FGFR1 Knockdown Research Articles

Abstract P24: Elucidating the Mechanism of Action of Lenvatinib in Estrogen Receptor-Positive (ER+) Breast Cancer

Abstract Background: ER+ breast cancer is the most common subtype of breast cancer. Although most patients respond initially to endocrine therapy (ET), endocrine resistance inevitably develops, leading to disease progression. Lenvatinib, a multi-kinase inhibitor, demonstrated clinical benefit in the treatment of patients with advanced ER+ breast cancer who have progressed on ET in a phase Ib/II clinical trial from our group. Our study aims to elucidate the molecular mechanism of action of lenvatinib in ER+ breast cancer. Methods: A cell viability assay was performed to assess the sensitivity of four ER+ cell lines to lenvatinib. Western blot was used to elucidate its effect on downstream pathway signalling, and cell cycle arrest investigated through flow cytometry. A series of functional assays, including BrdU, colony formation and wound-healing assays assessed proliferative, clonogenic and migratory activity respectively. The effect of lenvatinib on epithelial-mesenchymal transition (EMT) was analysed using RT-qPCR, and compared with siRNA knockdown (KD) of Fgfr1 and Fgfr2. Results: ER+ cell lines demonstrated sensitivity to lenvatinib in the micromolar range. Western blot showed downregulation of FGFR1, FGFR2, pERK and pAKT expression post-treatment. Lenvatinib was found to induce a G1 phase arrest, suppressed cell proliferation in a dose-dependent manner, and diminished both the clonogenic ability and migratory activity of ER+ cells. Lenvatinib treatment reduced expression of Fgfr1, Fgfr2 and the mesenchymal markers N-cad and Fibronectin, while increasing the expression of the epithelial markers E-cad and Claudin-1, indicating potential inhibition of EMT. A similar change in EMT marker expression was observed in Fgfr1 KD but not Fgfr2 KD cells. Conclusion: Lenvatinib demonstrated potent anti-cancer activity and induction of G1 phase arrest, leading to decreased cell proliferation, reduction of clonogenic ability and cell migration. EMT inhibition was also observed, possibly through the FGFR1 signalling pathway. Citation Format: Natasha Gandasasmita, Gauri Vaidya, Arpita Datta, Charlie Marvalim, Joline Si Jing Lim, Soo Chin Lee. Elucidating the Mechanism of Action of Lenvatinib in Estrogen Receptor-Positive (ER+) Breast Cancer [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr P24.

Expression of fibroblast growth factor receptor 1 correlates inversely with the efficacy of single-agent fibroblast growth factor receptor-specific inhibitors in pancreatic cancer.

Elevated fibroblast growth factor receptor (FGFR) activity correlates with pancreatic adenocarcinoma (PDAC) progression and poor prognosis. However, its potential as a therapeutic target remains largely unexplored. The mechanisms of action and therapeutic effects of selective pan-FGFR inhibitors (pan-FGFRi) were explored using in vitro and in vivo PDAC models ranging from gemcitabine-sensitive to highly gemcitabine-resistant (GemR). Gain-/loss-of-function investigations were employed to define the role of individual FGFRs in cell proliferation, migration, and treatment response and resistance. The pan-FGFRi NVP-BGJ398 significantly inhibited cell proliferation, migration, and invasion, and downregulated key cell survival- and invasiveness markers in multiple PDAC cell lines. Gemcitabine is a standard-of-care for PDAC, but development of resistance to gemcitabine (GemR) compromises its efficacy. Acquired GemR was modelled experimentally by developing highly GemR cells using escalating gemcitabine exposure in vitro and in vivo. FGFRi treatment inhibited GemR cell proliferation, migration, GemR marker expression, and tumour progression. FGFR2 or FGFR3 loss-of-function by shRNA knockdown failed to decrease cell growth, whereas FGFR1 knockdown was lethal. FGFR1 overexpression promoted cell migration more than proliferation, and reduced FGFRi-mediated inhibition of proliferation and migration. Single-agent FGFRi suppressed the viability and growth of multiple patient-derived xenografts inversely with respect to FGFR1 expression, underscoring the influence of FGFR1-dependent tumour responses to FGFRi. Importantly, secondary data analysis showed that PDAC tumours expressed FGFR1 at lower levels than in normal pancreas tissue. Single-agent FGFR inhibitors mediate selective, molecularly-targeted suppression of PDAC proliferation, and their effects are greatest in PDAC tumours expressing low-to-moderate levels of FGFR1.

