FGFR1 mRNA Levels Research Articles

MiRNA-470-5p suppresses epithelial-mesenchymal transition of embryonic palatal shelf epithelial cells by targeting Fgfr1 during palatogenesis.

MicroRNAs (miRNAs) have been identified as crucial modulators of gene expression and to play a role in palatogenesis. The aim of this study was to explore the potential role and regulatory mechanisms of miRNAs during palatogenesis. RNA-sequencing was performed to compare the RNA expression profiles of mouse embryonic palatal shelf (MEPS) tissue between an all-trans retinoic acid (ATRA)-induced group and control group, followed by reverse transcription-quantitative polymerase chain reaction for validation, demonstrating upregulated expression of miRNA-470-5p and downregulated expression of Fgfr1 in the ATRA-induced group. The specific binding sites of miRNA-470-5p that potentially govern Fgfr1 expression were predicted by miRanda and TargetScan. The relationship between miRNA-470-5p and Fgfr1 was validated in HEK293T cells by luciferase reporter assays, confirming that miR-470-5p acts directly on the Fgfr1 3'-untranslated region. Fgfr1 mRNA and FGFR1 protein levels were markedly downregulated in MEPS epithelial cells over-expressing miRNA-470-5p. Functional experiments in vitro with CCK-8, cell colony formation, and 5-ethynyl-2'-deoxyuridine (EdU) staining assays revealed that upregulated miRNA-470-5p expression could inhibit the epithelial-mesenchymal transition (EMT) of MEPS epithelial cells by targeting Fgfr1. These findings provide a new molecular mechanism of cleft palate formation, which can inform the development of new treatment and/or prevention targets.

EFFECT OF THYMOQUINONE IN COMBINATION WITH NIVOLUMAB ON EXPERIMENTAL RENAL CELL CANCER MODELS

Objectives: Thymoquinone a bioactive component extracted from the seeds of Nigella sativa, has been proved to have antiinflammatory, antioxidant properties anti antitumor efficiency against breast, prostate, bladder, and renal cell cancer. We aimed to investigate the synergistic cytotoxic and apoptotic effects of thymoquinone in combination with nivolumab, an immuno-control inhibitor, and its mechanism in renal cell cancer models. Materials and Methods: In vitro studies: Caki-1 and Renca renal cancer cell lines with mononuclear cells were cocultured and thymoquinone and nivolumab effect were analyzed by MTT and Annexin V + PI. In vivo studies: Renal cell cancer model was provided by subcutaneous injection of RENCA cells to C57BL/6, male, 8 weeks old mice in four groups. Control group, thymoquinone (1,4,7,14days,20mg/kgIP), nivolumab administered (1,6,13days10mg/ kgIP), thymoquinone and nivolumab. Seven animals was randomized in each experimental group. Apoptosis, necrosis, CD4,CD8,C20,CD34,CD31,VEGF-A by immunohistochemistry, FGFR-1,PDGFRbeta,VEGFR-2,c-kit,RET,c-Met,AXL,Flt-3,TIE2, ICAM-1,VCAM-1 and E-selectin mRNA levels by quantitative real-timePCR were evaluated on tumor tissue. Liver, kidney, brain, lung and heart tissues was evaluated for side effects. Plasma biochemistry tests were performed. Statistical analysis was be performed with Mann-Whitney U test with p <0.05 value. Results: Thymoquinone showed antitumor effect in vitro and in vivo. It also showed increase in immune cell death of nivolumab. Tumor tissue showed prominent necrosis or complete response to therapy in combination group. Conclusions: We conclude that thymoquinone has antitumor effect on renal cell carcinoma and increases the effect of nivolumab immune, cell death effects. Besides, it does not have side effect on other organs whether alone or in combination. 

Abstract 6199: Fusion genes: Novel therapeutic targets for gastric signet ring cell carcinoma

