Michelle Vaz

A Fra-1-dependent, matrix metalloproteinase driven EGFR activation promotes human lung epithelial cell motility and invasion

We and others have shown a persistently high induction of Fra‐1 transcription factor (a dimeric partner of AP‐1) levels by respiratory carcinogens in pulmonary epithelial cells. Fra‐1 is frequently overexpressed in various human tumors and cancer cells. We have recently shown that Fra‐1 significantly promotes growth, motility, and invasion of human pulmonary epithelial cells, the precise molecular mechanisms by which this enhancement occurs are unclear. Because matrix metalloproteinases (MMPs) play key roles in wound healing and lung tumor metastasis, we tested the hypothesis that Fra‐1 promotes lung epithelial cell motility and invasion via MMP activation. We show here that MMP‐9 and MMP‐2 activated signaling plays a critical role in regulating Fra‐1‐induced lung epithelial cell growth and invasion. Ectopic Fra‐1 markedly stimulates MMP‐2 and MMP‐9 mRNA expression. Inhibition of MMP‐2 and MMP‐9 activity significantly attenuated Fra‐1‐driven cell motility and invasion. Furthermore, Fra‐1 induced EGFR phosphorylation in an MMP‐dependent manner, and an EGFR‐specific inhibitor was able to block Fra‐1‐enhanced cell motility and invasion. Taken together, our data suggest that Fra‐1 enhances lung cancer epithelial cell motility and invasion by inducing the activity of MMPs, in particular MMP‐2 and MMP‐9, and EGFR‐activated signaling. J. Cell. Physiol. 216: 405–412, 2008.

Fra-1/AP-1 transcription factor negatively regulates pulmonary fibrosis in vivo

The Fra-1/AP-1 transcription factor plays a key role in tumor epithelial cell progression; however, its role in pathogenic lung fibrosis remains unclear. In the present study, using a genetic approach (Fra-1 deficient mice), we have demonstrated a novel regulatory (protective) role for Fra-1 in lung fibrosis. We found greater levels of progressive interstitial fibrosis, characterized by increased levels of inflammation, collagen accumulation, and profibrotic and fibrotic gene expression in the lungs of Fra-1 Δ/Δ mice than in those of Fra-1+/+ mice following bleomycin treatment. Fra-1 knockdown in human lung epithelial cells caused the upregulation of mesenchymal marker N-cadherin, concomitant with a downregulation of the epithelial phenotype marker E-cadherin, under basal conditions and in response to bleomycin and TGF-β1. Furthermore, Fra-1 knockdown caused an enhanced expression of type 1 collagen and the downregulation of collagenase (MMP-1 and MMP-13) gene expression in human lung epithelial cells. Collectively, our findings demonstrate that Fra-1 mediates anti-fibrotic effects in the lung through the modulation of proinflammatory, profibrotic and fibrotic gene expression, and suggests that the Fra-1 transcription factor may be a potential target for pulmonary fibrosis, a progressive disorder with poor prognosis and treatment.

ERK signaling regulates tumor promoter induced c-Jun recruitment at the Fra-1 promoter.

Fra-1 as an integral part of AP-1 (Jun/Fos) drives transcriptional programs involved in several physiologic and pathologic processes. It is also critical for tumor cell motility and metastasis. We have previously shown that two critical elements of Fra-1 promoter, the upstream TPA response element (TRE) and the serum response element (SRE), are necessary for its induction in response to phorbol esters in human pulmonary epithelial cell lines. Here, we have investigated the roles of various MAP kinases in regulating Fra-1 expression in response to TPA. Using pharmacologic and genetic tools, we demonstrate a prominent role for ERK1/2, but not JNK1/2 and p38, signaling in the TPA-induced activation of specific transcription factors that bind to the AP1 site and the SRE. Inhibition of ERK1/2 pathway suppresses Elk1 activation, and c-Jun and Fra-2 recruitment to the promoter.

