Lynn E. Heasley

Mechanisms of resistance to crizotinib in patients with ALK gene rearranged non-small cell lung cancer.

Purpose: Patients with anaplastic lymphoma kinase (ALK) gene rearrangements often manifest dramatic responses to crizotinib, a small-molecule ALK inhibitor. Unfortunately, not every patient responds and acquired drug resistance inevitably develops in those who do respond. This study aimed to define molecular mechanisms of resistance to crizotinib in patients with ALK+ non–small cell lung cancer (NSCLC). Experimental Design: We analyzed tissue obtained from 14 patients with ALK+ NSCLC showing evidence of radiologic progression while on crizotinib to define mechanisms of intrinsic and acquired resistance to crizotinib. Results: Eleven patients had material evaluable for molecular analysis. Four patients (36%) developed secondary mutations in the tyrosine kinase domain of ALK. A novel mutation in the ALK domain, encoding a G1269A amino acid substitution that confers resistance to crizotinib in vitro, was identified in two of these cases. Two patients, one with a resistance mutation, exhibited new onset ALK copy number gain (CNG). One patient showed outgrowth of epidermal growth factor receptor (EGFR) mutant NSCLC without evidence of a persistent ALK gene rearrangement. Two patients exhibited a KRAS mutation, one of which occurred without evidence of a persisting ALK gene rearrangement. One patient showed the emergence of an ALK gene fusion–negative tumor compared with the baseline sample but with no identifiable alternate driver. Two patients retained ALK positivity with no identifiable resistance mechanism. Conclusions: Crizotinib resistance in ALK+ NSCLC occurs through somatic kinase domain mutations, ALK gene fusion CNG, and emergence of separate oncogenic drivers. Clin Cancer Res; 18(5); 1472–82. ©2012 AACR.

Autocrine and paracrine signaling through neuropeptide receptors in human cancer.

Autocrine and paracrine signaling leading to stimulation of tumor cell growth is a common theme in human cancers. In addition to polypeptide growth factors such as EGF family members which signal through receptor tyrosine kinases, accumulating evidence supports the autocrine and paracrine involvement of specific neuropeptides with defined physiologic actions as neurotransmitters and gut hormones in lung, gastric, colorectal, pancreatic and prostatic cancers. These neuropeptides, including gastrin-releasing peptide, neuromedin B, neurotensin, gastrin, cholecystokinin and arginine vasopressin bind seven transmembrane-spanning receptors that couple to heterotrimeric G proteins. Studies with human small cell lung cancer (SCLC) cells support a requirement for balanced signaling through Gq and G12/13 proteins leading to intracellular Ca2+ mobilization, PKC activation and regulation of the ERK and JNK MAP kinase pathways. While specific neuropeptide antagonists offer promise for interrupting the single neuropeptide autocrine systems operating in pancreatic and prostatic cancers, SCLC is exemplified by multiple, redundant neuropeptide autocrine systems such that tumor growth cannot be inhibited with a single specific antagonist. However, a novel class of neuropeptide derivatives based on the substance P sequence have been defined that exhibit broad specificity for neuropeptide receptors and induce apoptosis in SCLC by functioning as biased agonists that stimulate discordant signal transduction. Thus, interruption of autocrine and paracrine neuropeptide signaling with specific antagonists or broad-spectrum biased agonists offer promising new therapeutic approaches to the treatment of human cancers.

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.

GTPase-deficient G alpha 16 and G alpha q induce PC12 cell differentiation and persistent activation of cJun NH2-terminal kinases.

Persistent stimulation of specific protein kinase pathways has been proposed as a key feature of receptor tyrosine kinases and intracellular oncoproteins that signal neuronal differentiation of rat pheochromocytoma (PC12) cells. Among the protein serine/threonine kinases identified to date, the p42/44 mitogen-activated protein (MAP) kinases have been highlighted for their potential role in signalling PC12 cell differentiation. We report here that retrovirus-mediated expression of GTPase-deficient, constitutively active forms of the heterotrimeric Gq family members, G alpha qQ209L and G alpha 16Q212L, in PC12 cells induces neuronal differentiation as indicated by neurite outgrowth and the increased expression of voltage-dependent sodium channels. Differentiation was not observed after cellular expression of GTPase-deficient forms of alpha i2 or alpha 0, indicating selectivity for the Gq family of G proteins. As predicted, overexpression of alpha qQ209L and alpha 16Q212L constitutively elevated basal phospholipase C activity approximately 10-fold in PC12 cells. Significantly, little or no p42/44 MAP kinase activity was detected in PC12 cells differentiated with alpha 16Q212L or alpha qQ209L, although these proteins were strongly activated following expression of constitutively active cRaf-1. Rather, a persistent threefold activation of the cJun NH2-terminal kinases (JNKs) was observed in PC12 cells expressing alpha qQ209L and alpha 16Q212L. This level of JNK activation was similar to that achieved with nerve growth factor, a strong inducer of PC12 cell differentiation. Supportive of a role for JNK activation in PC12 cell differentiation, retrovirus-mediated overexpression of cJun, a JNK target, in PC12 cells induced neurite outgrowth. The results define a p42/44 MAP kinase-independent mechanism for differentiation of PC12 cells and suggest that persistent activation of the JNK members of the proline-directed protein kinase family by GTPase-deficient G alpha q and G alpha 16 subunits is sufficient to induce differentiation of PC12 cells.