SPATIAL DISTRIBUTION AND FUNCTIONAL RELEVANCE OF FGFR1 AND FGFR2 EXPRESSION FOR GLIOBLASTOMA TUMOUR INVASION

Abstract AIMS Glioblastoma (GBM) is the most common brain tumour in adults. The GBM microenvironment is made up of heterogeneous GBM cell populations, but how microenvironmental signalling pathways contribute to GBM het- erogeneity and specific GBM pathological features remains incompletely understood. Here, we compare expression of fibroblast growth factor receptors (FGFRs) within the tumour core and the invasion front, specifically investigating the functional relevance of FGFR1 and FGFR2 for in GBM invasion in vitro and in vivo. METHOD Quantification of the relative expression levels of FGFR1 and FGFR2 was determined in GBM primary patient- derived cell lines and xenograft models. Additional transcriptional profiling using RNA sequencing identified potential gene-regulatory networks involved in FGFR1-driven GBM tumour invasion. RESULTS We found FGFR1 distributed across the tumour mass and expressed in invasive GBM cells, while FGFR2 expression is confined to the tumour mass only. Loss of FGFR1, but not FGFR2, significantly reduced cell migration in vitro and tumour invasion in human GBM xenografts. Comparative analysis of RNA-sequencing data of FGFR1 and FGFR2 knockdown GBM cells revealed a FGFR1-specific gene regulatory network associated with tumour invasion. CONCLUSIONS Differential expression of FGFR1 and FGFR2 on invasive GBM cells identified FGFR1 is functionally relevant for tumour invasion in GBM. New gene candidates linked to FGFR1-mediated GBM invasion have been revealed and warrant further investigation to identify how FGFR1 regulates GBM invasion.

249-LB: CRISPR/Cas9-Mediated Deletion of FGF Receptor 1 in AgRP Neurons Results in Obesity and Impaired Glucose Metabolism

Obesity remains a growing epidemic as current treatment approaches have limited success. We have recently identified hypothalamic α-Klotho as a novel metabolic regulator; however, the downstream pathways remain unclear. Using a Cre-dependent CRISPR/Cas9 system alongside transgenic mice to investigate the role of FGFR1 in defined neuron populations of adult mice, we show that deletion of FGFR1 in AgRP neurons results in increased weight gain and adiposity, worsened glucose tolerance, and increased liver lipid accumulation. Mechanistically, deletion of FGFR1 negates pERK signaling and the ability of α-Klotho to reduce fasting-induced AgRP neuron activity in mice. While in hypothalamic cells, knockdown of FGFR1 prevents FOXO1 phosphorylation by α-Klotho, providing evidence of an α-Klotho-FGFR1-PI3K-FOXO1 signaling axis in AgRP neurons. Taken together, our data demonstrate the metabolic role of FGFR1 in AgRP neurons and identify it as a critical regulator of energy and glucose homeostasis. Disclosure D. Shookster: None. H. Huang: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DK121215 to H.H.)

ATF4 renders human T-cell acute lymphoblastic leukemia cell resistance to FGFR1 inhibitors through amino acid metabolic reprogramming.

Abnormalities of FGFR1 have been reported in multiple malignancies, suggesting FGFR1 as a potential target for precision treatment, but drug resistance remains a formidable obstacle. In this study, we explored whether FGFR1 acted a therapeutic target in human T-cell acute lymphoblastic leukemia (T-ALL) and the molecular mechanisms underlying T-ALL cell resistance to FGFR1 inhibitors. We showed that FGFR1 was significantly upregulated in human T-ALL and inversely correlated with the prognosis of patients. Knockdown of FGFR1 suppressed T-ALL growth and progression both in vitro and in vivo. However, the T-ALL cells were resistant to FGFR1 inhibitors AZD4547 and PD-166866 even though FGFR1 signaling was specifically inhibited in the early stage. Mechanistically, we found that FGFR1 inhibitors markedly increased the expression of ATF4, which was a major initiator for T-ALL resistance to FGFR1 inhibitors. We further revealed that FGFR1 inhibitors induced expression of ATF4 through enhancing chromatin accessibility combined with translational activation via the GCN2-eIF2α pathway. Subsequently, ATF4 remodeled the amino acid metabolism by stimulating the expression of multiple metabolic genes ASNS, ASS1, PHGDH and SLC1A5, maintaining the activation of mTORC1, which contributed to the drug resistance in T-ALL cells. Targeting FGFR1 and mTOR exhibited synergistically anti-leukemic efficacy. These results reveal that FGFR1 is a potential therapeutic target in human T-ALL, and ATF4-mediated amino acid metabolic reprogramming contributes to the FGFR1 inhibitor resistance. Synergistically inhibiting FGFR1 and mTOR can overcome this obstacle in T-ALL therapy.

Abstract 412: Drug combination screen identifies pemigatinib, an FGFR inhibitor, as a mechanism to overcome KRASG12C inhibitor resistance in lung cancer