Oxidant-Induced Cell Death and Nrf2-Dependent Antioxidative Response Are Controlled by Fra-1/AP-1

ABSTRACT AP-1 (Jun/Fos) transcription factors play key roles in various biological processes, including cell death. Here we report a novel role for Fra-1 in oxidant-induced cell death controlled by modulating antioxidant gene expression. Fra-1-deficient (Fra-1Δ/Δ) mouse embryonic fibroblasts (MEFs) and primary lung fibroblasts (PLFs) were remarkably resistant to H2O2- and diquat-induced cell death, compared to their wild-type (Fra-1+/+) counterparts. Fra-1 deficiency ablated oxidant-induced mitochondrion-dependent apoptosis. Fra-1Δ/Δ cells had elevated basal levels of antioxidant enzymes and intracellular glutathione (GSH), which were further stimulated by oxidants. Loss of Fra-1 led to an increased half-life of transcription factor Nrf2 and increased recruitment of this protein to the promoters of antioxidant genes and increased their expression. Depletion of intracellular GSH or RNA interference (RNAi)-mediated knockdown of Nqo1, Hmox1, and Nrf2 restored oxidant-induced cell death in Fra-1Δ/Δ cells. Thus, Fra-1 appears to increase susceptibility to oxidants and promotes cell death by attenuating Nrf2-driven antioxidant responses.

Genetic Disruption of Fra-1 Decreases Susceptibility to Endotoxin-Induced Acute Lung Injury and Mortality in Mice

The activator protein-1 (AP-1) transcription factor, comprising Jun and Fos family proteins, distinctly regulates various cellular processes, including those involved in inflammation. FOS like antigen 1 (Fra-1), a member of the Fos family, dimerizes with members of the Jun family and regulates gene expression in a context-dependent manner. Although respiratory toxicants are known to stimulate the expression of Fra-1 in the lung, whether Fra-1 promotes or decreases susceptibility to the development and progression of toxicant-induced lung disease in vivo is not well established. To determine the role of Fra-1 in LPS-induced acute lung injury and mortality, we administered LPS either intraperitoneally or intratracheally to Fra-1-sufficient (Fra-11(+/+)) and Fra-1-deficient (Fra-1(Δ/Δ)) mice. LPS-induced mortality, lung injury, inflammation, cytokine measurements, and AP-1 and NF-κB activities were then assessed in these mice. Fra-1(Δ/Δ) mice showed a greater resistance to LPS-induced mortality than did their Fra-1(+/+) counterparts. Consistent with this result, LPS-induced lung injury and inflammatory responses were markedly lower in Fra-1(Δ/Δ) mice than in Fra-1(+/+) mice. Compared with Fra-1(+/+) mice, Fra-1(Δ/Δ) mice showed a reduced influx of neutrophils into the lungs, accompanied by a decreased expression of proinflammatory cytokines in response to treatment with LPS. The decreased inflammatory responses in Fra-1(Δ/Δ) mice coincided with diminished and increased levels of NF-κB and c-Jun/AP-1 binding, respectively. These results demonstrate that Fra-1/AP-1 plays a key role in promoting LPS-induced injury and mortality in mice, and they suggest that targeting (i.e., inhibiting) this transcription factor may be a useful approach to dampening the adverse effects of exposure to endotoxins.

FOSL1 Promotes Kras-induced Lung Cancer through Amphiregulin and Cell Survival Gene Regulation

&NA; The FOSL1/AP‐1 transcription factor regulates gene expression, thereby controlling various pathophysiological processes. It is a major effector of RAS‐ERK1/2 signaling and is activated in human lung epithelia by tumorigenic stimuli. Recent evidence shows an inverse correlation between FOSL1 expression and the survival of patients with lung cancer and adenocarcinomas; however, its role in lung tumorigenesis remains elusive. In this work, we sought to determine the role of FOSL1 in Kras‐induced lung adenocarcinoma in vivo and its downstream effector mechanisms. We used mice expressing the Kras oncogene in the lung with concomitant Fosl1 deletion, Kras‐activated murine alveolar epithelial cells (mAECs) with Fosl1 deletion, and KRAS mutant human lung adenocarcinoma (HLAC) cells with FOSL1 deficiency, and performed cell proliferation and gene expression analyses. Mutant Kras induced Fosl1 expression in vitro (mAECs) and in vivo (lung tissue), and mice with Fosl1 deletion showed reduced levels of mutant Kras‐induced lung tumorigenesis and survived longer than Fosl1‐sufficient mice. Studies with mutant Kras‐activated mAECs and KRAS‐mutant HLAC cells revealed that FOSL1 regulates mutant KRAS‐induced gene expression, thereby controlling cell proliferation and survival. In contrast, FOSL1 depletion in non‐KRAS‐mutant HLAC cells and nonmalignant human lung epithelia had no effect. Our data support the notion that FOSL1‐mediated expression of amphiregulin and apoptotic and antioxidative genes plays a role in regulating HLAC cell proliferation and survival. FOSL1 is a determinant of lung cancer in vivo and regulates HLAC cell proliferation and survival, largely in the context of KRAS mutations. Activation of FOSL1 in adenocarcinomas may be a prognostic marker and potential target for human lung cancer with KRAS mutations.