Abstract Background: Gastric cancer (GC) is one of the most common lethal malignancies in the world. In recent years, clinicopathological characteristics of GC are changing, marked by increasing incidence of gastric signet ring cell carcinoma (GSRCC) histology. However, at present, no effective therapeutic targets for GSRCC. Fusion genes are considered as ideal therapeutic targets for various cancers with the development of sequencing technique. We attempted to target fusion genes to treat GSRCC. Methods: We used STAR-Fusion, STAR-Seqr, Arriba to get fusion genes in 417 GC samples in The Cancer Genome Atlas (TCGA). Fusion genes identified simultaneously by at least two software are considered “real” fusion genes. Next, we included 556 GC samples of our specimen bank, including 64 GSRCC in this study. All histopathologic diagnoses were reviewed by at least two senior pathologists independently. Clinical information was retrospectively collected and the overall survival (OS) was measured from the date of surgery to the date of death or the last follow-up visit. Formalin fixed paraffin-embedded (FFPE) tissue were collected for next-generation sequencing (NGS) in a 450-gene panel assay. Results: Among 417 GC samples in TCGA, we detected fusion genes in 283 (68%) samples. On average, each GC samples contained 7 fusion genes. And, a single sample contained a maximum of 85 fusion genes. We noticed that one GC sample had FGFR2-TACC2 fusion, which is recurrent and meaningful in intrahepatic cholangiocarcinoma and urothelium carcinoma. On the other hand, among 556 GC samples from our specimen bank, GSRCC patients had lower tumor mutation burden (TMB-L) (P=0.001) than non-SRCC patients. However, we detected gene fusions in 20.3% (13/64) GSRCC patients. GSRCC patients with gene fusions had a higher N stage (P=0.001) and a higher tumor stage (P=0.010) defined by 8th American Joint Committee on Cancer (AICC) criterion. Moreover, GSRCC patients harboring gene fusions (n=9) had poorer overall survival (OS) in comparison with patients without any gene fusion (n=19) (16.0 months vs 21.0 months, P=0.043). In addition, FGFR2 gene fusions (FGFR2-VTI1A and TACC2-FGFR2) were recurrently detected in 3.1% GSRCC tumor samples. We stably expressed TACC2-FGFR2 in gastric cancer cell lines. Using qRT-PCR, we found FGFR2 mRNA levels were increased in TACC2-FGFR2-expressing GC cells and IHC showed TACC2-FGFR2 upregulated FGFR2 protein expression in TACC2-FGFR2-expressing GC cells. Also, TACC2-FGFR2-expressing GC cells were more sensitive to FGFR2 inhibitors. Conclusions: Our observation revealed that fusion genes were recurrent in GSRCC and might associate to shorter OS. FGFR2 fusion genes were recurrent in GSRCC and FGFR2 inhibitors might be a new therapeutic target for GSRCC. Citation Format: Yue Wang, Tao Shi, Xuan Wang, Jinwei Hu, Lixia Yu, Qin Liu, Nandie Wu, Baorui Liu, Jia Wei. Fusion genes: Novel therapeutic targets for gastric signet ring cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6199.

1957-P: Intermittent Ketogenic Diet Ameliorates Diet-Induced Obesity and Fatty Liver with Improved FGF21 Signaling

Recently, ketogenic diet has been found to be effective on improving obesity. Fibroblast growth factor 21 (FGF-21) is an endocrine factor mainly synthesized in the liver and shown to impact the metabolism of multiple tissues. Here, we investigated the effect of intermittent ketogenic diet on obesity and the sensitivity of FGF-21. Eight-week-old C57BL/6 mice were subjected to a chow diet (CD group), a high fat diet (HFD group), an intermittent chow diet (iCD group) or an intermittent ketogenic diet (iKD group) for 16 weeks. The intermittent diet intervention began with an HFD and was alternated with a chow diet or a ketogenic diet biweekly. Although the average energy intake of iKD group was highest in the four groups, the body weight, fasting blood glucose, the body fat mass and the weight of liver, epididymal adipose tissues (eWAT), subcutaneous adipose tissue (sWAT) and brown adipose tissue (BAT) of iKD group were ameliorated remarkably when compared to HFD group. The insulin sensitivity of iKD group was greatly improved as well. Both the histological results and the quantification of liver triglyceride showed that hepatic steatosis was significantly alleviated in iKD group. Western blotting showed that lipid metabolism in liver was greatly improved by iKD. What’s more, the cold exposure test showed that the iKD group had the best cold tolerance in the four groups. The mRNA expression of FGF-21 was greatly upregulated and hepatic inflammation was ameliorated in livers of iKD group. HE staining showed that the adipocytes in eWAT and sWAT were smaller in iKD group. The whitening of BAT was also improved in iKD group. Both mRNA and protein levels of UCP1 and PGC1α were remarkably upregulated by iKD in eWAT, sWAT and BAT. The mRNA levels of FGFR1 and KLB, which are related to FGF-21 sensitivity, were observed to be improved in eWAT and sWAT. Our data demonstrated that iKD ameliorates diet-induced obesity and fatty liver, and improved insulin sensitivity and cold tolerance with improved FGF-21 signaling. Disclosure W. Guo: None. W. Wang: None. W. Li: None.

Resistance to MET/VEGFR2 Inhibition by Cabozantinib Is Mediated by YAP/TBX5-Dependent Induction of FGFR1 in Castration-Resistant Prostate Cancer

The overall goal of this study was to elucidate the role of FGFR1 induction in acquired resistance to MET and VEGFR2 inhibition by cabozantinib in prostate cancer (PCa) and leverage this understanding to improve therapy outcomes. The response to cabozantinib was examined in mice bearing patient-derived xenografts in which FGFR1 was overexpressed. Using a variety of cell models that reflect different PCa disease states, the mechanism underpinning FGFR1 signaling activation by cabozantinib was investigated. We performed parallel investigations in specimens from cabozantinib-treated patients to confirm our in vitro and in vivo data. FGFR1 overexpression was sufficient to confer resistance to cabozantinib. Our results demonstrate transcriptional activation of FGF/FGFR1 expression in cabozantinib-resistant models. Further analysis of molecular pathways identified a YAP/TBX5-driven mechanism of FGFR1 and FGF overexpression induced by MET inhibition. Importantly, knockdown of YAP and TBX5 led to decreased FGFR1 protein expression and decreased mRNA levels of FGFR1, FGF1, and FGF2. This association was confirmed in a cohort of hormone-naïve patients with PCa receiving androgen deprivation therapy and cabozantinib, further validating our findings. These findings reveal that the molecular basis of resistance to MET inhibition in PCa is FGFR1 activation through a YAP/TBX5-dependent mechanism. YAP and its downstream target TBX5 represent a crucial mediator in acquired resistance to MET inhibitors. Thus, our studies provide insight into the mechanism of acquired resistance and will guide future development of clinical trials with MET inhibitors.

