responsive to BITC treatment, including miR‑99a‑5p. In addition, the induction of miR‑99a‑5p decreased IGF1R, mTOR and FGFR3 expression in BITC‑treated BC cells. The current study provided novel insight into the antitumor mechanism by which BITC restores miR‑99a‑5p expression and decreases cancer cell survival.

Alpha6-Integrin Regulates FGFR1 Expression through the ZEB1/YAP1 Transcription Complex in Glioblastoma Stem Cells Resulting in Enhanced Proliferation and Stemness.

Glioblastoma (GBM) is the most lethal primary brain tumor in adults and is known to be particularly aggressive and resistant to anti-cancer therapies, mainly due to the presence of GBM stem cells (GBMSC). By in vitro approaches supported by analysis from patients’ databases, we determined how α6-integrin and Fibroblast Growth Factor Receptor 1 (FGFR1) work in concert to regulate proliferation and stemness of GBMSC. We showed that α6-integrin regulates the expression of FGFR1 and its target gene Fokhead Box M1 (FOXM1) via the ZEB1/YAP1 transcription complex. These results were in accordance with the positive correlation observed in GBM between α6-integrin expression and its target genes ZEB1/YAP1, FGFR1, and FOXM1 in the databases, TCGA and Rembrandt. In addition, the clinical data demonstrate that GBM patients with high levels of the five genes signature, including α6-integrin, ZEB1/YAP1, FGFR1 and FOXM1, have a significantly shorter overall survival. In vitro, we observed a similar decrease in the expression of stemness-related factors, neurospheres forming capacity, as well as spheroids growth when α6-integrin or FGFR1 was blocked individually with specific siRNA, whereas the combination of both siRNA led to a significantly higher inhibition of spheres formation. These data suggest that co-administration of anti-FGFR1 and anti-α6-integrin could provide an improved therapeutic response in GBMSC.

FGFR1 Expression and Role in Migration in Low and High Grade Pediatric Gliomas.

The heterogeneous and invasive nature of pediatric gliomas poses significant treatment challenges, highlighting the importance of identifying novel chemotherapeutic targets. Recently, recurrent Fibroblast growth factor receptor 1 (FGFR1) mutations in pediatric gliomas have been reported. Here, we explored the clinical relevance of FGFR1 expression, cell migration in low and high grade pediatric gliomas and the role of FGFR1 in cell migration/invasion as a potential chemotherapeutic target. A high density tissue microarray (TMA) was used to investigate associations between FGFR1 and activated phosphorylated FGFR1 (pFGFR1) expression and various clinicopathologic parameters. Expression of FGFR1 and pFGFR1 were measured by immunofluorescence and by immunohistochemistry (IHC) in 3D spheroids in five rare patient-derived pediatric low-grade glioma (pLGG) and two established high-grade glioma (pHGG) cell lines. Two-dimensional (2D) and three-dimensional (3D) migration assays were performed for migration and inhibitor studies with three FGFR1 inhibitors. High FGFR1 expression was associated with age, malignancy, tumor location and tumor grade among astrocytomas. Membranous pFGFR1 was associated with malignancy and tumor grade. All glioma cell lines exhibited varying levels of FGFR1 and pFGFR1 expression and migratory phenotypes. There were significant anti-migratory effects on the pHGG cell lines with inhibitor treatment and anti-migratory or pro-migratory responses to FGFR1 inhibition in the pLGGs. Our findings support further research to target FGFR1 signaling in pediatric gliomas.

Thalidomide and Its Analogs Differentially Target Fibroblast Growth Factor Receptors: Thalidomide Suppresses FGFR Gene Expression while Pomalidomide Dampens FGFR2 Activity.