Myeloid-Specific Fos-Related Antigen-1 Regulates Cigarette Smoke–Induced Lung Inflammation, Not Emphysema, in Mice

Heightened lung inflammation is a cardinal feature of chronic obstructive pulmonary disease (COPD). Cigarette smoke (CS)-induced macrophage recruitment and activation, accompanied by abnormal secretion of a number of inflammatory cytokines and matrix metalloproteinases, play a major role in the pathophysiology of COPD. The Fos-related antigen-1 (Fra-1) transcription factor differentially regulates several cellular processes that are implicated in COPD, such as inflammation and immune responses, cell proliferation and death, and extracellular remodeling. Although CS stimulates Fra-1 expression in the lung, the precise role of this transcription factor in the regulation of CS-induced lung inflammation in vivo is poorly understood. Here, we report that myeloid-specific Fra-1 signaling is important for CS-induced lung macrophagic inflammatory response. In response to chronic CS exposure, mice with Fra-1 specifically deleted in myeloid cells showed reduced levels of CS-induced lung macrophagic inflammation, accompanied by decreased expression levels of proinflammatory cytokines compared with their wild-type counterparts. Consistent with this result, bone marrow-derived Fra-1-null macrophages treated with CS showed decreased levels of proinflammatory mediators and matrix metalloproteinases. Interestingly, deletion of Fra-1 in myeloid cells did not affect the severity of emphysema. We propose that Fra-1 plays a key role in promoting chronic CS-induced lung macrophagic inflammation in vivo, and that targeting this transcription factor may be useful in dampening persistent lung inflammation in patients with COPD.

Abstract LB-098: Sequential azacitidine and histone deacetylase inhibition induces a potent antitumor response in Kras G12D mouse model of NSCLC

Combination epigenetic treatment as a therapeutic approach holds much promise. However, to unlock the true potential of these combinatorial paradigms, further optimization is required. We have elucidated in our current study, a treatment schedule combining Azacitidine with Histone Deacetylase Inhibition (HDACi) which considers both schedule and isoform specificity. We have deployed a sequential schedule with HDACi administered post Azacitidine in a chronic low dose manner. This schedule takes advantage of two key parameters altered by Azacitidine: dsRNA induced immune signature and MYC depletion. In our system, HDACis amplify an existing immune signature and the MYC depleted environment acts as a potent sensitizer. HDACi induced augmentation of immune gene transcription seems driven predominantly through the inhibition of HDAC1/2 and HDAC6 isoforms, with broad spectrum HDACi inhibitors demonstrating the greatest potency. While the combination treatment induced perturbation of proliferation is driven by inhibition of HDAC1/2/3 isoforms. Applying this concept to a genetically engineered mouse model of NSCLC we observe a potent anti-tumor response as evidenced by a reduction in tumor burden, progression and proliferation. These phenotypic parameters are correlated with modification of both immune and proliferative gene signatures as well as alteration of key immune populations in the tumor microenvironment. We propose that this combinatorial alteration of both immune and proliferative parameters holds the promise to be a more robust treatment for NSCLC. Note: This abstract was not presented at the meeting. Citation Format: Michael Topper, Michelle Vaz, Katherine Chiappinelli, Christina DeStefano Shields, Alyssa Wenzel, Cynthia Zahnow, Pam Strissel, Reiner Strick, Stephen Baylin. Sequential azacitidine and histone deacetylase inhibition induces a potent antitumor response in Kras G12D mouse model of NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-098. doi:10.1158/1538-7445.AM2017-LB-098

Abstract 4653: Combination Azacitidine and histone deacetylase inhibition induces a multi factorial synergistic anti-tumor response in non-small cell lung cancer (NSCLC)

