FGF2 Treatment Research Articles

Paradoxical cancer cell proliferation after FGFR inhibition through decreased p21 signaling in FGFR1-amplified breast cancer cells

Fibroblast growth factors (FGFs) control various cellular functions through fibroblast growth factor receptor (FGFR) activation, including proliferation, differentiation, migration, and survival. FGFR amplification in ER + breast cancer patients correlate with poor prognosis, and FGFR inhibitors are currently being tested in clinical trials. By comparing three-dimensional spheroid growth of ER + breast cancer cells with and without FGFR1 amplification, our research discovered that FGF2 treatment can paradoxically decrease proliferation in cells with FGFR1 amplification or overexpression. In contrast, FGF2 treatment in cells without FGFR1 amplification promotes classical FGFR proliferative signaling through the MAPK cascade. The growth inhibitory effect of FGF2 in FGFR1 amplified cells aligned with an increase in p21, a cell cycle inhibitor that hinders the G1 to S phase transition in the cell cycle. Additionally, FGF2 addition in FGFR1 amplified cells activated JAK-STAT signaling and promoted a stem cell-like state. FGF2-induced paradoxical effects were reversed by inhibiting p21 or the JAK-STAT pathway and with pan-FGFR inhibitors. Analysis of patient ER + breast tumor transcriptomes from the TCGA and METABRIC datasets demonstrated a strong positive association between expression of FGF2 and stemness signatures, which was further enhanced in tumors with high FGFR1 expression. Overall, our findings reveal a divergence in FGFR signaling, transitioning from a proliferative to stemness state driven by activation of JAK-STAT signaling and modulation of p21 levels. Activation of these divergent signaling pathways in FGFR amplified cancer cells and paradoxical growth effects highlight a challenge in the use of FGFR inhibitors in cancer treatment.

Abstract 575: Identification of SORL1 as a key regulator of chemoresistance in ovarian cancer

Abstract This study aimed to understand the molecular mechanism of platinum chemoresistance in recurrent ovarian tumors with a focus on the SORL1-regulated pathways. SORL1 is an intracellular sorting receptor that binds to HER2 in breast cancer; this promotes HER2 recycling to the cell surface, which ultimately impacts the progression and treatment of breast cancer. The role of SORL1 in ovarian cancer has not been extensively studied. Method: RNA was extracted from 15 pairs of matched primary and recurrent high grade serous ovarian tumors collected from patients. Human transcriptomic microarray was used to identify the differentially expressed genes. The identified genes were validated by RT-QPCR and western blot in five ovarian cancer cell lines that were treated or untreated with carboplatin. To study the effects of identified target gene SORL1, overexpression and knockdown in ovarian cancer cell lines were established using SORL1 plasmid and shRNA. Proliferation and response to carboplatin were analyzed using celltiter Glo and caspase-3/7 activity assays respectively. EGFR and FGFR4 were identified as the interacting proteins of SORL1 in ovarian cancer cells through FGF1 and EGF1 treatment and co-immunoprecipitation. A proximity ligation assay was performed to validate their interactions. Finally, a xenograft mouse model was used to compare the tumor forming abilities and the response to carboplatin of SORL1-knockdown ovarian cancer cells and the control cancer cells in vivo. Protein expression of SORL1, EGFR, and FGFR4 in the collected tumors was determined via western blot. Result: Transcriptomic analysis of matched human primary and recurrent ovarian tumors identified SORL1 as an upregulated gene in recurrent tumors compared to primary samples. SORL1 expression was also upregulated in ovarian cancer cells surviving carboplatin treatment. Proliferation assays demonstrated that SORL1-overexpressing and knockdown cell lines had increased and inhibited proliferative capabilities respectively. Caspase-3 activity assays showed that SORL1-overexpressing and knockdown cell lines had reduced and increased sensitivity to carboplatin treatment respectively. SORL1 was found to bind to and promote the expression of EGFR and FGFR4. When injected subcutaneously into nude mice, SORL1-knockdown cells had reduced tumor forming capabilities and increased sensitivity to carboplatin. SORL1-knockdown caused the tumors to express lower levels of SORL1, EGFR, and FGFR4 proteins in comparison with the control tumors. Conclusion: This study demonstrates that SORL1 regulates the EGFR and FGF4 pathways to promote the growth and carboplatin resistance of ovarian cancer cells. Targeting this pathway has potential to aid in the treatment of platinum-resistant ovarian cancer. Citation Format: Miranda Mansolf, Ziyan Jian, Fangfang Bi, Tobias M. Hartwich, Yang Yang-Hartwich. Identification of SORL1 as a key regulator of chemoresistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 575.