Abstract Background: Lung cancer is one of the most common tumors, accounting for approximately 1.8 million deaths worldwide in 2020. KRAS is a frequently mutated oncogene, with mutations reported in roughly 20-25% of non-small cell lung cancer cases. Specifically, KRAS mutations at amino acid 12, resulting in a glycine to cysteine (G12C) substitution, occur in approximately 13% and 3% of lung and colon cancers, respectively, and less frequently in other solid tumors. Recently, the development of covalent KRASG12C inhibitors has shown meaningful anticancer activity in patients. However, many patients with KRASG12C do not respond and/or develop resistance to single-agent treatment. Here we investigate combination therapies that may overcome resistance and broaden patient response to KRASG12C inhibitors. Methods: In vitro preclinical KRASG12C models including lung and colon cancer cell lines were treated with a panel of small molecule and covalent KRASG12C inhibitors. The activity of single-agent versus combination treatments was measured in cell viability assays, and screen hits were validated by in vitro mechanism of action studies. Results: A curated set of 152 compounds was used in combination with screen monitoring cell-viability assays across 12 KRAS mutant lung and colon cancer cell lines. Compounds tested included kinase inhibitors targeting oncogenic signaling pathways, epigenetics modifiers, regulators of apoptosis, chemotherapeutics, and other anticancer agents. The screen identified synergy between covalent KRASG12C and several other inhibitors including those for FGFR, EGFR, and SHP2. Notably, pemigatinib, a potent and selective FGFR1-3 inhibitor, had a significantly high Bliss synergy score. Selectivity and siRNA knockdown experiments were performed to identify the specific FGFR isoform involved in synergistic anticancer activity. Inhibition of FGFR1 activity was shown to be essential, whereas an FGFR2-3 specific inhibitor demonstrated only modest activity in combination with KRASG12C inhibitors. Additionally, FGFR1 knockdown combined with KRASG12C inhibition resulted in increased drug sensitivity; in contrast, knockdown of other FGFR family members did not demonstrate similar increase in sensitivity to KRASG12C inhibition. Conclusions: Cell-viability combination screening identified strong synergy between KRASG12C covalent inhibitors and the FGFR1-3 inhibitor pemigatinib in KRASG12C cancer cell lines. The synergistic effect of pemigatinib and KRASG12C inhibition was notably strongest in mesenchymal cancer cell lines. This effect was further validated in in vitro selectivity and siRNA studies. Together, our results support pemigatinib as a promising agent for combination therapy with KRAS inhibitors in lung cancer. Citation Format: Angela Abdollahi, Margaret Favata, Darlise DiMatteo, Sean Schuette, Erika Boarder, Mark Rupar, Ricardo Macarron, Aidan Gilmartin, Hui Wang, Alejandro Amador-Arjona. Drug combination screen identifies pemigatinib, an FGFR inhibitor, as a mechanism to overcome KRASG12C inhibitor resistance in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 412.

FGFR1 promotes tumor immune evasion via YAP-mediated PD-L1 expression upregulation in lung squamous cell carcinoma

BackgroundVariations in FGFR1 are common driver mutations of LSQCC. And immune checkpoint inhibitors targeting PD-1 and PD-L1 are powerful anticancer weapons. Activation of FGFR1 leads to tumorigenesis through multiple downstream molecules, including YAP, but whether and how FGFR1 regulates tumor immune evasion remain largely unclear. MethodsLSQCC cells were modified to increase or decrease the expression of FGFR1, YAP and PD-L1, as assessed by molecular assays. After FGFR1 knockdown, cancer cells were assessed after cocultured with Jurkat T cells in vitro, and the tumor microenvironment were analyzed in C57BL/6 mice. The effect of the combination of FGFR1 knockdown and PD-1 blockade was also explored. ResultsIn human LSQCC, activation of FGFR1 was positively correlated with transcription of PD-L1. In H520 and HCC95 cells, FGFR1 upregulated PD-L1 expression via YAP, and YAP initiated the transcription of PD-L1 after binding to its promoter region. FGFR1 knockdown decreased tumor growth, reduced immune escape and induced reactivation of CD8+ T cells. The combination of FGFR1 knockdown and PD-1 blockade synergistically exerted antitumor effects. ConclusionsThe FGFR1/YAP/PD-L1 regulatory axis mediates tumor-associated immune suppression in lung squamous cell carcinoma, and FGFR1 knockdown reactivates T cells in the tumor microenvironment. Synergistic inhibition of both FGFR1 and PD-1/PD-L1 pathways may be a possible treatment for lung cancer patients.

Nuclear Localization of Fibroblast Growth Factor Receptor 1 in Breast Cancer Cells Interacting with Cancer Associated Fibroblasts.

Cancer-associated fibroblasts (CAFs) represent a major component of the tumor microenvironment and interplay with cancer cells by secreting cytokines, growth factors and extracellular matrix proteins. When estrogen receptor-negative breast cancer MDA-MB-231 cells were treated with the CAF-conditioned medium (CAF-CM), Akt and STAT3 involved in cell proliferation and survival were activated through phosphorylation. CAFs secrete fibroblast growth factor 2 (FGF2), thereby stimulating breast cancer cell progression. Akt activation induced by CAF-CM in MDA-MB-231 cells was abolished when FGF2-neutralizing antibody was added. Treatment of MDA-MB-231 cells directly with FGF2 enhanced the phosphorylation of Akt and the FGF receptor (FGFR) substrate, FRS2α. These events were abrogated by siRNA-mediated silencing of FGFR1. In a xenograft mouse model, co-injection of MDA-MB-231 cells with activated fibroblasts expressing FGF2 dramatically enhanced activation of Akt. Stable knockdown of FGFR1 blunted Akt phosphorylation in xenograft tumors. MDA-MB-231 cells co-cultured with CAFs or directly stimulated with FGF2 exhibited enhanced nuclear localization of FGFR1. Notably, FGF2 stimulation produced reactive oxygen species (ROS) accumulation in MDA-MB-231 cells, and FGF2-induced nuclear accumulation of FGFR1 was abrogated by the ROS scavenging agent, N-acetylcysteine.

