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Lynn E Heasley

Publications and source records attributed to Lynn E Heasley.

13 recordsLinked to original sources
PubMed2006

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 gamma.

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 gamma (PPARgamma) 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 PPARgamma. We found that Wnt 7a and Fzd 9 expression led to increased PPARgamma 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 PPARgamma activation in NSCLC. SR 202, a known PPARgamma inhibitor, blocked the increase in PPARgamma 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 PPARgamma represents a major effector pathway mediating the anti-tumorigenic effects of Wnt 7a and Fzd 9 in NSCLC.

Antineoplastic Agents↗
PubMed2006

JNK regulation of oncogenesis.

The literature provides strong precedent for both pro-tumorigenic and tumor suppressor roles for the c-Jun N-terminal kinases (JNKs) in the setting of oncogenesis. Clearly, JNKs are activated by numerous oncogenes and growth factors and the literature documents a role for these MAP kinases in cell proliferation and transformation. By contrast, JNKs mediate signals from diverse stimuli that result in cell death or differentiation and a role for JNKs as tumor suppressors has emerged. This enigmatic nature of the JNKs in the setting of oncogenesis is considered herein. Further illumination of the complex and context-dependent functions of the JNKs in cancer cells is of obvious importance for the rational use of small molecule JNK inhibitors as therapeutics.

Animals↗
PubMed2005

Restoration of Wnt-7a expression reverses non-small cell lung cancer cellular transformation through frizzled-9-mediated growth inhibition and promotion of cell differentiation.

The Wnt signaling pathway is critical in normal development, and mutation of specific components is frequently observed in carcinomas of diverse origins. However, the potential involvement of this pathway in lung tumorigenesis has not been established. In this study, analysis of multiple Wnt mRNAs in non-small cell lung cancer (NSCLC) cell lines and primary lung tumors revealed markedly decreased Wnt-7a expression compared with normal short-term bronchial epithelial cell lines and normal uninvolved lung tissue. Wnt-7a transfection in NSCLC cell lines reversed cellular transformation, decreased anchorage-independent growth, and induced epithelial differentiation as demonstrated by soft agar and three-dimensional cell culture assays in a subset of the NSCLC cell lines. The action of Wnt-7a correlated with expression of the specific Wnt receptor Frizzled-9 (Fzd-9), and transfection of Fzd-9 into a Wnt-7a-insensitive NSCLC cell line established Wnt-7a sensitivity. Moreover, Wnt-7a was present in Fzd-9 immunoprecipitates, indicating a direct interaction of Wnt-7a and Fzd-9. In NSCLC cells, Wnt-7a and Fzd-9 induced both cadherin and Sprouty-4 expression and stimulated the JNK pathway, but not beta-catenin/T cell factor activity. In addition, transfection of gain-of-function JNK strongly inhibited anchorage-independent growth. Thus, this study demonstrates that Wnt-7a and Fzd-9 signaling through activation of the JNK pathway induces cadherin proteins and the receptor tyrosine kinase inhibitor Sprouty-4 and represents a novel tumor suppressor pathway in lung cancer that is required for maintenance of epithelial differentiation and inhibition of transformed cell growth in a subset of human NSCLCs.

Base Sequence↗
PubMed2005

Ablation of MEKK4 kinase activity causes neurulation and skeletal patterning defects in the mouse embryo.

Skeletal disorders and neural tube closure defects represent clinically significant human malformations. The signaling networks regulating normal skeletal patterning and neurulation are largely unknown. Targeted mutation of the active site lysine of MEK kinase 4 (MEKK4) produces a kinase-inactive MEKK4 protein (MEKK4(K1361R)). Embryos homozygous for this mutation die at birth as a result of skeletal malformations and neural tube defects. Hindbrains of exencephalic MEKK4(K1361R) embryos show a striking increase in neuroepithelial cell apoptosis and a dramatic loss of phosphorylation of MKK3 and -6, mitogen-activated protein kinase kinases (MKKs) regulated by MEKK4 in the p38 pathway. Phosphorylation of MAPK-activated protein kinase 2, a p38 substrate, is also inhibited, demonstrating a loss of p38 activity in MEKK4(K1361R) embryos. In contrast, the MEK1/2-extracellular signal-regulated kinase 1 (ERK1)/ERK2 and MKK4-Jun N-terminal protein kinase pathways were unaffected. The p38 pathway has been shown to regulate the phosphorylation and expression of the small heat shock protein HSP27. Compared to the wild type, MEKK4(K1361R) fibroblasts showed significantly reduced phosphorylation of p38 and HSP27, with a corresponding heat shock-induced instability of the actin cytoskeleton. Together, these data demonstrate MEKK4 regulation of p38 and that substrates downstream of p38 control cellular homeostasis. The findings are the first demonstration that MEKK4-regulated p38 activity is critical for neurulation.