Direct programming of human pluripotent stem cells into endothelial progenitors with SOX17 and FGF2

Transcription factors (TFs) are pivotal in guiding stem cell behavior, including their maintenance and differentiation. Using single-cell RNA sequencing, we investigated TFs expressed in endothelial progenitors (EPs) derived from human pluripotent stem cells (hPSCs) and identified upregulated expression of SOXF factors SOX7, SOX17, and SOX18 in the EP population. To test whether overexpression of these factors increases differentiation efficiency, we established inducible hPSC lines for each SOXF factor and found only SOX17 overexpression robustly increased the percentage of cells expressing CD34 and vascular endothelial cadherin (VEC). Conversely, SOX17 knockdown via CRISPR-Cas13d significantly compromised EP differentiation. Intriguingly, we discovered SOX17 overexpression alone was sufficient to generate CD34+VEC+CD31- cells, and, when combined with FGF2 treatment, more than 90% of CD34+VEC+CD31+ EP was produced. These cells are capable of further differentiating into endothelial cells. These findings underscore an undiscovered role of SOX17 in programming hPSCs toward an EP lineage, illuminating pivotal mechanisms in EP differentiation.

FGF10 attenuates allergic airway inflammation in asthma by inhibiting PI3K/AKT/NF-κB pathway

BackgroundThe effect of fibroblast growth factor 10 (Fgf10) against allergic asthma has remained unclear, despite its importance in lung development and homeostasis maintenance. The purpose of this study was to investigate the protective effect and potential mechanism of Fgf10 on asthma. MethodHouse Dust Mite (HDM)-induced asthma mice were administered recombinant Fgf10 intranasally during activation. Flow cytometry and ELISA were performed to determine type of inflammatory cells and type 2 cytokines levels in bronchoalveolar lavage fluid (BALF). Hematoxylin and eosin (H&E) and periodic acid - Schiff (PAS) staining of lung sections were conducted to evaluate histopathological assessment. Transcriptome profiling was analyzed using RNA-seq, followed by bioinformatics and network analyses to investigate the potential mechanisms of Fgf10 in asthma. RT-qPCR was also used to search for and validate differentially expressed genes in human Peripheral Blood Mononuclear Cells (PBMCs). ResultsExogenous administration of Fgf10 alleviated HDM-induced inflammation and mucus secretion in lung tissues of mice. Fgf10 also significantly inhibited the accumulation of eosinophils and type 2 cytokines (IL-4, IL-5, and IL-13) in BALF. The PI3K/AKT/NF-κB pathway may mediate the suppressive impact of Fgf10 on the asthma inflammation. Through RNA-seq analysis, the intersection of 71 differentially expressed genes (DEGs) was found between HDM challenge and Fgf10 treatment. GO and KEGG enrichment analyses indicated a strong correlation between the DEGs and different immune response. Immune infiltration analysis predicted the differential infiltration of five types of immune cells, such as NK cells, dendritic cells, monocytes and M1 macrophages. PPI analysis determined hub genes such as Irf7, Rsad2, Isg15 and Rtp4. Interestingly, above genes were consistently altered in human PBMCs in asthmatic patients. ConclusionAsthma airway inflammation could be attenuated by Fgf10 in this study, suggesting that it could be a potential therapeutic target.

Fibroblast growth factor 10 reverses cigarette smoke- and elastase-induced emphysema and pulmonary hypertension in mice.

COPD is an incurable disease and a leading cause of death worldwide. In mice, fibroblast growth factor (FGF)10 is essential for lung morphogenesis, and in humans, polymorphisms in the human FGF10 gene correlate with an increased susceptibility to develop COPD. We analysed FGF10 signalling in human lung sections and isolated cells from healthy donor, smoker and COPD lungs. The development of emphysema and PH was investigated in Fgf10+/- and Fgfr2b+/- (FGF receptor 2b) mice upon chronic exposure to cigarette smoke. In addition, we overexpressed FGF10 in mice following elastase- or cigarette smoke-induced emphysema and pulmonary hypertension (PH). We found impaired FGF10 expression in human lung alveolar walls and in primary interstitial COPD lung fibroblasts. In contrast, FGF10 expression was increased in large pulmonary vessels in COPD lungs. Consequently, we identified impaired FGF10 signalling in alveolar walls as an integral part of the pathomechanism that leads to emphysema and PH development: mice with impaired FGF10 signalling (Fgf10+/- and Fgfr2b+/- ) spontaneously developed lung emphysema, PH and other typical pathomechanistic features that generally arise in response to cigarette smoke exposure. In a therapeutic approach, FGF10 overexpression successfully restored lung alveolar and vascular structure in mice with established cigarette smoke- and elastase-induced emphysema and PH. FGF10 treatment triggered an initial increase in the number of alveolar type 2 cells that gradually returned to the basal level when the FGF10-mediated repair process progressed. Therefore, the application of recombinant FGF10 or stimulation of the downstream signalling cascade might represent a novel therapeutic strategy in the future.