Sulforaphane ameliorates non-alcoholic fatty liver disease in mice by promoting FGF21/FGFR1 signaling pathway.

Most studies regarding the beneficial effect of sulforaphane (SFN) on non-alcoholic fatty liver disease (NAFLD) have focused on nuclear factor E2-related factor 2 (Nrf2). But the molecular mechanisms underlying the beneficial effect of SFN in the treatment of NAFLD remain controversial. Fibroblast growth factor (FGF) 21 is a member of the FGF family expressed mainly in liver but also in adipose tissue, muscle and pancreas, which functions as an endocrine factor and has been considered as a promising therapeutic candidate for the treatment of NAFLD. In the present study we investigated whether FGF21 was involved in the therapeutic effect of SFN against NAFLD. C57BL/6J mice were fed a high-fat diet (HFD) for 12 weeks to generate NAFLD and continued on the HFD for additional 6 weeks with or without SFN treatment. We showed that administration of SFN (0.56 g/kg) significantly ameliorated hepatic steatosis and inflammation in NAFLD mice, along with the improved glucose tolerance and insulin sensitivity, through suppressing the expression of proteins responsible for hepatic lipogenesis, while enhancing proteins for hepatic lipolysis and fatty acids oxidation. SFN administration significantly increased hepatic expression of FGFR1 and fibroblast growth factor 21 (FGF21) in NAFLD mice, along with decreased phosphorylation of p38 MAPK (the downstream of FGF21). HepG2 cells were treated in vitro with FFAs (palmitic acid and oleic acid) followed by different concentrations of SFN. We showed that the effects of SFN on FGF21 and FGFR1 protein expression were replicated in FFAs-treated HepG2 cells. Moreover, the increased FGFR1 protein occurred earlier than increased FGF21 protein. Interestingly, the rapid effect of SFN on FGFR1 protein was not regulated by the FGFR1 gene transcription. Knockdown of FGFR1 and p38 genes weakened SFN-reduced lipid deposition in FFAs-treated HepG2 cells. SFN administration in combination with rmFGF21 (1.5 mg/kg, i.p., every other day) for 3 weeks further suppressed hepatic steatosis in NAFLD mice. In conclusion, SFN ameliorates lipid metabolism disorders in NAFLD mice by upregulating FGF21/FGFR1 pathway. Our results verify that SFN may become a promising intervention to treat or relieve NAFLD.

Metabolic flexibility maintains proliferation and migration of FGFR signaling–deficient lymphatic endothelial cells

Metabolic flexibility is the capacity of cells to alter fuel metabolism in response to changes in metabolic demand or nutrient availability. It is critical for maintaining cellular bioenergetics and is involved in the pathogenesis of cardiovascular disease and metabolic disorders. However, the regulation and function of metabolic flexibility in lymphatic endothelial cells (LECs) remain unclear. We have previously shown that glycolysis is the predominant metabolic pathway to generate ATP in LECs and that fibroblast growth factor receptor (FGFR) signaling controls lymphatic vessel formation by promoting glycolysis. Here, we found that chemical inhibition of FGFR activity or knockdown of FGFR1 induces substantial upregulation of fatty acid β-oxidation (FAO) while reducing glycolysis and cellular ATP generation in LECs. Interestingly, such compensatory elevation was not observed in glucose oxidation and glutamine oxidation. Mechanistic studies show that FGFR blockade promotes the expression of carnitine palmitoyltransferase 1A (CPT1A), a rate-limiting enzyme of FAO; this is achieved by dampened extracellular signal–regulated protein kinase activation, which in turn upregulates the expression of the peroxisome proliferator–activated receptor alpha. Metabolic analysis further demonstrates that CPT1A depletion decreases total cellular ATP levels in FGFR1-deficient rather than wildtype LECs. This result suggests that FAO, which makes a negligible contribution to cellular energy under normal conditions, can partially compensate for energy deficiency caused by FGFR inhibition. Consequently, CPT1A silencing potentiates the effect of FGFR1 knockdown on impeding LEC proliferation and migration. Collectively, our study identified a key role of metabolic flexibility in modulating the effect of FGFR signaling on LEC growth.