Animals↗
PubMed2005

Inhibited neurogenesis in JNK1-deficient embryonic stem cells.

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.

Animals↗
PubMed2004

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 beta 1 (TGF beta 1) 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.

Animals↗
PubMed2002

gamma-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 beta-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 beta-catenin was highly and uniformly expressed in a panel of NSCLC cell lines and primary lung tumors. By contrast, gamma-catenin was weakly expressed or absent in several NSCLC cell lines and immunohistochemical analysis of primary NSCLC tumors revealed negligible to weak gamma-catenin staining in approximately 30% of the specimens. Treatment of NSCLC cells expressing reduced gamma-catenin protein with 5-aza-2'-deoxycytidine (5aza2dc), a DNA methylation inhibitor, or trichostatin A (TSA), a histone deacetylase inhibitor, increased gamma-catenin protein content in NSCLC cells with low gamma-catenin expression. Significantly, the activity of a beta-catenin/TCF-dependent luciferase reporter was markedly elevated in the NSCLC cell lines that underexpressed gamma-catenin relative to those lines that highly expressed gamma-catenin. Moreover, transfection of these cells with a gamma-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 gamma-catenin can function as an inhibitor of beta-catenin/TCF-dependent gene transcription and highlights gamma-catenin as a potentially novel tumor suppressor protein in a subset of human NSCLC cancers.

Carcinoma, Non-Small-Cell Lung↗
PubMed2002

Differential gene regulation by specific gain-of-function JNK1 proteins expressed in Swiss 3T3 fibroblasts.

The c-Jun N-terminal kinases (JNKs) are encoded by three genes that yield 10 isoforms through alternative mRNA splicing. The roles of each JNK isoform in the many putative biological responses where the JNK pathway is activated are still unclear. To examine the cellular responses mediated by different JNK isoforms, gain-of-function JNK1 polypeptides were generated by fusing the upstream mitogen-activated protein kinase kinase, MKK7, with p46JNK1alpha or p46JNK1beta. The MKK7-JNK fusion proteins, which exhibited constitutive activity in 293T cells, were stably expressed in Swiss 3T3 fibroblasts using retrovirus-mediated gene transfer. Swiss 3T3 cells expressing either of the MKK7-JNK polypeptides were equally sensitized to induction of cell death following serum withdrawal. To search for other cellular responses that may be selectively regulated by the JNK1 isoforms, the gene expression profiles of Swiss 3T3 cells expressing MKK7-JNK1alpha or MKK7-JNK1beta were compared with empty vector-transfected control cells. Affymetrix Genechips identified 46 genes for which expression was increased in MKK7-JNK-expressing cells relative to vector control cells. Twenty genes including those for c-Jun, MKP-7, interluekin-1 receptor family member ST2L/ST2, and c-Jun-binding protein were induced similarly by MKK7-JNK1alpha and MKK7-JNK1beta proteins, whereas 13 genes were selectively increased by MKK7-JNK1alpha and 13 genes were selectively increased by MKK7-JNK1beta. The set of genes selectively induced by MKK7-JNK1beta included a number of known interferon-stimulated genes (ISG12, ISG15, IGTP, and GTPI). Consistent with these gene expression changes, Swiss 3T3 cells expressing MKK7-JNK1beta exhibited increased resistance to vesicular stomatitis virus-induced cell death. These findings reveal evidence for JNK isoform-selective gene regulation and support a role for distinct JNK isoforms in specific cellular responses.

3T3 Cells↗
PubMed2002

Regulation of platinum-compound cytotoxicity by the c-Jun N-terminal kinase and c-Jun signaling pathway in small-cell lung cancer cells.

Cytotoxic platinum compounds including cisplatin are standard cancer chemotherapeutics and are also activators of stress-signaling pathways. In this study, we tested the role of the c-Jun N-terminal kinase (JNK) family of mitogen-activated protein kinases and their transcription factor target, c-Jun, in the cytotoxic response of small-cell lung cancer (SCLC) cells to cisplatin and its less effective trans-isomer, transplatin. Both agents stimulated JNK activity; the transplatin response was rapid and transient, whereas JNK activation by cisplatin was delayed and sustained. Despite the differential kinetics of JNK activation, expression of nonphosphorylatable JNK mutants sensitized the SCLC cells to killing by cisplatin or transplatin, suggesting that JNK activation in response to these agents signals a protective response. Consistent with this finding, overexpression of the JNK target, c-Jun, significantly protected SCLC cells from platinum compounds, whereas expression of a c-Jun mutant encoding only the DNA binding domain increased the sensitivity of the SCLC cells to these drugs. These findings support the hypothesis that activation of the JNKs by platinum compounds controls c-Jun-dependent transcriptional events that promote a protective response in SCLC cells. Oligonucleotide array analysis identified genes encoding a variety of signaling proteins whose expression was reciprocally changed by c-Jun and c-Jun-DBD (c-Jun-DNA binding domain). It is noteworthy that genes whose products are involved in DNA repair, glutathione synthesis, or drug accumulation did not exhibit altered expression by c-Jun or c-Jun-DBD. The findings indicate that inhibition of the JNK pathway is a potential means to enhance the sensitivity of SCLC cells to platinum compounds.