Fibroblast growth factor 10 ameliorates neurodegeneration in mouse and cellular models of Alzheimer's disease via reducing tau hyperphosphorylation and neuronal apoptosis.

Alzheimer's disease (AD) is characterized with senile plaques formed by Aβ deposition, and neurofibrillary tangles composed of hyperphosphorylated tau protein, which ultimately lead to cognitive impairment. Despite the heavy economic and life burdens faced by the patients with AD, effective treatments are still lacking. Previous studies have reported the neuroprotective effects of FGF10 in CNS diseases, but its role in AD remains unclear. In this study, we demonstrated that FGF10 levels were reduced in the serum of AD patients, as well as in the brains of 3xTg-AD mice and APPswe-transfected HT22 cells, suggesting a close relationship between FGF10 and AD. Further investigations revealed that intranasal delivery of FGF10 improved cognitive functions in 3xTg-AD mice. Additionally, FGF10 treatment reduced tau hyperphosphorylation and neuronal apoptosis, thereby mitigating neuronal cell damage and synaptic deficits in the cortex and hippocampus of 3xTg-AD mice, as well as APPswe-transfected HT22 cells. Furthermore, we evaluated the therapeutic potential of FGF10 gene delivery for treating AD symptoms and pathologies. Tail vein delivery of the FGF10 gene using AAV9 improved cognitive and neuronal functions in 3xTg-AD mice. Similarly, endogenous FGF10 overexpression ameliorated tau hyperphosphorylation and neuronal apoptosis in the cortex and hippocampus of 3xTg-AD mice. Importantly, we confirmed that the FGFR2/PI3K/AKT signaling pathway was activated following intranasal FGF10 delivery and AAV9-mediated FGF10 gene delivery in 3xTg-AD mice and APPswe-transfected HT22 cells. Knockdown of FGFR2 attenuated the protective effect of FGF10. Collectively, these findings suggest that intranasal delivery of FGF10 and AAV9-mediated FGF10 gene delivery could be a promising disease-modifying therapy for AD.

Elucidating Fibroblast Growth Factor–Induced Kinome Dynamics Using Targeted Mass Spectrometry and Dynamic Modeling

Fibroblast growth factors (FGFs) are paracrine or endocrine signaling proteins that, activated by their ligands, elicit a wide range of health and disease-related processes, such as cell proliferation and the epithelial-to-mesenchymal transition. The detailed molecular pathway dynamics that coordinate these responses have remained to be determined. To elucidate these, we stimulated MCF-7 breast cancer cells with either FGF2, FGF3, FGF4, FGF10, or FGF19. Following activation of the receptor, we quantified the kinase activity dynamics of 44 kinases using a targeted mass spectrometry assay. Our system-wide kinase activity data, supplemented with (phospho)proteomics data, reveal ligand-dependent distinct pathway dynamics, elucidate the involvement of not earlier reported kinases such as MARK, and revise some of the pathway effects on biological outcomes. In addition, logic-based dynamic modeling of the kinome dynamics further verifies the biological goodness-of-fit of the predicted models and reveals BRAF-driven activation upon FGF2 treatment and ARAF-driven activation upon FGF4 treatment.

Fibroblast growth factor 2 reduces endoplasmic reticulum stress and apoptosis in in-vitro Non-Alcoholic Fatty Liver Disease model.