The Regulatory Activity of GIPC1 on RhoA‐Mediated bFGF Angiogenic Functions

Introduction Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are the best characterized pro-angiogenic factors involved in tumor angiogenesis. Acquired resistance upon anti-VEGF therapy remains a serious disadvantage of current approaches. Here we show that the small GTPase RhoA, which is known to regulate VEGF-induced angiogenesis, also regulates bFGF-induced angiogenesis and we further explore the role of the scaffolding protein GAIP Interacting Protein C-terminus (GIPC1) as a regulator of RhoA activation and bFGF angiogenic outcomes. Methods We performed in vitro angiogenesis assays (cell migration, tube formation, spheroid sprouting) to assess the impact of pharmacological inhibition or knockdown of endothelial RhoA on bFGF-induced angiogenesis using human umbilical vein endothelial cells (HUVECs). RhoA and GEF Pull-down experiments, along with other biochemical assays were performed to identify the molecular mechanism(s) of bFGF-induced RhoA activation and regulatory function of GIPC1. In vivo, endothelial-specific inducible RhoA-deficient mice with a fluorescent reporter were used in a modified matrigel plug angiogenesis assay and in the ex vivo aortic ring assay. Results In vitro, bFGF-induced angiogenesis was abrogated by pharmacological RhoA inhibition or RhoA knockdown. In vivo, tamoxifen-induced endothelial RhoA-deficiency blocked bFGF-induced angiogenesis. Mechanistically, bFGF activates RhoA, through the RhoA-specific GEFs GEFH1, P115, LARG and PDZ-RhoGEF. Among the bFGF receptors, FGFR1 is predominantly expressed in HUVECs and FGFR1 knockdown abrogated bFGF-induced RhoA activation and angiogenesis. The scaffolding protein GIPC1 interacts with FGFR1 and has an inhibitory effect, since its downregulation induced RhoA activation as well as basal and bFGF-induced angiogenesis. Ongoing experiments are aiming to further delineate the FGFR1-GIPC1-RhoA relationship. Conclusions Our data suggest that GIPC1 is an important element of bFGF-induced angiogenesis through its regulatory effect on endothelial RhoA.

Atractylenolide III predisposes miR-195-5p/FGFR1 signaling axis to exert tumor-suppressive functions in liver cancer.

Antineoplastic activity of atractylenolide III (ATL) has been reported in several malignant tumors. However, its activity has not been completely clarified in hepatocellular carcinoma (HCC). Herein, anticancer effects and underlying molecular mechanisms of ATL were investigated in HCC cells in vitro. Cell viability was evaluated by CCK-8 assay. Cell migration and invasion were evaluated using the transwell assay. TUNEL staining was performed to evaluate cell apoptosis. Protein expression was measured by western blotting analysis. Online database TargetScan and luciferase reporter gene analysis were performed to validate FGFR1 as a target of miR-195-5p. HepG2 and SMMC7721 cell growth, migration, and invasion were inhibited by ATL treatment in a dose-dependent pattern. ATL treatment-induced apoptosis of HepG2 and SMMC7721 cells. Intriguingly, ATL treatment unexpectedly inhibited FGFR1 protein expression in HepG2 and SMMC7721 cells. Knockdown of FGFR1 inhibited proliferation, migration, and invasion, and evoked apoptosis of HepG2 and SMMC7721 cells. We also found that ATL treatment could increase the expression of miR-195-5p, which as a posttranscriptional targeted FGFR1. In HCC tissues, miR-195-5p expression is negatively correlated with FGFR1. Furthermore, the antiproliferative and proapoptotic roles of miR-195-5p were neutralized by overexpressed FGFR1 in HCC cells. ATL effectively repressed growth and induced apoptosis of human HCC cells through the upregulation of miR-195-5p to downregulate FGFR1 expression. Atractylenolide III as a bioactive anticancer adjuvant medication will provide chemosensitization strategy for reversing the drug resistance of HCC.

Integrated Analysis of Key Genes for FGFR1 Knockdown in Mantle Cell Lymphoma Cell Line (Z-138)

Background:Mantle cell lymphoma (MCL) is the secondary common B cell lymphoma subtype that comprises 6 to 8% of non-Hodgkin's lymphoma, and is closely related to the poor clinical outcomes. Previous studies have focussed on the mechanisms mediating ibrutinib resistance, the first-in-class oral covalent inhibitor of Bruton's tyrosine kinase (BTK). The aims of the this study is to identify key genes related to the FGFR1 Knockdown in Mantle cell lymphoma cell line (Z-138) that has been proved to play a vital role in MCL progression. Methods:GSE138127 mRNA microarray datasets from Gene Expression Omnibus (GEO) were analysed to obtain differentially expressed genes (DEGs) between vector control and the Knockdown of FGFR1 charactered by ibrutinib resistance. The GO and GSEA were carried out by WEB-based GEne SeT AnaLysis Toolkit (WebGestalt) to do the functional enrichment analysis. The network analysis of protein-protein interactions (PPIs), TF-gene interaction were carried out by Network Analyst 3.0 to identify hub genes. And the main hub gene was probed using Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis. Results:In total, 175 DEGs were obtained, of which 87 and 88 were up- and down-regulated, respectively. Three hub genes (CDK1, CCND1) were identified and associated to cell cycle and DNA replication. FOXC1 were identified as the potential Transcription factors in the biological process. Conclusion:CDK1, CCND1 may affect the cell cycle regulated by FOXC1, and represent the new candidate molecular markers of the occurrence of ibrutinib resistance. Keywords: Mantle cell lymphoma (MCL), ibrutinib resistance, Network Analyst, Microarray, Protein-protein interactions, Molecular markers Disclosures No relevant conflicts of interest to declare.

