Lindsay Marek

Fibroblast Growth Factor (FGF) and FGF Receptor-Mediated Autocrine Signaling in Non-Small-Cell Lung Cancer Cells

Despite widespread expression of epidermal growth factor (EGF) receptors (EGFRs) and EGF family ligands in non-small-cell lung cancer (NSCLC), EGFR-specific tyrosine kinase inhibitors (TKIs) such as gefitinib exhibit limited activity in this cancer. We propose that autocrine growth signaling pathways distinct from EGFR are active in NSCLC cells. To this end, gene expression profiling revealed frequent coexpression of specific fibroblast growth factors (FGFs) and FGF receptors (FGFRs) in NSCLC cell lines. It is noteworthy that FGF2 and FGF9 as well as FGFR1 IIIc and/or FGFR2 IIIc mRNA and protein are frequently coexpressed in NSCLC cell lines, especially those that are insensitive to gefitinib. Specific silencing of FGF2 reduced anchorage-independent growth of two independent NSCLC cell lines that secrete FGF2 and coexpress FGFR1 IIIc and/or FGFR2 IIIc. Moreover, a TKI [(±)-1-(anti-3-hydroxy-cyclopentyl)-3-(4-methoxy-phenyl)-7-phenylamino-3,4-dihydro-1H-pyrimido-[4,5-d]pyrimidin-2-one (RO4383596)] that targets FGFRs inhibited basal FRS2 and extracellular signal-regulated kinase phosphorylation, two measures of FGFR activity, as well as proliferation and anchorage-independent growth of NSCLC cell lines that coexpress FGF2 or FGF9 and FGFRs. By contrast, RO4383596 influenced neither signal transduction nor growth of NSCLC cell lines lacking FGF2, FGF9, FGFR1, or FGFR2 expression. Thus, FGF2, FGF9 and their respective high-affinity FGFRs comprise a growth factor autocrine loop that is active in a subset of gefitinib-insensitive NSCLC cell lines.

A mechanism of resistance to gefitinib mediated by cellular reprogramming and the acquisition of an FGF2-FGFR1 autocrine growth loop.

Despite initial and often dramatic responses of epidermal growth factor receptor (EGFR)-addicted lung tumors to the EGFR-specific tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, nearly all develop resistance and relapse. To explore novel mechanisms mediating acquired resistance, we employed non-small-cell lung cancer (NSCLC) cell lines bearing activating mutations in EGFR and rendered them resistant to EGFR-specific TKIs through chronic adaptation in tissue culture. In addition to previously observed resistance mechanisms including EGFR-T790M ‘gate-keeper’ mutations and MET amplification, a subset of the seven chronically adapted NSCLC cell lines including HCC4006, HCC2279 and H1650 cells exhibited marked induction of fibroblast growth factor (FGF) 2 and FGF receptor 1 (FGFR1) mRNA and protein. Also, adaptation to EGFR-specific TKIs was accompanied by an epithelial to mesenchymal transition (EMT) as assessed by changes in CDH1, VIM, ZEB1 and ZEB2 expression and altered growth properties in Matrigel. In adapted cell lines exhibiting increased FGF2 and FGFR1 expression, measures of growth and signaling, but not EMT, were blocked by FGFR-specific TKIs, an FGF-ligand trap and FGFR1 silencing with RNAi. In parental HCC4006 cells, cell growth was strongly inhibited by gefitinib, although drug-resistant clones progress within 10 days. Combined treatment with gefitinib and AZD4547, an FGFR-specific TKI, prevented the outgrowth of drug-resistant clones. Thus, induction of FGF2 and FGFR1 following chronic adaptation to EGFR-specific TKIs provides a novel autocrine receptor tyrosine kinase-driven bypass pathway in a subset of lung cancer cell lines that are initially sensitive to EGFR-specific TKIs. The findings support FGFR-specific TKIs as potentially valuable additions to existing targeted therapeutic strategies with EGFR-specific TKIs to prevent or delay acquired resistance in EGFR-driven NSCLC.