P65 Targets FGFR1 to Regulate the Survival of Ovarian Granulosa Cells.

In female mammals, the abnormal apoptosis of ovarian granulosa cells (GCs) impairs follicular development and causes reproductive dysfunction. Many studies have indicated that the FGFR1 gene of the PI3K signaling pathway and the p65 subunit of the transcription factor NF-κB may regulate the proliferation and apoptosis of GCs involved in follicular development. However, little is known about whether p65 regulates the transcription of FGFR1, as well as the biological effects of p65 and FGFR1 on the survival of GCs and follicular development. In porcine follicles and GCs, we found that p65 and FGFR1 were exclusively expressed in the GCs of follicles, and the mRNA and protein levels of p65 and FGFR1 significantly increased from small to large follicles. Both p65 and FGFR1 were found to activate the PI3K signaling pathway, and the expressions of proliferation markers (PCNA and MKI67) and the anti-apoptotic gene BCL2 were significantly increased by p65 and FGFR1. Furthermore, both p65 and FGFR1 were observed to promote cell proliferation and inhibit the cell apoptosis of GCs, and p65 was confirmed to bind at the −348/−338 region of FGFR1 to positively regulate its transcription. Moreover, p65 was further found to enhance the pro-proliferation and anti-apoptotic effects of FGFR1. Taken together, p65 may target the −348/−338 region of FGFR1, promote the transcription of FGFR1, and enhance the pro-proliferation effect and anti-apoptotic effect of FGFR1 to facilitate the growth of follicles. This study will provide useful information for further investigations on the p65-mediated-FGFR1 signaling pathway during folliculogenesis in mammals.

OR01-3 MicroRNA-34a-Mediated FGF21 Resistance in the Adipose Tissue Contributes to Insulin Resistance and Hypoadiponectinemia in Diet-Induced Obesity

Background and Objective: Fibroblast growth factor 21 (FGF21) is a metabolic hormone with multiple beneficial effects on glucose and lipid homeostasis, in part mediated through its stimulatory effect on adiponectin secretion. However, FGF21 circulating levels are paradoxically elevated in obesity, type 2 diabetes and other obesity-related disorders, suggesting the presence of FGF21 resistance in obesity. MicroRNA-34a (miR-34a) is the most highly elevated microRNA in white adipose tissues of obese mice and a conserved miR-34a seed sequence had been detected within the 3’UTR of FGFR1 and β-klotho. Here we sought to investigate the mediatory role of miR-34a in FGF21resistance. Methods: miR-34a expression and the corresponding changes in the expression of FGFR1 and β-klotho were assessed in visceral adipose tissue from mice with or without diet-induced obesity (DIO). Luciferase assay was used to examine the binding capacity of miR-34a to the FGF21 receptor complex. The ability to reverse FGF21 resistance via inhibiting adipose miR-34a in vivo was evaluated by both lentivirus-mediated miR-34a sponge and adipose tissue specific genetic depletion. Results: A consistent elevation of miR-34a expression in the adipose tissue was observed during the development of DIO, accompanied by a progressive increase in serum FGF21 levels. Following a transient increase at 4 weeks, mRNA levels of FGFR1 and β-klotho markedly decreased with DIO, accompanied by progressive resistance to the actions of exogenous FGF21, in terms of lowering of hyperglycemia and hyperinsulinemia, and adiponectin stimulation. Site-directed mutagenesis and luciferase activity studies showed that miR-34a suppressed the expression of FGFR1 and β-klotho and also adiponectin secretion. Blocking miR-34a actions by in vivo lentivirus-mediated miR-34a sponge or adipose tissue specific gene knock-out counteracted the reduction of FGFR1 and β-klotho expressions in mice with DIO and restored the ability of FGF21 to induce adiponectin secretion in their adipose tissue. Conclusion: During the development of obesity, a progressive increase of miR-34a in visceral adipose tissue is accompanied by a decline in FGF21 sensitivity, leading to hypoadiponectinemia and insulin resistance. The possibility of selective inhibition of miR-34a in the adipose tissue, as a therapeutic approach to obesity-related metabolic disorders, warrants further investigation. Acknowledgement: This work was supported by a grant from the Health & Medical Research Fund (02132836).

Preselection of lung cancer cases using FGFR1 mRNA and gene copy number for treatment with ponatinib.

12095Background: NSCLC and SCLC cell line data suggested high FGFR1 mRNA levels and FGFR1 amplification (FGFR1-AMP) could predict sensitivity to FGFR TKIs. Overlap with KRAS mutations did not precl...