Anlotinib can overcome acquired resistance to EGFR-TKIs via FGFR1 signaling in non-small cell lung cancer without harboring EGFR T790M mutation.

BackgroundAlthough many studies have defined mechanisms of resistance to EGFR‐TKIs, acquired resistance remains the major limitation of monotherapy with EGFR‐TKIs.MethodsCell viability was analyzed using a Cell Counting Kit‐8 (CCK‐8) assay. EGFR T790M mutation was sequenced on a HiSeq 4000 platform. mRNAs from HCC827 and HCC827 gefitinib‐resistant (GR) cells were analyzed by genome analyzer‐based deep sequencing. The effect of anlotinib on apoptosis and cell cycle arrest of HCC827 GR was detected by fluorescence‐activated cell sorting (FACS) analysis. A mouse xenograft model was used to assess the effect of anlotinib on HCC827 GR cells.ResultsThe T790M mutation was found in the PC‐9 GR cell line but not in the HCC827 GR cell line. Anlotinib could suppress the growth of HCC827 GR cells by inhibiting FGFR1 in vitro and in a mouse xenograft model. Moreover, FGFR1 was overexpressed in HCC827 GR cells, and the knockdown of FGFR1 reversed gefitinib resistance in HCC827 GR cells. Furthermore, anlotinib induced apoptosis and cell cycle arrest in HCC827 GR cells by increasing the activity of Caspase‐3.ConclusionsFGFR1 overexpression could be the mechanism of EGFR‐TKI acquired resistance and anlotinib can suppresse the growth of EGFR‐TKI‐resistant NSCLC cells without T790M mutation.

Fibrosis growth factor 23 is a promoting factor for cardiac fibrosis in the presence of transforming growth factor-β1.

Myocardial fibrosis is often associated with cardiac hypertrophy; indeed, fibrosis is one of the most critical factors affecting prognosis. We aimed to identify the molecules involved in promoting fibrosis under hypertrophic stimuli. We previously established a rat model of cardiac hypertrophy by pulmonary artery banding, in which approximately half of the animals developed fibrosis in the right ventricle. Here, we first comprehensively analyzed mRNA expression in the right ventricle with or without fibrosis in pulmonary artery banding model rats by DNA microarray analysis (GSE141650 at NCBI GEO). The expression levels of 19 genes were up-regulated more than 1.5-fold in fibrotic hearts compared with non-fibrotic hearts. Among them, fibrosis growth factor (FGF) 23 showed one of the biggest increases in expression. Real-time PCR analysis also revealed that, among the FGF receptor (FGFR) family, FGFR1 was highly expressed in fibrotic hearts. We then found that FGF23 was expressed predominantly in cardiomyocytes, while FGFR1 was predominantly expressed in fibroblasts in the rat ventricle. Next, we added FGF23 and transforming growth factor (TGF)-β1 (10-50 ng/mL of each) to isolated fibroblasts from normal adult rat ventricles and cultured them for three days. While FGF23 itself did not directly affect the expression levels of any fibrosis-related mRNAs, FGF23 enhanced the effect of TGF-β1 on increasing the expression levels of α-smooth muscle actin (α-SMA) mRNA. This increase in xx-SMA mRNA levels due to the combination of TGF-β1 and FGF23 was attenuated by the inhibition of FGFR1 or the knockdown of FGFR1 in fibroblasts. Thus, FGF23 synergistically promoted the activation of fibroblasts with TGF-β1, transforming fibroblasts into myofibroblasts via FGFR1. Thus, we identified FGF23 as a paracrine factor secreted from cardiomyocytes to promote cardiac fibrosis under conditions in which TGF-β1 is activated. FGF23 could be a possible target to prevent fibrosis following myocardial hypertrophy.