Non-Alcoholic fatty liver disease is characterized by the accumulation of excess fat in the liver, chronic inflammation, and cell death, ranging from simple steatosis to fibrosis, and finally leads to cirrhosis and hepatocellular carcinoma. The effect of Fibroblast growth factor 2 on apoptosis and ER stress inhibition has been investigated in many studies. In this study, we aimed to investigate the effect of FGF2 on the NAFLD in-vitro model in the HepG2 cell line. The in-vitro NAFLD model was first induced on the HepG2 cell line using oleic acid and palmitic acid for 24h and evaluated by ORO staining and Real-time PCR. The cell line was then treated with various concentrations of fibroblast growth factor 2 for 24h, total RNA was extracted and cDNA was consequently synthesized. Real-time PCR and flow cytometry was applied to evaluate gene expression and apoptosis rate, respectively. It was shown that fibroblast growth factor 2 ameliorated apoptosis in the NAFLD in-vitro model by reducing the expression of genes involved in the intrinsic apoptosis pathway, including caspase 3 and 9. Moreover, endoplasmic reticulum stress was decreased following upregulating the protective ER-stress genes, including SOD1 and PPARα. FGF2 significantly reduced ER stress and intrinsic apoptosis pathway. Our data suggest that FGF2 treatment could be a potential therapeutic strategy for NAFLD.

FGF1 ameliorates obesity-associated hepatic steatosis by reversing IGFBP2 hypermethylation.

Obesity is a major contributing factor for metabolic-associated fatty liver disease (MAFLD). Fibroblast growth factor (FGF) 1 is the first paracrine FGF family member identified to exhibit promising metabolic regulatory properties capable of conferring glucose-lowering and insulin-sensitizing effect. This study explores the role and molecular underpinnings of FGF1 in obesity-associated hepatic steatosis. In a mouse high-fat diet (HFD)-induced MAFLD model, chronic treatment with recombinant FGF1(rFGF1) was found to effectively reduce the severity of insulin resistance, hyperlipidemia, and inflammation. FGF1 treatment decreased lipid accumulation in the mouse liver and palmitic acid-treated AML12 cells. These effects were associated with decreased mature form SREBF1 expression and its target genes FASN and SCD1. Interestingly, we uncovered that rFGF1 significantly induced IGFBP2 expression at both mRNA and protein levels in HFD-fed mouse livers and cultured hepatocytes treated with palmitic acid. Adeno-associated virus-mediated IGFBP2 suppression significantly diminished the therapeutic benefit of rFGF1 on MAFLD-associated phenotypes, indicating that IGFBP2 plays a crucial role in the FGF1-mediated reduction of hepatic steatosis. Further analysis revealed that rFGF1 treatment reduces the recruitment of DNA methyltransferase 3 alpha to the IGFBP2 genomic locus, leading to decreased IGFBP2 gene methylation and increased mRNA and protein expression. Collectively, our findings reveal FGF1 modulation of lipid metabolism via epigenetic regulation of IGFBP2 expression, and unravel the therapeutic potential of the FGF1-IGFBP2 axis in metabolic diseases associated with obesity.

Spatiotemporal gene targeting in the mouse corneal endothelium.

The inducible Cre-ERT2 recombinase system allows for temporal control of gene targeting, and it is useful to studying adult function of genes that have critical developmental roles. The Zeb1flox/flox: UBC-CreERT2 mouse was generated to conditionally target Zeb1 to investigate its role in mesenchymal transition in the mouse corneal endothelium in vivo. Hemizygous UBC-CreERT2 mice were crossed with homozygous mice harboring loxP-flanked Zeb1 alleles (Zeb1flox/flox) to generate the Zeb1flox/flox: UBC-CreERT2 mouse. 4-hydroxytamoxifen (4-OHT) exposure leads to excision of exon 6 of Zeb1, resulting in a loss function allele in the Zeb1flox/flox: UBC-CreERT2 mouse. Intracameral 4-OHT injection further isolates Zeb1 targeting to the anterior chamber. Mesenchymal transition and induction of Zeb1 expression in the corneal endothelium was achieved using FGF2 in ex vivo organ culture. Gene expression was analyzed by semi-quantitative reverse transcription-polymerase chain reaction and by immunoblotting in the mouse corneal endothelium in vivo. Following Cre-mediated targeting of Zeb1 by intracameral 4-OHT injection in Zeb1flox/flox: UBC-CreERT2 mice, FGF2 treatment in ex vivo organ culture resulted in abrogation of Zeb1 mRNA and protein expression in the corneal endothelium. The data show Zeb1, a critical mediator of fibrosis in corneal endothelial mesenchymal transition, can be targeted by intracameral injection of 4-OHT in the mouse corneal endothelium in vivo. These results suggest that genes with critical developmental roles can be targeted at a specific time in the corneal endothelium to study its role in adult disease using an inducible Cre-Lox strategy.