Rapidly Acquired Resistance to EGFR Tyrosine Kinase Inhibitors in NSCLC Cell Lines through De-Repression of FGFR2 and FGFR3 Expression

Despite initial and sometimes dramatic responses of specific NSCLC tumors to EGFR TKIs, nearly all will develop resistance and relapse. Gene expression analysis of NSCLC cell lines treated with the EGFR TKI, gefitinib, revealed increased levels of FGFR2 and FGFR3 mRNA. Analysis of gefitinib action on a larger panel of NSCLC cell lines verified that FGFR2 and FGFR3 expression is increased at the mRNA and protein level in NSCLC cell lines in which the EGFR is dominant for growth signaling, but not in cell lines where EGFR signaling is absent. A luciferase reporter containing 2.5 kilobases of fgfr2 5′ flanking sequence was activated after gefitinib treatment, indicating transcriptional regulation as a contributing mechanism controlling increased FGFR2 expression. Induction of FGFR2 and FGFR3 protein as well as fgfr2-luc activity was also observed with Erbitux, an EGFR-specific monoclonal antibody. Moreover, inhibitors of c-Src and MEK stimulated fgfr2-luc activity to a similar degree as gefitinib, suggesting that these pathways may mediate EGFR-dependent repression of FGFR2 and FGFR3. Importantly, our studies demonstrate that EGFR TKI-induced FGFR2 and FGFR3 are capable of mediating FGF2 and FGF7 stimulated ERK activation as well as FGF-stimulated transformed growth in the setting of EGFR TKIs. In conclusion, this study highlights EGFR TKI-induced FGFR2 and FGFR3 signaling as a novel and rapid mechanism of acquired resistance to EGFR TKIs and suggests that treatment of NSCLC patients with combinations of EGFR and FGFR specific TKIs may be a strategy to enhance efficacy of single EGFR inhibitors.

Fibroblast Growth Factor Receptors Are Components of Autocrine Signaling Networks in Head and Neck Squamous Cell Carcinoma Cells

Purpose: We previously reported that a fibroblast growth factor (FGF) receptor (FGFR) signaling pathway drives growth of lung cancer cell lines of squamous and large cell histologies. Herein, we explored FGFR dependency in cell lines derived from the tobacco-related malignancy, head and neck squamous cell carcinoma (HNSCC). Experimental Design: FGF and FGFR mRNA and protein expression was assessed in nine HNSCC cell lines. Dependence on secreted FGF2 for cell growth was tested with FP-1039, an FGFR1-Fc fusion protein. FGFR and epidermal growth factor receptor (EGFR) dependence was defined by sensitivity to multiple inhibitors selective for FGFRs or EGFR. Results: FGF2 was expressed in eight of the nine HNSCC cell lines examined. Also, FGFR2 and FGFR3 were frequently expressed, whereas only two lines expressed FGFR1. FP-1039 inhibited growth of HNSCC cell lines expressing FGF2, identifying FGF2 as an autocrine growth factor. FGFR inhibitors selectively reduced in vitro growth and extracellular signal-regulated kinase signaling in three HNSCC cell lines, whereas three distinct lines exhibited responsiveness to both EGFR and FGFR inhibitors. Combinations of these drugs yielded additive growth inhibition. Finally, three cell lines were highly sensitive to EGFR tyrosine kinase inhibitors (TKI) with no contribution from FGFR pathways. Conclusions: FGFR signaling was dominant or codominant with EGFR in six HNSCC lines, whereas three lines exhibited little or no role for FGFRs and were highly EGFR dependent. Thus, the HNSCC cell lines can be divided into subsets defined by sensitivity to EGFR and FGFR-specific TKIs. FGFR inhibitors may represent novel therapeutics to deploy alone or in combination with EGFR inhibitors in HNSCC. Clin Cancer Res; 17(15); 5016–25. ©2011 AACR.

FGFR1 mRNA and Protein Expression, not Gene Copy Number, Predict FGFR TKI Sensitivity across All Lung Cancer Histologies