Endothelial Small GTPase RhoA: A Potential Target for Anti‐angiogenic Therapy

ObjectiveTumor vessels display abnormal structure and function due to imbalanced angiogenesis. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) are the best known pro‐angiogenic factors in this process. The goal of antiangiogenic therapy is to inhibit tumor growth by oxygen and nutrient deprivation. Current antiangiogenic therapies target VEGF/VEGFR signaling pathways. However development of resistance due to upregulation of bFGF remains a serious disadvantage of current approaches, leading to efforts of simultaneous targeting of both VEGF and FGF pathways. A small GTPase RhoA has been reported to regulate VEGF‐induced angiogenesis. In the present study we investigate the role of endothelial RhoA in bFGF‐induced angiogenesis to identify whether RhoA can be considered a target for both VEGF‐ and FGF‐induced angiogenesis.MethodsWe performed RhoA pulldown assays to identify RhoA activation downstream of bFGF treatment. We used Boyden chamber cell migration assay, 2D tube formation assay using matrigel and 3D sprouting assay with collagen to assess angiogenic potential. Endothelial RhoA was knocked down by siRNAs or blocked by pharmacological inhibitors. Biochemical assays were performed to identify the molecular mechanism of bFGF‐induced RhoA activation. In vivo, endothelial‐specific inducible RhoA‐deficient mice with a fluorescent reporter were used in a newly modified matrigel plug angiogenesis model allowing identification of endothelial RhoA excision efficiency.ResultsbFGF treatment activates RhoA in human umbilical vein endothelial Cells (HUVEC). RhoA inhibition with C3 toxin or RhoA knockdown abrogated bFGF‐induced endothelial cell migration and tube formation in vitro. In vivo, endothelial RhoA deficiency blocked bFGF‐induced angiogenesis in matrigel plug assay. Among the bFGF receptors, FGFR1 is predominantly expressed in HUVECs and FGFR1 knockdown abrogated bFGF‐induced RhoA activation and angiogenesis. RhoA inhibition partially blocked bFGF‐induced JNK phosphorylation but did not affect ERK and p38 activation. Ongoing experiments are aiming to delineate the precise role of endothelial RhoA in the FGF downstream pathway.ConclusionCollectively, our data suggest that RhoA pathway participates in bFGF‐induced angiogenesis and can be a potential target for anti angiogenic therapy.

FGF signaling in mammary gland fibroblasts regulates multiple fibroblast functions and mammary epithelial morphogenesis.

Fibroblast growth factor (FGF) signaling is crucial for mammary gland development. Although multiple roles for FGF signaling in the epithelium have been described, the function of FGF signaling in mammary stroma has not been elucidated. In this study, we investigated FGF signaling in mammary fibroblasts. We found that murine mammary fibroblasts express FGF receptors FGFR1 and FGFR2 and respond to FGF ligands. In particular, FGF2 and FGF9 induce sustained ERK1/2 signaling and promote fibroblast proliferation and migration in 2D cultures. Intriguingly, only FGF2 induces fibroblast migration in 3D extracellular matrix (ECM) through regulation of actomyosin cytoskeleton and promotes force-mediated collagen remodeling by mammary fibroblasts. Moreover, FGF2 regulates production of ECM proteins by mammary fibroblasts, including collagens, fibronectin, osteopontin and matrix metalloproteinases. Finally, using organotypic 3D co-cultures we show that FGF2 and FGF9 signaling in mammary fibroblasts enhances fibroblast-induced branching of mammary epithelium by modulating paracrine signaling, and that knockdown of Fgfr1 and Fgfr2 in mammary fibroblasts reduces branching of mammary epithelium. Our results demonstrate a pleiotropic role for FGF signaling in mammary fibroblasts, with implications for regulation of mammary stromal functions and epithelial branching morphogenesis.

Delayed recanalization at 3 days after permanent MCAO attenuates neuronal apoptosis through FGF21/FGFR1/PI3K/Caspase-3 pathway in rats

Reperfusion exceeded time window may induce ischemia/reperfusion injury, increase hemorrhagic transformation, and deteriorate neurological outcomes in ischemic stroke models. However, the increasing clinical evidences supported that reperfusion even within 6–24 h may salvage ischemic tissue and improve neurological outcomes in selected large vessel occlusion patients, without inducing serious ischemia/reperfusion injury and hemorrhagic transformation. The underlying molecular mechanisms are less clear. In present study, we demonstrated that delayed recanalization at 3 days after permanent middle cerebral artery occlusion (MCAO) decreased infarct volumes and improved neurobehavioral deficits in rats, with no increasing animal mortality and intracerebral hemorrhage. Meanwhile, we observed that endogenous neuroprotective agent fibroblast growth factor 21 (FGF21) significantly increased in serum after MCAO, but which did not synchronously increase in penumbra due to permanent MCAO. Recanalization dramatically increased the endogenous FGF21 expression on neurons in penumbra after MCAO. We confirmed that FGF21 activated the FGFR1/PI3K/Caspase-3 signaling pathway, which attenuated neuronal apoptosis in penumbra. Conversely, knockdown of FGFR1 via FGFR1 siRNA abolished the anti-apoptotic effects of FGF21, and in part abrogated beneficial effects of recanalization on neurological outcomes. These findings suggested that delayed recanalization at 3 days after MCAO improved neurological outcomes in rats via increasing endogenous FGF21 expression and activating FGFR1/PI3K/Caspase-3 pathway to attenuate neuronal apoptosis in penumbra. Delayed recanalization at 3 days after ischemic stroke onset may be a promising treatment strategy in selected patients.