Overexpression of miR-133 decrease primary endothelial cells proliferation and migration via FGFR1 targeting

Angiogenesis is one of the essential hallmarks of cancer that is controlled by the balance between positive and negative regulators. FGFR1 signaling is crucial for the execution of bFGF-induced proliferation, migration, and tube formation of endothelial cells (ECs) and onset of angiogenesis on tumors. The purpose of this study is to identify whether or not miR-133 regulates FGFR1 expression and accordingly hypothesize if it plays a crucial role in modulating bFGF/FGFR1 activity in ECs and blocking tumor angiogenesis through targeting FGFR1. The influences of miR-133 overexpression on bFGF stimulated endothelial cells were assessed by cell growth curve, MTT assaying, tube formation, and migration assays. Forced expression of miR-133 caused significant reductions in bFGF-induced proliferation and migratory ability of ECs. MiR-133 Expression was negatively correlated with both mRNA and protein levels of FGFR1 in the transfected ECs isolated from peripheral blood. Moreover, overexpression of miR-133 drastically reduced the rate of cell division and disturbed capillary network formation of transfected ECs. These findings suggest that miR-133 plays an important function in bFGF-induced angiogenesis processes in ECs and provides a rationale for new therapeutic approaches to suppress tumor angiogenesis and cancer.

Fibroblast growth factor 21 attenuates calcification of vascular smooth muscle cells in vitro.

Vascular calcification is a dysfunction of the vasculature. Recent findings indicate that fibroblast growth factor21 (FGF21), a protector of the cardiovascular system, is related to the mineral deposition of bone and enhances the osteogenic activity of bone morphogenic protein (BMP)-2. In this study, we explored whether FGF21 suppresses vascular calcification. A calcifying model was established by culturing primary rat vascular aortic smooth muscle cells (VSMCs) in a beta-glycerophosphate (BGP)-containing calcifying medium for 14days. In addition, recombinant human FGF21 was applied to protect against VSMC calcification. In the presence of BGP, the expression levels of osteoblastic genes, including alkaline phosphatase (ALP), BMP-2 and runt-related transcription factor (RUNX)-2, were significantly upregulated on day 3, an effect that was maintained through day 14 (P<0.001). A concomitant increase in ALP protein expression was observed through day 9 (P<0.05). The incubation of VSMCs with calcifying medium for 14days increased ALP activity (P<0.05) and led to the formation of visible calcium nodules over the course of the protocol. β-klotho expression was unaltered in BGP-induced VSMCs for the 14-day culture period. The culturing of VSMCs with calcifying medium led to opposing trends in the expression of FGFRs, namely, an increase in FGFR1 and FGFR4 mRNA levels (P<0.001) and a decrease in FGFR2 and FGFR3 mRNA levels (P<0.01). Reduced mineral deposition, in combination with decreased ALP activity (P<0.001) and ALP protein expression (P<0.001), was noted in VSMCs treated with varying doses of FGF21 and BGP in a dose-dependent manner. In addition, FGF21 downregulated osteoblastic-promoting gene expression, including ALP (P<0.001), BMP-2 (P<0.001) and RUNX-2 (P<0.001). Furthermore, FGF21 enhanced β-klotho expression (P<0.05) and increased FGFR1 and FGFR3 mRNA levels (P<0.001). FGFR-1 inhibitor SU5402 blocked partial inhibition of FGF21 on the expression of BMP-2 (P<0.001) and RUNX-2 (P<0.05). Furthermore, FGF21 suppressed the phosphorylation of P38, while P38 inhibitor, SB203580, attenuated the downregulation of RUNX-2 (P<0.05). These data demonstrate FGF21 attenuates VSMC calcification invitro via an FGF21/FGFR1/3/β-klotho/P38MAPK/RUNX-2 signalling pathway.

In situ analysis of FGFR2 mRNA and comparison with FGFR2 gene copy number by dual-color in situ hybridization in a large cohort of gastric cancer patients

BackgroundFibroblast growth factor receptor (FGFR2) has been proposed as a target in gastric cancer. However, appropriate methods to select patients for anti-FGFR2 therapies have not yet been established.MethodsWe used in situ techniques to investigate FGFR2 mRNA expression and gene amplification in a large cohort of 1036 Japanese gastric cancer patients. FGFR2 mRNA expression was determined by RNAscope. FGFR2 gene amplification was determined by dual-color in situ hybridization (DISH).ResultsWe successfully analyzed 578 and 718 samples by DISH and RNAscope, respectively; 2% (12/578) showed strong FGFR2 gene amplification (FGFR2:CEN10 >10); moderate FGFR2 gene amplification (FGFR2:CEN10 <10; ≥2) was detected in 8% (47/578); and high FGFR2 mRNA expression of score 4 (>10 dots/cell and >10% of positive cells with dot clusters under a 20× objective) was seen in 4% (29/718). For 468 samples, both mRNA and DISH data were available. FGFR2 mRNA expression levels were associated with gene amplification; FGFR2 mRNA levels were highest in the highly amplified samples (n = 12). All highly amplified samples showed very strong FGFR2 mRNA expression (dense clusters of the signal visible under a 1× objective). Patients with very strong FGFR2 mRNA expression showed more homogeneous FGFR2 mRNA expression compared to patients with lower FGFGR2 mRNA expression. Gastric cancer patients with tumors that had an FGFR2 mRNA expression score of 4 had shorter RFS compared with score 0–3 patients.ConclusionRNAscope and DISH are suitable methods to evaluate FGFR2 status in gastric cancer. Formalin-fixed paraffin-embedded (FFPE) tissue slides allowed evaluation of the intratumor heterogeneity of these FGFR2 biomarkers.