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Epigenetic therapy holds much promise as an emerging paradigm for the treatment of many disparate types of human cancer. However, the data in solid malignancies, particularly, would suggest further optimization is required to extract the true potential of these therapies. In our current study, we have deployed at low, clinically achievable doses, chronic combinatorial administration of the DNA methyltransferase inhibitor Azacitidine in conjunction with both benzamide and hydroxyamic acid based histone deacetylase inhibitors (HDACi) in NSCLC. We have developed our paradigms by sequentially employing chronic HDACi administration after Azacitidine. This strategy achieves durable alteration of the cell transcriptome with corresponding induction of both cell cycle arrest and apoptosis. These events appear driven by alterations, both transcriptionally and post transcriptionally of Myc and Myc regulatory proteins. Epigenetic treatment induced perturbation of Myc is facilitated by Wnt pathway inhibition through induction of Wnt antagonists, namely DKK proteins. Importantly, as part of this scenario of altering a Myc regulatory axis, there is a synergistic cytotoxic response mediated at least in part through downregulation of a critical effector, the Skp2 oncogene. Additionally, we observe significant induction of an interferon responsive viral defense gene signature as the result of epigenetic treatment. This gene signature has been shown to correlate with response to immune checkpoint blockade in the setting of metastatic melanoma. We propose that this combination of viral defense gene signature augmentation in conjunction with potent direct epithelial cancer cytotoxicity holds promise to be a more complete therapeutic paradigm for the treatment of NSCLC. Citation Format: Michael Topper, Christin Hanigan, Michelle Vaz, Katherine Chiappinelli, Julin Justin, Lauren Murphy, Cynthia Zahnow, Stephen Baylin. Combination Azacitidine and histone deacetylase inhibition induces a multi factorial synergistic anti-tumor response in non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4653.

Abstract 2773: Chronic cigarette smoke exposure of bronchial epithelial cells induces progressive epigenomic changes leading to transformation

Herein, we define a model for how cigarette smoke and chronic inflammation induce human lung cancer via evolution of a co-ordinated pattern of progressive cancer-associated epigenetic abnormalities which prime cells for addiction to a single key genetic alteration, KRAS mutation. Non-clonogenic, non-tumorigenic, epigenetically stable human bronchial epithelial cells (HBEC) were exposed to cigarette smoke condensate (CSC) for 15 months. Earlier studies have established a requirement for the simultaneous disruption of three oncogenes to fully transform these cells. Genome-wide DNA methylation, expression, chromatin changes, as well as binding to chromatin of key epigenetic regulators and cell phenotypic features were examined over time in exposed versus non-exposed cells. CSC exposure acutely causes, within 10 days, a change we have previously associated with DNA damage, tightening of DNA methyltransferase 1 (DNMT1) and EZH2 to chromatin. While the EZH2 binding decreases with prolonged exposure, DNMT1 remains tightly bound to chromatin. Chronic exposure causes progressive, but stochastically variable, global DNA methylation changes which begin by six months and progress over the time course of the study to include hypermethylation of gene promoters which are frequent in human lung cancer. ChIP-seq analyses reveal, preceding the above methylation changes, that promoters of such methylated genes have an initial recruitment of EZH2, which begins at 10 days and then decreases with time. In contrast, recruitment of EZH2 increases with time and remains dominant for these same genes in the non-exposed controls. Following 10 months of exposure, CSC treated cells begin to clone in soft agar, often a feature of transformation, yet do not form tumors in immunodeficient mice. At this time point, gene expression studies show the top signaling pathway change is strong activation of MAP-kinase and KRAS pathways. Yet genome-wide, exome sequencing reveals no known lung cancer driver gene mutations. Remarkably, overexpression of mutant KRAS alone now markedly enlarges the soft agar colonies, and the cells are now fully transformed and form tumors in mice. Our study reveals, in a chronic cigarette exposure model relevant to the time course for evolution of KRAS mutant human lung adenocarcinoma, a key initial role for smoking induced epigenetic changes which facilitate addiction to the oncogene. Citation Format: Michelle P. Vaz, Stephen Y. Hwang, Ashwini Patil, Jillian Phallen, Lauren Murphy, Cynthia A. Zahnow, Victor E. Velculescu, Hariharan Easwaran, Stephen B. Baylin. Chronic cigarette smoke exposure of bronchial epithelial cells induces progressive epigenomic changes leading to transformation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2773.