Abstract 3818: BGJ398, a pan-FGFR inhibitor, overcomes paclitaxel resistance in urothelial carcinoma with FGFR1 overexpression

Abstract Introduction: Paclitaxel (PTX) is commonly used to treat urothelial carcinoma (UC) after platinum-based chemotherapy has failed. However, single-agent taxane therapy is not sufficient to inhibit tumor progression and drug resistance in advanced UC. Epithelial-to-mesenchymal transition (EMT) induced by fibroblast growth factor receptor (FGFR)1 signaling has been proposed as a mechanism of PTX resistance, but it is unclear whether this can be overcome by FGFR1 inhibition. The present study investigated whether FGFR1 overexpression contributes to PTX resistance and whether FGFR inhibition can enhance PTX efficacy in UC. Materials and Methods: UC cell lines: UMUC-14, RT4, T24, J82, HTB5 and HTB9 cells were used. Cells were treated with various concentration of PTX (0 to 20 nM) and BGJ398 (0 to 6 μM). The half-maximal inhibitory concentrations (IC50) were measured by the CellTiter-Glo assay. Cell cycle was analyzed after propidium iodide (PI) staining via flow cytometry. Changes in cell cycle, apoptosis and EMT-associated protein levels were assessed by Western Blot analysis. The migratory and colony formative property were analyzed by wound healing assay and colony forming assay, respectively. RNA interference was performed to assess the dependence of PTX sensitivity on pro-apoptotic proteins. Results: We showed that mesenchymal-type T24 and J82 cell lines were highly resistant to PTX (0 to 20 nM) or BGJ398 (0 to 6 μM) monotherapy compared to FGFR3-positive epithelial-type (i.e., RT4 and UMUC-14) UC cell lines. We further found that PTX (10 nM) combined with BGJ398 (6 μM) caused synergistic effects on sub G1 accumulation and apoptosis compared to PTX or BGJ398 monotherapy in T24 and J82 cells, which were accompanied by downregulation of cyclin D1 protein and upregulation of gamma-histone 2A family member X, cleaved caspase-9 and cleaved poly (ADP-ribose) polymerase (PARP). Additionally, 10 nM of PTX combined with 6 μM of BGJ398 synergistically suppressed mesenchymal-type UC cell migration and colony formation via downregulation of EMT-associated factors including snail, slug and ZEB1. FGFR1 knockdown enhanced the antitumor effect of PTX (10 nM) by upregulation of apoptosis-associated markers, cleaved caspase-9 and PARP. Conclusions: We investigated the mechanism of resistance to PTX in UC mediated by FGFR1 and found that combined treatment with BGJ398 can enhance the efficacy of PTX in UC cell lines with mesenchymal features. Our results demonstrate that combination with FGFR1-targeted therapy improves the antitumor efficacy of standard cytotoxic chemotherapy, which is a strategy that warrants further investigation in clinical trials in selected UC patients with FGFR1 overexpression. Citation Format: Haram Ryu, Se Hyun Kim, Chan-Young Ock, Koung Jin Suh, Ji Yun Lee, Ji-Won Kim, Jin Won Kim, Jeong-Ok Lee, Yu Jung Kim, Keun-Wook Lee, Soo-Mee Bang, Jee Hyun Kim, Jong Seok Lee, Joong Bae Ahn, Kui-Jin Kim, Sun Young Rha. BGJ398, a pan-FGFR inhibitor, overcomes paclitaxel resistance in urothelial carcinoma with FGFR1 overexpression [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 3818.

Abstract 5170: Targeting FGFR signaling to disrupt cellular cross-talk in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis with a 5 year survival rate of less than 5%. PDAC tumors consist of a desmoplastic stroma, which limits the effectiveness of chemotherapy. Pancreatic stellate cells (PSCs), which form a key part of this stroma, become activated in response to tumor development. Activated PSCs enter a cross-talk with cancer cells to induce tumor cell proliferation and invasion, leading to metastatic spread. Nuclear fibroblast growth factor receptor 1 (nFGFR1) has been found in PSCs at the invasive edge of PDAC tumors. Inhibition of FGFR1 prevents its nuclear translocation in PSCs, which results in decreased invasion in 3D in vitro PDAC models. Nuclear translocation of FGFR1 in PSCs appears to be a vital mechanism that triggers the transcription of key proteins involved in PDAC invasion. We have used a powerful combination of chromatin immunoprecipitation (ChIP-seq) and sub-cellular mass spectrometry to determine the transcriptional targets of nFGFR1 and consequent sub-cellular protein flux. These techniques have allowed us to dissect the functional consequences of FGFR1 knockdown or inhibition in the PSCs. Candidate drivers of invasion are being validated in state-of-the-art 3D in vitro PDAC models. We have extended these functional studies to combination therapy with the clinical agent gemcitabine (targeting cancer cells) and all-trans retinoic acid (ATRA, modulating PSCs), providing translational relevance for our findings. We are embarking on validating this novel strategy using in vivo co-culture xenograft models with specific reference to FGFR1. Effectively disrupting the cross-talk between the tumor and stroma, either alone or in combination with other therapies, could translate to improved therapeutic responses in PDAC patients in the clinic. Citation Format: Abigail Coetzee, Edward Carter, James Heward, Faraz Mardakheh, Richard Grose, Hemant Kocher. Targeting FGFR signaling to disrupt cellular cross-talk in pancreatic cancer [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 5170.