Abstract 1079: Preclinical activity of the FGFRinhibitor BAY 1163877 alone or in combination with antihormonal therapy in breast cancer

Abstract BAY 1163877 is an orally available, highly potent and selective pan fibroblast growth factor receptor (FGFR) inhibitor. In an ongoing Phase 1 clinical trial (NCT01976741) BAY 1163877 showed clinical responses at exceptional tolerability in patients suffering from different tumor types including urothelial bladder carcinoma or lung tumors, which were selected based on elevated FGFR1-3 mRNA expression. In the preclinical phase, the compound demonstrated significant single agent anti-tumor activity in various tumor models with different FGFR alterations leading to FGFR overexpression (e.g. FGFR gene amplifications or mutations). Genetic alterations of FGFRs can also be found in breast cancer with 7.5 - 17% of all tumors harboring a FGFR1 gene amplification. Elevated FGFR1 mRNA levels can be found in up to 22% of breast cancer cell lines as well as clinical samples. Other FGFR alterations include FGFR2 or FGFR4 gene amplifications as well as elevated FGFR mRNA levels, which were reported in all breast cancer subtypes. We therefore investigated BAY1163877 monotherapy in various breast cancer models. Due to the favorable clinical safety profile of BAY1163877, we also examined a combination treatment with early line antihormonal therapies in hormone receptor positive breast cancer. In vitro profiling of BAY 1163877 in a number of breast cancer cell lines showed a clear association of efficacy with expression levels of different FGFR isoforms. The efficacy was further investigated in several patient- or cell line-derived breast cancer in vivo models. For instance, BAY 1163877 alone dosed 38mg/kg twice daily induced tumor growth inhibition of greater than 90% in a subcutaneous mouse syngeneic 4T1 breast cancer model expressing elevated levels of FGFR2. Resistance to endocrine therapy appears associated with FGFR1 gene amplification and may explain the poor prognosis of FGFR1 overexpressing tumors treated with adjuvant tamoxifen. We therefore investigated the combination of the panFGFR-inhibitor BAY 1163877 with the clinically used antihormonal compound fulvestrant in selected luminal breast cancer PDX models. Some of these models showed resistance to antihormonal treatment in monotherapy but improved in vivo efficacy in combined treatment using BAY 1163877 and fulvestrant. These data may warrant further clinical investigation of BAY1163877alone or in combination with antihormonal therapy in patients with FGFR overexpressing breast cancer. Citation Format: Oliver Politz, Peter Ellinghaus, Sebastian Bender, Sylvia Gruenewald, Franziska Siegel, Marie-Pierre Collin, Sabine Zitzmann-Kolbe, Dominik Mumberg, Karl Ziegelbauer. Preclinical activity of the FGFRinhibitor BAY 1163877 alone or in combination with antihormonal therapy in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1079. doi:10.1158/1538-7445.AM2017-1079

Abstract P6-07-16: Quantitative proteomic analysis of FGFR by mass spectrometry may improve identification of FGFR amplified tumors sensitive to inhibitor therapy

Abstract Background: The fibroblast growth factor receptors 1 and 2 (FGFR1 and FGFR2) have been reported as amplified in multiple solid tumors, including lung, breast and gastric cancers. The FGFR family of proteins is an attractive target for therapy due to the importance of FGFR signaling in the pathogenesis of diverse tumor types. However, pan-FGFR inhibitor have shown only modest efficacy in patients with FGFR1 or FGFR2 gene amplification as determined by fluorescence in situ hybridization (FISH). Gene copy number alterations may not be the optimal predictive biomarker since the targets of these drugs are FGFR proteins; recent findings suggest that direct measurement of the protein targets of FGFR inhibitor therapies may be necessary to assess their treatment efficacy. We therefore developed quantitative FGFR protein assays using selected reaction monitoring mass spectrometry (SRM-MS). We sought to correlate levels of FGFR1, FGFR2 and pan-FGFR (FGFR1-4) proteins as measured by SRM-MS with FGFR gene amplification status as determined by FISH. Methods: Formalin-fixed, paraffin-embedded (FFPE) tissue sections from breast (n=18), esophageal (n=1), gastric (n=1), lung (n=2), and endometrial (n=1) tumors were obtained. A board-certified pathologist marked the tumor area for laser microdissection. Tumor cell proteins were extracted using the Liquid Tissue® process and subjected to SRM-MS for analysis of protein expression levels of FGFR1, FGFR2 and FGFR1-4, as well as other targetable proteins including MET, EGFR, and PD-L1. We compared FGFR protein levels with FGFR amplification defined as FGFR to CEP FISH ratio &amp;gt;2.2. Results: Of 23 tumor samples analyzed, the pan-FGFR1-4 proteomic assay detected FGFR protein in 11 cases (protein expression range: 217.8-3199.5 amol/ug). Ten of these 11 samples (91%) showed FGFR gene amplification. Only a single non-amplified case showed protein expression (235.6 amol/ug). FGFR protein (of 1, 2, and 1-4) was undetectable in 12 samples, of which 5 (42%) harbored FGFR1 amplification. Two of 2 (100%) of FGFR2-amplified cases (average copy number=38) showed high FGFR2 protein expression (3063.0 and 3199.5 amol/ug). Sensitivity of the pan-FGFR1-4 assay was superior to single FGFR assays. Conclusion: A subset of FGFR-amplified tumors does not express FGFR protein when assessed by highly-sensitive SRM-MS. Patients whose tumors do not express FGFR protein are not likely to respond to FGFR inhibitor therapy, as supported by previous findings in squamous cell lung tumors, in which FGFR1 mRNA and/or protein expression levels greatly outperformed FGFR1 gene copy number in predicting sensitivity to a pan-FGFR inhibitors. An approach combining quantitative proteomics and FISH analyses may accurately identify patients most likely to respond to anti-FGFR protein agents. Citation Format: Schwartz S, Tian Y, Fasani R, Diaz Delgado M, Hierro C, Rodon J, Sellappan S, Cecchi F, Hembrough T, Nuciforo P. Quantitative proteomic analysis of FGFR by mass spectrometry may improve identification of FGFR amplified tumors sensitive to inhibitor therapy [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-16.