Exogenous FGF-1 Differently Regulates Oligodendrocyte Replenishment in an SCI Repair Model and Cultured Cells.

We studied the phenotypes in an oligodendrocyte genesis site at the acute stage of spinal cord injury, when we observed regenerated ascending neurites. Pan-oligodendrocyte marker OLIG2+ cells were more in fibroblast growth factor (FGF)-1-treated rats (F group) than in non-treated (T group) in this site, while the number of NG2+OX42- oligodendrocyte progenitor cell (OPC), CNPase+ OPC, Nkx2.2+ OPC, and APC+ remyelinating oligodendrocytes was less in the F group. Paradoxically, when we label the rats with pulsed bromodeoxyuridine (BrdU), we found that the mitotic NKX2.2+ OPC cells are more in the F group than in the T group. We tested the embryonic spinal cord mixed culture. FGF treatment resulted in more NG2(+) CNPase (+) than non-FGF-1-treated culture, while the more mature NG2(-) CNPase(+) cell numbers were reduced. When we block the FGF receptor in the injured rat model, the NG2+OX42- cell numbers were increased to be comparable to non-FGF-1 rats, while this failed to bring back the APC+ mature oligodendrocyte cell numbers. As migration of OPC toward injury is a major factor that was absent from the cell culture, we tested 8 mm away from the injury center, and found there were more NG2+ cells with FGF-1 treatment. We proposed that it was possibly a combination of migration and proliferation that resulted in a reduction in the NG2+ OPC population at the oligodendrocyte genesis site when FGF-1 was added to the spinal cord injury in vivo.

REPS2 downregulation facilitates FGF-induced adhesion and migration in human lens epithelial cells through FAK/Cdc42 signaling and contributes to posterior capsule opacification

Posterior capsular opacification (PCO) can cause postoperative visual loss after cataract surgery. Residual human lens epithelial cell (HLEC) proliferation, migration, epithelial-mesenchymal transition (EMT) and synthesis of extracellular matrix (ECM) are the entitative reasons for PCO. Low expression of Ral-binding protein 1-associated Eps domain-containing 2 (REPS2) and high levels of basic fibroblast growth factor (b-FGF) were observed in the lens and postoperative aqueous humor of cataract patients. REPS2 was identified as a negative regulator in growth factor signaling; however, its function in HLECs is unknown. This was first investigated in the present study by evaluating REPS2 expression in anterior lens capsules from cataract patients, a mouse cataract model, and HLE-b3 cells. The biological function of REPS2 in HLE-B3 cells was assessed by REPS2 silencing and Cell Counting Kit 8, wound healing, Transwell migration, F-actin staining, G-protein pulldown and western blot assays. In the present study, REPS2 was significantly downregulated in human and mouse cataract capsules and H2O2-treated HLE-B3 cells. REPS2 knockdown increased fibronectin, type I collagen, and α-smooth muscle actin expression levels and stimulated HLECs proliferation and migration; these effects were enhanced by FGF treatment and accompanied with focal adhesion kinase (FAK) phosphorylation, cell division cycle 42 (Cdc42) activation, focal adhesion protein upregulation, and F-actin cytoskeleton reorganization. However, treatment with the FAK inhibitor PF573228 abolished these effects. Thus, REPS2 downregulation in cataract HLECs induces their proliferation and facilitates FGF-induced ECM synthesis, EMT, cell adhesion and migration by activating FAK/Cdc42 signaling, which may underlie PCO pathogenesis.

A Stage-Specific OTX2 Regulatory Network and Maturation-Associated Gene Programs Are Inherent Barriers to RPE Neural Competency.