Purpose: FGFR1 gene copy number (GCN) is being evaluated as a biomarker for FGFR tyrosine kinase inhibitor (TKI) response in squamous cell lung cancers (SCC). The exclusive use of FGFR1 GCN for predicting FGFR TKI sensitivity assumes increased GCN is the only mechanism for biologically relevant increases in FGFR1 signaling. Herein, we tested whether FGFR1 mRNA and protein expression may serve as better biomarkers of FGFR TKI sensitivity in lung cancer. Experimental Design: Histologically diverse lung cancer cell lines were submitted to assays for ponatinib sensitivity, a potent FGFR TKI. A tissue microarray composed of resected lung tumors was submitted to FGFR1 GCN, and mRNA analyses and the results were validated with The Cancer Genome Atlas (TCGA) lung cancer data. Results: Among 58 cell lines, 14 exhibited ponatinib sensitivity (IC50 values ≤ 50 nmol/L) that correlated with FGFR1 mRNA and protein expression, but not with FGFR1 GCN or histology. Moreover, ponatinib sensitivity associated with mRNA expression of the ligands, FGF2 and FGF9. In resected tumors, 22% of adenocarcinomas and 28% of SCCs expressed high FGFR1 mRNA. Importantly, only 46% of SCCs with increased FGFR1 GCN expressed high mRNA. Lung cancer TCGA data validated these findings and unveiled overlap of FGFR1 mRNA positivity with KRAS and PIK3CA mutations. Conclusions: FGFR1 dependency is frequent across various lung cancer histologies, and FGFR1 mRNA may serve as a better biomarker of FGFR TKI response in lung cancer than FGFR1 GCN. The study provides important and timely insight into clinical testing of FGFR TKIs in lung cancer and other solid tumor types. Clin Cancer Res; 20(12); 3299–309. ©2014 AACR.

Inhibited Neurogenesis in JNK1-Deficient Embryonic Stem Cells

ABSTRACT The JNKs are components of stress signaling pathways but also regulate morphogenesis and differentiation. Previously, we invoked a role for the JNKs in nerve growth factor (NGF)-stimulated PC12 cell neural differentiation (L. Marek et al., J. Cell. Physiol. 201:459-469, 2004; E. Zentrich et al., J. Biol. Chem. 277:4110-4118, 2002). Herein, the role for JNKs in neural differentiation and transcriptional regulation of the marker gene, NFLC, modeled in mouse embryonic stem (ES) cells was studied. NFLC-luciferase reporters revealed the requirement for NFLC promoter sequences encompassing base pairs −128 to −98 relative to the transcriptional start site as well as a proximal cyclic AMP response element-activating transcription factor binding site at −45 to −38 base pairs for transcriptional induction in NGF-treated PC12 cells and neurally differentiated ES cells. The findings reveal common promoter sequences that integrate conserved signal pathways in both PC12 cell and ES cell systems. To test the requirement for the JNK pathway in ES cell neurogenesis, ES cell lines bearing homozygous disruptions of the jnk1, jnk2, or jnk3 genes were derived and submitted to an embryoid body (EB) differentiation protocol. Neural differentiation was observed in wild-type, JNK2−/−, and JNK3−/− cultures but not in JNK1−/− EBs. Rather, an outgrowth of cells with epithelial morphology and enhanced E-cadherin expression but low NFLC mRNA and protein was observed in JNK1−/− cultures. The expression of wnt-4 and wnt-6, identified inhibitors of ES cell neurogenesis, was significantly elevated in JNK1−/− cultures relative to wild-type, JNK2−/−, and JNK3−/− cultures. Moreover, the Wnt antagonist, sFRP-2, partially rescued neural differentiation in JNK1−/− cultures. Thus, a genetic approach using JNK-deficient ES cells reveals a novel role for JNK1 involving repression of Wnt expression in neural differentiation modeled in murine ES cells.

Multiple signaling conduits regulate global differentiation-specific gene expression in PC12 cells

PC12 cells serve as a model for exploring nerve growth factor (NGF)‐stimulated signal pathways that mediate neural differentiation. We previously demonstrated that neurofilament light chain (NFLC) gene induction by NGF requires collaborative extracellular signal‐regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) signaling. Herein, we investigate the broader requirement for integrated ERK and JNK signaling in NGF‐stimulated gene expression. NGF stimulates differentiation as well as maintenance of cell viability while insulin‐like growth factor‐1 (IGF‐1) stimulates only trophic actions in PC12 cells. Affymetrix Genechips were used to identify genes whose expression specifically increased in response to NGF, but not IGF‐1. From the set of NGF‐specific genes, the induction by NGF of ten genes with diverse predicted cellular functions was tested for ERK and JNK pathway requirements using the protein kinase inhibitors, PD98059 and SP600125, respectively. Like NFLC, induction of urokinase plasminogen activator (uPAR), transin/matrix metalloproteinase 3 (MMP3), Fra‐1 and transforming growth factor β1 (TGFβ1) required collaborative ERK and JNK signaling while the increased expression of cortexin, rat collapsin response mediator protein 4 (rCRMP4), rat growth and transformation‐dependent protein (RGT), and synapsin II required neither mitogen‐activated protein kinase (MAPK) pathway. NGF‐induction of the bradykinin B2 receptor and c‐Ret mRNAs was partially inhibited by SP600125, but not PD98059. Reporter constructs containing the promoters for ERK/JNK‐dependent genes (NFLC, transin, uPAR) as well as an ERK/JNK‐independent gene (synapsin II) revealed that both sets of genes required functional Ras signaling for activation by NGF. Integrated signaling through the ERK and JNK MAPKs, therefore, represents a general conduit for NGF‐dependent gene expression, but additional Ras‐dependent signaling pathways distinct from the ERKs and JNKs must contribute as well. Thus, multiple signaling conduits control global differentiation‐specific gene expression in PC12 cells.