FGFR1 Expression Levels Predict BGJ398 Sensitivity of FGFR1-Dependent Head and Neck Squamous Cell Cancers.

FGFR1 copy-number gain (CNG) occurs in head and neck squamous cell cancers (HNSCC) and is used for patient selection in FGFR-specific inhibitor clinical trials. This study explores FGFR1 mRNA and protein levels in HNSCC cell lines, primary tumors, and patient-derived xenografts (PDX) as predictors of sensitivity to the FGFR inhibitor, NVP-BGJ398. FGFR1 status, expression levels, and BGJ398 sensitive growth were measured in 12 HNSCC cell lines. Primary HNSCCs (n = 353) were assessed for FGFR1 CNG and mRNA levels, and HNSCC TCGA data were interrogated as an independent sample set. HNSCC PDXs (n = 39) were submitted to FGFR1 copy-number detection and mRNA assays to identify putative FGFR1-dependent tumors. Cell line sensitivity to BGJ398 is associated with FGFR1 mRNA and protein levels, not FGFR1 CNG. Thirty-one percent of primary HNSCC tumors expressed FGFR1 mRNA, 18% exhibited FGFR1 CNG, 35% of amplified tumors were also positive for FGFR1 mRNA. This relationship was confirmed with the TCGA dataset. Using high FGFR1 mRNA for selection, 2 HNSCC PDXs were identified, one of which also exhibited FGFR1 CNG. The nonamplified tumor with high mRNA levels exhibited in vivo sensitivity to BGJ398. FGFR1 expression associates with BGJ398 sensitivity in HNSCC cell lines and predicts tyrosine kinase inhibitor sensitivity in PDXs. Our results support FGFR1 mRNA or protein expression, rather than FGFR1 CNG as a predictive biomarker for the response to FGFR inhibitors in a subset of patients suffering from HNSCC.

Abstract 558: Correlations of FGFR1 with ligand signaling in small-cell lung cancer

Abstract Introduction: Small cell lung cancer (SCLC) accounts for 15% of all lung cancers and has been under-studied relative to novel therapies. Signaling through fibroblast growth factors (FGF2, FGF9) and their high-affinity receptor (FGFR1) has recently emerged as a contributing factor in the pathogenesis and progression of non-small cell lung cancer (NSCLC). However, knowledge of FGFR1 and ligand expression in SCLC is limited. Methods: FGFR1 gene copy number, mRNA levels and protein expression were determined by silver in situ hybridization (SISH), mRNA ISH and immunohistochemistry (IHC) in primary tumors from 78 SCLC patients. Expression of the ligands FGF2 and FGF9 mRNA and protein were determined by mRNA ISH and IHC, respectively. Spearman's correlations were conducted using SAS 9.4. Results: FGFR1 protein expression by IHC demonstrated a significant correlation with FGFR1 gene copy number (p = 0.03) and FGFR1 mRNA levels (p&amp;lt;0.0001). The positivity of FGFR1 mRNA in the SCLC cohort was 21%. FGFR1 mRNA expression correlated with both FGF2 (p = 0.0001) and FGF9 (p = 0.0018) mRNA levels, as well as with FGF2 (0.0105) and FGF9 (0.0012) protein levels. There was no significant association between FGFR1 and ligands with clinical characteristics or prognosis. Conclusions: FGFR1 amplification and mRNA levels correlate with FGFR1 protein expression. The SCLC cohort has a substantially higher proportion of FGFR1 mRNA positive cases (21%) compared to FGFR1 gene copy number positive cases (7.8%). Combined analysis of FGFR1 and ligand expression may be valuable to determine which SCLC patients may respond to FGFR1 inhibitor therapy. Key Words: small cell lung cancer; FGFR1; FGF2; FGF9 Citation Format: Hui Yu, Andrzej Badzio, Theresa Boyle, Xian Lu, Christopher J. Rivard, Ashley Kowalewski, Brad Rikke, Kim Ellison, Leslie Rozeboom, Biftu Hassan, Fred R. Hirsch. Correlations of FGFR1 with ligand signaling in small-cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 558. doi:10.1158/1538-7445.AM2015-558

Nonamplified FGFR1 is a growth driver in malignant pleural mesothelioma.