The retinal pigment epithelium (RPE) exhibits a diverse range of plasticity across vertebrates and is a potential source of cells for the regeneration of retinal neurons. Embryonic amniotes possess a transitory ability to regenerate neural retina through the reprogramming of RPE cells in an FGF-dependent manner. Chicken RPE can regenerate neural retina at embryonic day 4 (E4), but RPE neural competence is lost by embryonic day 5 (E5). To identify mechanisms that underlie loss of regenerative competence, we performed RNA and ATAC sequencing using E4 and E5 chicken RPE, as well as at both stages following retinectomy and FGF2 treatment. We find that genes associated with neural retina fate remain FGF2-inducible in the non-regenerative E5 RPE. Coinciding with fate restriction, RPE cells stably exit the cell cycle and dampen the expression of cell cycle progression genes normally expressed during regeneration, including E2F1. E5 RPE exhibits progressive activation of gene pathways associated with mature function independently of retinectomy or FGF2 treatment, including retinal metabolism, pigmentation synthesis, and ion transport. Moreover, the E5 RPE fails to efficiently repress OTX2 expression in response to FGF2. Predicted OTX2 binding motifs undergo robust accessibility increases in E5 RPE, many of which coincide with putative regulatory elements for genes known to facilitate RPE differentiation and maturation. Together, these results uncover widespread alterations in gene regulation that culminate in the loss of RPE neural competence and implicate OTX2 as a key determinant in solidifying the RPE fate. These results yield valuable insight to the basis of RPE lineage restriction during early development and will be of importance in understanding the varying capacities for RPE-derived retinal regeneration observed among vertebrates.

Morphometric Analysis of Rat Prostate Development: Roles of MEK/ERK and Rho Signaling Pathways in Prostatic Morphogenesis.

The molecular mechanisms underlying prostate development can provide clues for prostate cancer research. It has been demonstrated that MEK/ERK signaling downstream of androgen-targeted FGF10 signaling directly induces prostatic branching during development, while Rho/Rho-kinase can regulate prostate cell proliferation. MEK/ERK and Rho/Rho kinase regulate myosin light chain kinase (MLCK), and MLCK regulates myosin light chain phosphorylation (MLC-P), which is critical for cell fate, including cell proliferation, differentiation, and apoptosis. However, the roles and crosstalk of the MEK/ERK and Rho/Rho kinase signaling pathways in prostatic morphogenesis have not been examined. In the present study, we used numerical and image analysis to characterize lobe-specific rat prostatic branching during postnatal organ culture and investigated the roles of FGF10-MEK/ERK and Rho/Rho kinase signaling pathways in prostatic morphogenesis. Prostates exhibited distinctive lobe-specific growth and branching patterns in the ventral (VP) and lateral (LP) lobes, while exogenous FGF10 treatment shifted LP branching towards a VP branching pattern. Treatment with inhibitors of MEK1/2, Rho, Rho kinase, or MLCK significantly inhibited VP growth and blocked branching morphogenesis, further supporting critical roles for MEK/ERK and Rho/Rho kinase signaling pathways in prostatic growth and branching during development. We propose that MLCK-regulated MLC-P may be a central downstream target of both signaling pathways in regulating prostate morphogenesis.

Effect of Interleukin 17 and Epidermal Growth Factor 2 (FGF2) on the Biological Behavior of Endometrial Carcinoma Human Endometrial Cancer-1B Cells

The clinical prognosis of endometrial cancer is poor. We assessed the influence of IL-17 and FGF2 on HEC-1B cells to provide evidence for the clinical prevention and treatment. HEC-1B cells were assigned into control group, IL-17 group, FGF2 group, and IL-17+FGF2 group followed by analysis of cell viability, IL-17 and FGF2 protein and mRNA content by Western Blot and RT-PCR. Under co-culture, the cell viability, migration and invasion and FGF2 level in IL-17 group, FGF2 group, and IL-17+FGF2 group were found to be significantly higher than those in control Group (P <0.05), but the apoptosis rate was significantly lower than control group. The above changes were more significant in IL-17+ FGF2 group compared to other three groups (P < 0.05). IL-17 and FGF2 treatment in HEC-1B cells can significantly promote the viability, migration and invasion of malignant tumor cells with more significant changes in the IL-17+FGF2 intervention group, suggesting these two have a synergistic effect. In conclusion, IL-17 and FGF2 exerts synergistic effect on promoting endometrial cancer cell migration and invasion, indicating that they might be novel targets for the treatment of endometrial cancer.

Influence of surface modification of titanium implants on improving osseointegration: An in vitro study