Mitogen-activated protein kinase activation is insufficient for growth factor receptor-mediated PC12 cell differentiation.

When expressed in PC12 cells, the platelet-derived growth factor beta receptor (beta PDGF-R) mediates cell differentiation. Mutational analysis of the beta PDGF-R indicated that persistent receptor stimulation of the Ras/Raf/mitogen-activated protein (MAP) kinase pathway alone was insufficient to sustain PC12 cell differentiation. PDGF receptor activation of signal pathways involving p60c-src or the persistent regulation of phospholipase C gamma was required for PC12 cell differentiation. beta PDGF-R regulation of phosphatidylinositol 3-kinase, the GTPase-activating protein of Ras, and the tyrosine phosphatase, Syp, was not required for PC12 cell differentiation. In contrast to overexpression of oncoproteins involved in regulating the MAP kinase pathway, growth factor receptor-mediated differentiation of PC12 cells requires the integration of other signals with the Ras/Raf/MAP kinase pathway.

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.

γ-Catenin expression is reduced or absent in a subset of human lung cancers and re-expression inhibits transformed cell growth

Lung cancer is a heterogeneous disease categorized into multiple subtypes of cancers which likely arise from distinct patterns of genetic alterations and disruptions. Precedent exists for a role of β-catenin, a downstream component of the Wnt signaling pathway that serves as a transcriptional co-activator with TCF/LEF, in several human cancers including colon carcinomas. In this study, we observed that β-catenin was highly and uniformly expressed in a panel of NSCLC cell lines and primary lung tumors. By contrast, γ-catenin was weakly expressed or absent in several NSCLC cell lines and immunohistochemical analysis of primary NSCLC tumors revealed negligible to weak γ-catenin staining in ∼30% of the specimens. Treatment of NSCLC cells expressing reduced γ-catenin protein with 5-aza-2′-deoxycytidine (5aza2dc), a DNA methylation inhibitor, or trichostatin A (TSA), a histone deacetylase inhibitor, increased γ-catenin protein content in NSCLC cells with low γ-catenin expression. Significantly, the activity of a β-catenin/TCF-dependent luciferase reporter was markedly elevated in the NSCLC cell lines that underexpressed γ-catenin relative to those lines that highly expressed γ-catenin. Moreover, transfection of these cells with a γ-catenin expression plasmid reduced the elevated TCF activity by 85% and strongly inhibited cell growth on tissue culture plastic as well as anchorage-independent growth in soft agar. This study shows that γ-catenin can function as an inhibitor of β-catenin/TCF-dependent gene transcription and highlights γ-catenin as a potentially novel tumor suppressor protein in a subset of human NSCLC cancers.

Antitumorigenic Effect of Wnt 7a and Fzd 9 in Non-small Cell Lung Cancer Cells Is Mediated through ERK-5-dependent Activation of Peroxisome Proliferator-activated Receptor γ

The Wnt pathway is critical for normal development, and mutation of specific components is seen in carcinomas of diverse origins. The role of this pathway in lung tumorigenesis has not been clearly established. Recent studies from our laboratory indicate that combined expression of the combination of Wnt 7a and Frizzled 9 (Fzd 9) in Non-small Cell Lung Cancer (NSCLC) cell lines inhibits transformed growth. We have also shown that increased expression of peroxisome proliferator-activated receptor γ (PPARγ) inhibits transformed growth of NSCLC and promotes epithelial differentiation of these cells. The goal of this study was to determine whether the effects of Wnt 7a/Fzd 9 were mediated through PPARγ. We found that Wnt 7a and Fzd 9 expression led to increased PPARγ activity. This effect was not mediated by altered expression of the protein. Wnt 7a and Fzd 9 expression resulted in activation of ERK5, which was required for PPARγ activation in NSCLC. SR 202, a known PPARγ inhibitor, blocked the increase in PPARγ activity and restored anchorage-independent growth in NSCLC expressing Wnt 7a and Fzd 9. SR 202 also reversed the increase in E-cadherin expression mediated by Wnt 7a and Fzd 9. These data suggest that ERK5-dependent activation of PPARγ represents a major effector pathway mediating the anti-tumorigenic effects of Wnt 7a and Fzd 9 in NSCLC.

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.