Malignant pleural mesothelioma (MPM) is associated with asbestos exposure and is a cancer that has not been significantly affected by small molecule-based targeted therapeutics. Previously, we demonstrated the existence of functional subsets of lung cancer and head and neck squamous cell carcinoma (HNSCC) cell lines in which fibroblast growth factor receptor (FGFR) autocrine signaling functions as a nonmutated growth pathway. In a panel of pleural mesothelioma cell lines, FGFR1 and FGF2 were coexpressed in three of seven cell lines and were significantly associated with sensitivity to the FGFR-active tyrosine kinase inhibitor (TKI), ponatinib, both in vitro and in vivo using orthotopically propagated xenografts. Furthermore, RNAi-mediated silencing confirmed the requirement for FGFR1 in specific mesothelioma cells and sensitivity to the FGF ligand trap, FP-1039, validated the requirement for autocrine FGFs. None of the FGFR1-dependent mesothelioma cells exhibited increased FGFR1 gene copy number, based on a FISH assay, indicating that increased FGFR1 transcript and protein expression were not mediated by gene amplification. Elevated FGFR1 mRNA was detected in a subset of primary MPM clinical specimens and like MPM cells; none harbored increased FGFR1 gene copy number. These results indicate that autocrine signaling through FGFR1 represents a targetable therapeutic pathway in MPM and that biomarkers distinct from increased FGFR1 gene copy number such as FGFR1 mRNA would be required to identify patients with MPM bearing tumors driven by FGFR1 activity. FGFR1 is a viable therapeutic target in a subset of MPMs, but FGFR TKI-responsive tumors will need to be selected by a biomarker distinct from increased FGFR1 gene copy number, possibly FGFR1 mRNA or protein levels.

Twist1 correlates with poor differentiation and progression in gastric adenocarcinomaviaelevation of FGFR2 expression

To explore the correlation between Twist-related protein (Twist)1, fibroblast growth factor receptor (FGFR)2 and gastric adenocarcinoma differentiation and progression. We evaluated Twist1 and FGFR2 in 52 gastric adenocarcinoma samples by immunohistochemistry and quantitative real time polymerase chain reaction, and analyzed the correlation between Twist1, FGFR2 and cancer differentiation. We also detected Twist1 and FGFR2 expression in gastric adenocarcinoma cell lines, and evaluated Twist1 influence on FGFR2 expression. In addition, we studied the role of FGFR2 in Twist1-promoted cancer progression, including proliferation, invasion and epithelial-mesenchymal transition (EMT). Twist1 and FGFR2 were detected in almost all the gastric adenocarcinoma samples. Twist1 (P = 0.0213) and FGFR2 (P = 0.0310) mRNA levels had a significant association with gastric adenocarcinoma differentiation. Moreover, Twist1 and FGFR2 expression in poorly differentiated cells (SNU-1 and SNU-16) was notably higher than in well-differentiated cells (MKN-7 and MKN-28). In poorly differentiated gastric adenocarcinomas, FGFR2 mRNA level was significantly positively correlated with Twist1 mRNA level (P = 0.004). Twist1 was proved to promote FGFR2 by regulating Twist1 expression by knockdown and overexpression. Additionally, Twist1 could induce proliferation, invasion and EMT in gastric cancer; of these, FGFR2 was required for invasion and EMT, rather than proliferation. Twist1 and FGFR2 are highly associated with differentiation of gastric adenocarcinoma; Twist1 can facilitate invasion and EMT in gastric adenocarcinoma via promotion of FGFR2 expression.

A New Synthetic FGF Receptor Antagonist Inhibits Arteriosclerosis in a Mouse Vein Graft Model and Atherosclerosis in Apolipoprotein E-Deficient Mice

ObjectiveThe role of fibroblast growth factors (FGFs) in the development of vascular diseases remains incompletely understood. The objective of this study was to examine the effects of a new small-molecule multi-FGF receptor blocker with allosteric properties, SSR128129E, on neointimal proliferation after a vein graft procedure in mice and on the development of atherosclerosis in atherosclerosis-prone apolipoprotein E (apoE)-deficient mice.Methods and ResultsVein grafts were performed in 3 month-old male C57BL6 mice. Segments of the vena cava were interposed at the level of the carotid artery. In SSR128129E (50 mg/kg/d)-treated animals, a dramatic decrease in neointimal proliferation was observed 2 and 8 weeks after the graft (72.5 %, p<0.01, and 47.8 %, p<0.05, respectively). Four-week old male apoE-deficient mice were treated with SSR128129E (50 mg/kg/d) for 3 and 5 months in comparison with a control group. SSR128129E treatment resulted in a reduction of lesion size in the aortic sinus (16.4 % (ns) at 3 months and 42.9 % (p<0.01) at 5 months, without any change in serum lipids. SSR128129 significantly reduced FGFR2 mRNA levels in the aortic sinus (p<0.05, n=5-6), but did not affect the mRNA expression levels of other FGF receptors or ligands.ConclusionThese studies indicate that FGFs have an important role in the development of vascular diseases like atherosclerosis and graft arteriosclerosis. These data suggest that inhibition of FGF receptors by compounds like SSR128129E might be useful as a new therapeutic approach for these vascular pathologies.