Thalidomide is an infamous teratogen and it is continuously being explored for its anticancer properties. Fibroblast growth factor receptors (FGFRs) are implicated in embryo development and cancer pathophysiology. With striking similarities observed between FGFR implicated conditions and thalidomide embryopathy, we hypothesized thalidomide targets FGFRs. We utilized three different cell lines and chicken embryo model to investigate the effects of thalidomide and analogs on FGFR expression. We performed molecular docking, KINOMEscan analysis, and kinase activity assays to study the drug-protein interactions. The expression of FGFR1 and FGFR2 was differentially regulated by all the three drugs in cells as well as in developing organs. Transcriptome analysis of thalidomide-treated chick embryo strongly suggests the modulation of FGFR signaling and key transcription factors. Corroboration with previous studies suggests that thalidomide might affect FGFR expression through the transcription factor, E2F1. At the protein level, molecular docking predicted all three analogs to interact with lysine residue at 517th and 508th positions of FGFR2 and FGFR3, respectively. This lysine coordinates the ATP binding site of FGFR, thus hinting at the possible perturbation of FGFR activity by thalidomide. Kinome analysis revealed that kinase activities of FGFR2 and FGFR3 (G697C) reduced by 31% and 65%, respectively, in the presence of 10 μM thalidomide. Further, we checked and confirmed that the analogs inhibited the FGFR2 kinase activity in a dose-dependent manner. This study suggests that FGFRs could be potential targets of thalidomide and the two analogs, and also endorses the link between the teratogenicity and antitumor activities of the drugs.

Nuclear FGFR2 negatively regulates hypoxia-induced cell invasion in prostate cancer by interacting with HIF-1 and HIF-2

The fibroblast growth factor receptor 2 (FGFR2) is a membrane receptor that promotes cell proliferation and differentiation. FGFR2 is also present in the nucleus, which raises a question on a new role of FGFR2 in regulating gene expression. Hypoxia-inducible factors 1 and 2 (HIF-1 and HIF-2) are nuclear proteins that transactivate many genes essential for cancer survival and metastasis under hypoxic conditions. Here, we investigated if nuclear FGFR2 modulates the HIF-driven hypoxic response. Using the TCGA database, we found that FGFR2 downregulation is associated with poor prognosis in prostate cancer. A gene-set enrichment analysis showed that metastasis- and hypoxia-related genes are associated with a low expression of FGFR2 in prostate cancer. Thus, we tested the possibility that FGFR2 negatively regulates the hypoxia-triggered metastasis of prostate cancer. FGFR2 controls migration and invasion of prostate cancer cells under hypoxia by inhibiting the HIF-driven gene expression. FGFR2 and HIF proteins co-localize and associate in the nucleus under hypoxia. FGFR2 interacts with the transactivation domain of HIF-1α and blocks the recruitment of coactivator p300, resulting in repression of HIF target genes. Based on these results, we propose a novel function of FGFR2 as a metastasis suppressor by controlling HIF-mediated hypoxic responses.

Exercise Alleviates Obesity-Induced Metabolic Dysfunction via Enhancing FGF21 Sensitivity in Adipose Tissues

Exercise promotes adipose remodeling and improves obesity-induced metabolic disorders through mechanisms that remain obscure. Here, we identify the FGF21 signaling in adipose tissues as an obligatory molecular transducer of exercise conferring its metabolic benefits in mice. Long-term high fat diet-fed obese mice exhibit compromised effects of exogenous FGF21 on alleviation of hyperglycemia, hyperinsulinemia, and hyperlipidemia, accompanied with markedly reduced expression of FGF receptor-1 (FGFR1) and β-Klotho (KLB) in adipose tissues. These impairments in obese mice are reversed by treadmill exercise. Mice lacking adipose KLB are refractory to exercise-induced alleviation of insulin resistance, glucose dysregulation, and ectopic lipid accumulation due to diminished adiponectin production, excessive fatty acid release, and enhanced adipose inflammation. Mechanistically, exercise induces the adipose expression of FGFR1 and KLB via peroxisome proliferator-activated receptor-gamma-mediated transcriptional activation. Thus, exercise sensitizes FGF21 actions in adipose tissues, which in turn sends humoral signals to coordinate multi-organ crosstalk for maintaining metabolic homeostasis.