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

Purpose: 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. Experimental Design: 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. Results: 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. Conclusions: 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. Clin Cancer Res; 21(19); 4356–64. ©2015 AACR.

A receptor tyrosine kinase network composed of fibroblast growth factor receptors, epidermal growth factor receptor, v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, and hepatocyte growth factor receptor drives growth and survival of head and neck squamous carcinoma cell lines.

Our laboratory has previously shown that some gefitinib-insensitive head and neck squamous cell carcinoma (HNSCC) cell lines exhibit dominant autocrine fibroblast growth factor receptor (FGFR) signaling. Herein, we deployed a whole-genome loss-of-function screen to identify genes whose knockdown potentiated the inhibitory effect of the FGFR inhibitor, AZ8010, in HNSCC cell lines. Three HNSCC cell lines expressing a genome-wide small hairpin RNA (shRNA) library were treated with AZ8010 and the abundance of shRNA sequences was assessed by deep sequencing. Under-represented shRNAs in treated cells are expected to target genes important for survival with AZ8010 treatment. Synthetic lethal hits were validated with specific inhibitors and independent shRNAs. We found that multiple alternate receptors provided protection from FGFR inhibition, including receptor tyrosine kinases (RTKs), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (ERBB2), and hepatocyte growth factor receptor (MET). We showed that specific knockdown of either ERBB2 or MET in combination with FGFR inhibition led to increased inhibition of growth relative to FGFR tyrosine kinase inhibitor (TKI) treatment alone. These results were confirmed using specific small molecule inhibitors of either ERBB family members or MET. Moreover, the triple combination of FGFR, MET, and ERBB family inhibitors showed the largest inhibition of growth and induction of apoptosis compared with the double combinations. These results reveal a role for alternate RTKs in maintaining progrowth and survival signaling in HNSCC cells in the setting of FGFR inhibition. Thus, improved therapies for HNSCC patients could involve rationally designed combinations of TKIs targeting FGFR, ERBB family members, and MET.

Kinome RNAi Screens Reveal Synergistic Targeting of MTOR and FGFR1 Pathways for Treatment of Lung Cancer and HNSCC

The FGFR1 is a therapeutic target under investigation in multiple solid tumors and clinical trials of selective tyrosine kinase inhibitors (TKI) are underway. Treatment with a single TKI represents a logical step toward personalized cancer therapy, but intrinsic and acquired resistance mechanisms limit their long-term benefit. In this study, we deployed RNAi-based functional genomic screens to identify protein kinases controlling the intrinsic sensitivity of FGFR1-dependent lung cancer and head and neck squamous cell cancer (HNSCC) cells to ponatinib, a multikinase FGFR-active inhibitor. We identified and validated a synthetic lethal interaction between MTOR and ponatinib in non-small cell lung carcinoma cells. In addition, treatment with MTOR-targeting shRNAs and pharmacologic inhibitors revealed that MTOR is an essential protein kinase in other FGFR1-expressing cancer cells. The combination of FGFR inhibitors and MTOR or AKT inhibitors resulted in synergistic growth suppression in vitro. Notably, tumor xenografts generated from FGFR1-dependent lung cancer cells exhibited only modest sensitivity to monotherapy with the FGFR-specific TKI, AZD4547, but when combined with the MTOR inhibitor, AZD2014, significantly attenuated tumor growth and prolonged survival. Our findings support the existence of a signaling network wherein FGFR1-driven ERK and activated MTOR/AKT represent distinct arms required to induce full transformation. Furthermore, they suggest that clinical efficacy of treatments for FGFR1-driven lung cancers and HNSCC may be achieved by combining MTOR inhibitors and FGFR-specific TKIs.