Actionable Gene Expression-Based Patient Stratification for Molecular Targeted Therapy in Hepatocellular Carcinoma

BackgroundThe effectiveness of molecular targeted agents is modest in hepatocellular carcinoma (HCC). Efficacy of molecular targeted therapies has been better in cancer patients with high expression of actionable molecules defined as cognate target molecules. However, patient stratification based on the actionable molecules dictating the effectiveness of targeted drugs has remained understudied in HCC.Experimental Design & ResultsPaired tumor and non-tumoral tissues derived from a total of 130 HCC patients were studied. Real-time RT-PCR was used to analyze the mRNA expression of actionable molecules in the tissues. mRNA levels of EGFR, VEGFR2, PDGFRβ, FGFR1, and mTOR were up-regulated in tumors compared to non-tumors in 35.4, 42.3, 61.5, 24.6, and 50.0% of patients, respectively. Up-regulation of EGFR was observed at early stage and tended to gradually decrease toward late stages (BCLC stage A: 41.9%; B: 30.8%; C: 17.6%). Frequency of VEGFR2 expression in tumors at stage C was lower than that in the other stages (BCLC stage A: 45.9%; B: 41.0%; C: 29.4%). PDGFRβ and mTOR were observed to be up-regulated in more than 50% of tumors in all the stages whereas FGFR1 was up-regulated in only about 20% of HCC irrespective of stages. A cluster analysis of actionable gene expression revealed that HCC can be categorized into different subtypes that predict the effectiveness of molecular targeted agents and combination therapies in clinical trials. Analysis of in vitro sensitivity to sorafenib demonstrated that HCC cells with up-regulation of PDGFRβ and c-Raf mRNA are more susceptible to sorafenib treatment in a dose and time-dependent manner than cells with low expression of the genes.ConclusionsmRNA expression analysis of actionable molecules could provide the rationale for new companion diagnostics-based therapeutic strategies in the treatment of HCC.

Abstract 2407: Expression profiling of FGF-receptor pathway genes in squamous NSCLC tissue by Nanostring.

Abstract FGFR1 amplifications have recently been identified in 22% of squamous non-small cell lung cancers (sqNSCLC) (Weiss et al., Sci Transl Med., 2(62), 2010) and in pre-clinical models this amplification confers sensitivity to FGFR inhibitor induced cell growth arrest and death (Zhang et al., Clin. Can. Res., In press). Ongoing clinical trials of FGFR inhibitors are selecting patients based on FGFR1 amplifications, as determined by FISH. The development of additional biomarker assays to evaluate the expression of FGFRs, FGF ligands and other potential modulators of response to support analysis of clinical tissues is warranted. We validated the Nanostring platform to allow gene expression (mRNA) analysis of 194 genes, including FGFRs and FGF ligands, from one 5μm section of formalin fixed paraffin embedded clinical tumor tissue. Intra- and inter-assay variability was low, as was variability between adjacent sections from the same tumor sample. Further validation of a subset of these genes by RT-PCR revealed a good correlation between platforms. A panel of 50 sqNSCLC tumors were analysed for FGFR1 gene amplification by FISH and for gene expression by Nanostring. FGFR1 amplfied tumours were enriched for high FGFR1 mRNA expression (p&amp;lt;0.02) compared to non-amplified samples, although a subset of non-amplified tumours also demonstrated increased FGFR1 mRNA expression. Immunohistochemical analysis, employing an FGFR1 specific antibody, revealed a correlation between FGFR1 mRNA levels and protein expression in these tumors. Nanostring analysis of additional genes on the 8p12 region, revealed that several of these, for example PPAPDC1B, ASH2L, TACC1 and in particular the FGFR1 neighbouring gene WHSC1L1 are highly expressed in FGFR1 amplified tumors (p&amp;lt;0.0001 for WHSC1L1). In contrast, expression of LETM2, located between WHSCL1 and FGFR1, was not significantly increased in amplified tumors. An FGF ligand expression profile was generated for each tumor, showing that a-FGF, b-FGF and FGF7 were the most abundantly expressed ligands in the sqNSCLC samples, with increased b-FGF expression detected in high FGFR1 expressing tumours. The expression of a panel of potential mediators of resistance to FGFR inhibitor therapy such as c-MET, EGFR and IGF1R were also profiled across the samples, revealing differential expression of these factors across each of the tumors and an inverse correlation between FGFR1 expression and ERBB3 expression. Taken together, these data validate the utility of the Nanostring platform for expression analysis of FGFR pathway genes using mRNA isolated from FFPE tumor samples and this platform can be used to assess the level of expression of potential modulators of clinical outcome. Citation Format: Claire Rooney, Dawn Baker, Neil R. Smith, Victoria Williams, Elizabeth A. Harrington, Carl Barrett, Paul D. Smith, Elaine Kilgour. Expression profiling of FGF-receptor pathway genes in squamous NSCLC tissue by Nanostring. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2407. doi:10.1158/1538-7445.AM2013-2407