Statement of problemThe effect of aging and the surface treatment of implants on osseointegration needs to be evaluated. PurposeThe purpose of this in vitro study was to evaluate the effects of aging and the surface treatment of titanium with ultraviolet (UV) radiation and fibroblast growth factor (FGF) on hydrophilicity and cell growth and thus on osseointegration. Material and methodsA total of 28 specimens were divided into 2 groups to measure hydrophilicity (n=14) and cell growth (n=14). Each group was further divided into 4 groups according to surface modification. These include the control group (CG) (nascent specimens), aged group (AG) (nascent specimens aged for 4 weeks), photofunctionalized group (PG) (aged specimens UV-A treated), and mimed group (MG) (aged specimens UV-A and FGF2 treated). The PG and MG specimens were treated with UV-A light for 40 minutes. The biomimetic surface modification was performed for MG. Hydrophilicity was measured by using the contact angle in relation to the surface of titanium disks with the help of a drop shape analyzing device (KRUSS), and cell growth was measured by calculating the number of stem cells per cm2 with the help of a scanning electron microscope (SEM). The data obtained were subjected to statistical analysis with a statistical software program (α=.05). ResultsThe lowest contact angle values were found in PG (13.52 ±0.90 degrees) and the highest in AG (70.54 ±1.72 degrees). The highest number of cells per cm2 (2880 ±99.33) were found for MG, and the lowest number of cells per cm2 (760 ±9.17) for AG. ConclusionsAging decreased the hydrophilicity and cell adhesion, migration, and growth on the titanium surface. UV treatment improved the hydrophilicity, cell adhesion, migration, and growth for both CG and AG. FGF2 treatment increased the cell adhesion, migration, and growth for CG, AG, and PG.

Prediction of response to sunitinib in patients with advanced renal cell carcinoma (RCC) using mass spectrometry-based (phospho) proteomics.

e16556 Background: Sunitinib, a multi-targeted antiangiogenic tyrosine kinase inhibitor has improved the outcome of patients with advanced RCC considerably. Unfortunately, ± 30% of patients are intrinsically resistant, which underscores the urgent need to develop a clinically applicable method to select sunitinib treatment for patients. Since RCC is not single oncogene driven, but rather by multiple aberrantly activated kinase signaling pathways, we hypothesized that a functional read-out by large scale (phospho)proteomics can identify predictive biomarkers for sunitinib. Methods: Frozen tumor tissue of 26 patients (pts) with RCC, treated with sunitinib upon recurrence or progression, was obtained. Pts were classified as primary resistant (progression-free survival (PFS) < 3 months, n = 8) or sensitive (PFS ≥ 3 months, n = 18). Mass spectrometry-based tyrosine (pTyr) phosphoproteomics was performed by pTyr-immunoprecipitation followed by LC-MS/MS. Discriminatory phosphosites (p-sites) were identified (p < 0.05, fold-change (FC) > 2). Expression proteomics was performed by LC-MS/MS and differentially expressed proteins identified (p < 0.05, FC > 2, ≥ 50% data presence in group with highest abundance). Tumor biology was further analyzed by INferred Kinase Activity analysis, posttranslational modifications signature enrichment analysis (PTM-SEA, Krug et al 2019) and gene ontology mining. Results: pTyr-phosphoproteomics was successfully performed in 23 of 26 samples. Seventy-eight differential p-sites were identified, of which 22 (4 unique; BCAR3, NOP58, EIF4A2, GDI1) were upregulated in resistant and 56 in sensitive pts (35 unique). Supervised cluster analysis of these p-sites resulted in near-complete separation of the groups. EIF4A1 and its homolog EIF4A2 were differentially expressed in resistant pts both at the (phospho-)proteome and, in an independent cohort, at transcriptome level. Significantly higher inferred kinase activity of MAPK3 (p = 0.026) and EGFR (p = 0.045) was found in sensitive pts. PTM-SEA showed 3 p-site-centric signatures that were significantly enriched (p < 0.05) in resistant pts, including FGF1 and prolactin pathways. Fifteen signatures were significantly enriched (p < 0.05) in sensitive pts, including insulin, VEGF and FGF2 treatment and KIT receptor pathway. Expression proteomics revealed significantly higher expression (p < 0.05) of proteins related to vesicle mediated transport in resistant pts, while this was not the case in sensitive pts. Conclusions: This MS-based (phospho)proteomics analysis of RCC tissues revealed discriminatory phosphosite and protein signatures and differential kinase and pathway activities that are associated with sensitive and resistant tumors. These findings warrant validation in an independent cohort and the clinical utility for treatment selection remains to be demonstrated.