Carcinoma, Small Cell↗
PubMed2002

Peroxisome proliferator-activated receptor-gamma is a target of nonsteroidal anti-inflammatory drugs mediating cyclooxygenase-independent inhibition of lung cancer cell growth.

Nonsteroidal anti-inflammatory drugs (NSAIDs) inhibit the growth of different cancer cell types, suggesting a broad role for their cyclooxygenase (COX) targets and eicosanoid products in tumor cell growth. Sulindac sulfide, a COX inhibitor, inhibited the growth of non-small-cell lung cancers (NSCLC) both in soft agar and as xenografts in nude mice. Importantly, the concentration of sulindac sulfide required to inhibit NSCLC cell growth greatly exceeded the concentration required to inhibit prostaglandin (PG) E(2) synthesis in NSCLC cells, suggesting that NSAID inhibition of cell growth is mediated by additional targets distinct from COX. Both sulindac sulfide and ciglitazone, a defined peroxisome proliferator-activated receptor-gamma (PPARgamma) agonist, stimulated a promoter construct containing a PPAR response element linked to luciferase and potently inhibited NSCLC cell growth at similar concentrations, indicating a role for PPARgamma as a target of NSAID action in these cells. Overexpression of PPARgamma in NSCLC cells strongly inhibited the transformed growth properties of the cells, providing a molecular confirmation of the results obtained with the PPARgamma agonists. Increased expression of PPARgamma, as well as ciglitazone and sulindac sulfide induced expression of E-cadherin, which has been linked to increased differentiation of NSCLC. Despite the fact that SCLC cell lines expressed little or no cytosolic phospholipase A(2), COX-1, or COX-2, sulindac sulfide and PPARgamma agonists also inhibited the transformed growth of these lung cancer cells. We propose that PPARgamma serves as a target for NSAIDs that accounts for COX-independent inhibition of lung cancer cell growth.

Animals↗
PubMed2001

Collaboration of JNKs and ERKs in nerve growth factor regulation of the neurofilament light chain promoter in PC12 cells.

Nerve growth factor (NGF) induces transcription-dependent neural differentiation of PC12 cells, and the ERK family of MAPKs has been implicated as the dominant signal pathway that mediates this response. We employed a neurofilament light chain (NFLC) promoter-luciferase (NFLC-Luc) reporter to define the role of the ERKs as well as additional MAPK pathways in NGF induction of this neural specific gene. Constitutive active forms of c-Raf-1, MEKK1 and MKK6, proximal regulators of the ERKs, JNKs, and p38 MAPKs, respectively, all stimulated NFLC-Luc activity. NFLC-Luc activity stimulated by NGF, however, was partially (approximately 50%) inhibited by the MEK inhibitor, PD098059, or by co-transfection of kinase-inactive MEK1 but not by the p38 MAPK inhibitor, SB203580, indicating a role for the ERKs, but not the p38 MAPKs, in NGF regulation of the NFLC promoter. Importantly, a gain-of-function MKK7-JNK3 fusion protein stimulated NFLC-Luc and synergized with gain-of-function c-Raf-1 to activate the NFLC promoter. In addition, transfection of kinase-inactive forms of MEK1 and MKK7 produced an additive inhibition of NGF-stimulated NFLC-Luc relative to either inhibitor alone. These findings indicate that the ERK and JNK pathways collaborate downstream of the NGF receptor for regulation of the NFLC promoter. Truncation analysis and electromobility shift assays established the requirement for a cAMP-response element/activating transcription factor-like site in the NFLC promoter that minimally interacts with constitutively expressed cAMP-response element-binding protein and JunD as well as c-Jun which is induced by NGF in an ERK-dependent manner. Cumulatively, these findings indicate that the ERK pathway requires collaboration with the JNK pathway for maximal activation of the NFLC gene in PC12 cells through the integrated control of c-Jun function.

Animals↗