Long-term outcomes of basic fibroblast growth factor treatments in patients with vocal fold scarring, aged vocal fold, and sulcus vocalis

ObjectiveFibrotic changes in the vocal fold mucosa have been observed in patients with vocal fold scarring, aged vocal fold, and sulcus vocalis, which often lead to severe voice disorders. Previous research suggests that the basic fibroblast growth factor (b FGF) improves variations in vocal fold properties [1,2]. Although clinical studies on b FGF treatments have been conducted [3,4,5], these studies only demonstrated the efficacy of this drug over a short period. The present study is the first to investigate the long-term efficacy of b FGF treatment. Methodsb FGF injections were performed in six patients from January of 2016 to December of 2017 at our institution. Patient follow-up continued for at least two years after the last injection. Three patients had vocal fold scarring, two had aged vocal fold atrophy, and one patient had sulcus vocalis. Each vocal fold was injected with 10 µg of b FGF four times. Voice and stroboscopic examinations were performed after surgery (at one month, three months, six months, one year, two years). Fundamental frequency, maximum phonation time (MPT), mean flow rate (MFR), amplitude perturbation quotient (APQ), pitch perturbation quotient (PPQ), and noise-to-harmonic ratio (NHR), and voice handicap index-10 (VHI-10) were examined and compared statistically between the pretreatment time and at each posttreatment time point. ResultsThe speaking F0 had an obvious decreasing tendency, with significant differences suggesting the increase in volume in the vocal folds. Aerodynamic parameters also showed small improvements. The most remarkable improvement was observed in the acoustic parameters, indicating that the treatment could improve the vocal fold to make vibrations symmetrically and regularly for a long period. Achievement of symmetry and regularity on vocal fold vibrations suggested the property changes had happened in the vocal folds. Consequently, the score of VHI-10 had improved, indicating high patient satisfaction with this treatment. Conclusionb FGF injections could be a reliable treatment option for diseases that deteriorate the property of vocal fold.

Tumor necrosis factor-α promotes and exacerbates calcification in heart valve myofibroblast populations.

Pro-inflammatory cytokines play critical roles in regulating valvular interstitial cell (VIC) phenotypic changes that can cause heart valve fibrosis and calcification. Tumor necrosis factor alpha (TNF-α) is a cytokine known to influence VIC behavior and has been reported at high levels in calcified valves ex vivo. We sought to understand the specific effects of TNF-α on VIC phenotypes (eg, fibroblast, profibrotic activated myofibroblasts) and its link with heart valve disorders. We characterize human aortic valve tissue from patients with valve disorders and identify a high variability of fibrotic and calcific markers between tissues. These results motivated in vitro studies to explore the effects of TNF-α on defined VIC fibroblasts and profibrotic activated myofibroblasts, induced via FGF-2 and TGF-β1 treatment. Using 3D hydrogels to culture VICs, we measure the effect of TNF-α (0.1-10ng/mL) on key markers of fibrosis (eg, αSMA, COL1A1) and calcification (eg, RUNX2, BMP2, and calcium deposits). We observe calcification in TNF-α-treated VIC activated myofibroblasts and identify the MAPK/ERK signaling cascade as a potential pathway for TNF-α mediated calcification. Conversely, VIC fibroblasts respond to TNF-α with decreased calcification. Treatment of VIC profibrotic activated myofibroblast populations with TNF-α leads to increased calcification. Our in vitro findings correlate with findings in diseased human valves and highlight the importance of understanding the effect of cytokines and signaling pathways on specific VIC phenotypes. Finally, we reveal MAPK/ERK as a potential pathway involved in VIC-mediated matrix calcification with TNF-α treatment, suggesting this pathway as a potential pharmaceutical target for aortic valve disease.

Praja2 suppresses the growth of gastric cancer by ubiquitylation of KSR1 and inhibiting MEK-ERK signal pathways.

Gastric cancer (GC) is a common malignant tumor, which has a high incidence and fatality. Therefore, it is important to clarify the molecular mechanism of the occurrence and development for GC and to find more effective treatments and targeted drugs. In this study, we found that the kinase suppressor of Ras1 (KSR1) was increased in GC tissues and cell lines. Silencing of KSR1 inhibited the proliferation, migration and invasion of MKN-45 cells. E3 ligase Praja2 was downregulated in GC tissues and cell lines. In addition, praja2 promoted ubiquitylation of KSR1, but inhibited MEK-ERK signal pathways. Functional analysis indicated overexpression of praja2 inhibited the proliferation, migration and invasion of MKN-45 cells, while MG132 or FGF2 treatment removed the inhibitory effects of praja2 on GC progression. In vivo tumorigenesis experiments indicated praja2 inhibited tumor growth via KSR1-MEK-ERK axis. In conclusion, praja2 promoted the ubiquitylation and degradation of KSR1, which disturbed MEK- ERK signaling and inhibited GC progression. Our study might provide a novel target for GC clinical treatment.