Pavan Adiseshaiah

FRA-1 Proto-Oncogene Induces Lung Epithelial Cell Invasion and Anchorage-Independent Growth In vitro, but Is Insufficient to Promote Tumor Growth In vivo

FRA-1 forms activator protein-1 complexes in association with members of the JUN family and drives gene transcription. FRA-1 has been implicated in the development of airway squamous metaplasia and is frequently overexpressed in squamous cell carcinomas of the esophagus and stomach. We and others have shown a high level of persistent induction of FRA-1 by lung carcinogens, such as cigarette smoke and asbestos, in pulmonary epithelial cells. However, the exact roles of FRA-1 in regulating lung epithelial cell growth and invasion are poorly understood. To examine this aspect, we have stably overexpressed FRA-1 in human type-II-like alveolar malignant cell line (A549) and a nonmalignant bronchial epithelial cell line (BEAS-2B). FRA-1 greatly enhanced the rate of proliferation, motility, and invasion of A549 and BEAS-2B cells. In athymic nude mice, FRA-1, but not the control vector, rapidly enhanced tumor formation and metastasis by A549 cells. In contrast, FRA-1 failed to promote tumor formation by BEAS-2B. We suggest that FRA-1 can promote motility, invasion, and anchorage-independent growth of lung epithelial cells in vitro, but is insufficient for tumor formation.

Mitogen regulated induction of FRA-1 proto-oncogene is controlled by the transcription factors binding to both serum and TPA response elements.

FRA-1, a member of the FOS family of transcription factors, is overexpressed in a variety of human tumors, and contributes to tumor progression. In addition to mitogens, various toxicants and carcinogens persistently induce FRA-1 expression in vitro and in vivo. Although the mitogen induced expression of c-FOS is relatively well understood, it is poorly defined in the case of FRA-1. Our recent analysis of the FRA-1 promoter has shown a critical role for a TRE located at −318 in mediating the TPA-induced expression. The −379 to −283 bp promoter segment containing a critical TRE (−318), however, is insufficient for the induction of FRA-1 promoter. Here, we show that a 40-bp (−276/−237) segment, comprising a TCF binding site and the CArG box (collectively known as serum response element, SRE), and an ATF site, is also necessary for the FRA-1 induction by TPA and EGF. Interestingly, the −283 to +32 bp FRA-1 promoter fragment containing an SRE and an ATF site alone was also insufficient to confer TPA sensitivity to a reporter gene. However, in association with the −318 TRE, the SRE and ATF sites imparted a strong TPA-inducibility to the reporter. Similarly, EGF also required these motifs for the full induction of this gene. Using ChIP assays we show that, in contrast to c-Jun, SRF, Elk1, ATF1 and CREB proteins bind to SRE and ATF sites of the FRA-1 promoter, constitutively. RNAi-mediated knockdown of endogenous SRF, ELK1 and c-JUN protein expression significantly reduced TPA-stimulated FRA-1 promoter activity. Thus, a bipartite enhancer formed by an upstream TRE and the downstream SRE and ATF sites and the cognate factors is necessary and sufficient for the regulation of FRA-1 in response to mitogens.

A Phosphatidylinositol 3-Kinase-regulated Akt-Independent Signaling Promotes Cigarette Smoke-induced FRA-1 Expression

The FRA-1 proto-oncogene is overexpressed in a variety of human tumors and is known to up-regulate the expression of genes involved in tumor progression and invasion. The phosphatidylinositol 3-kinase (PI3K)-Akt pathway is also known to regulate these cellular processes. More importantly, respiratory toxicants and carcinogens activate both the PI3K-Akt pathway and FRA-1 expression in human bronchial epithelial (HBE) cells. In this study we investigated a potential link between the PI3K-Akt pathway and the cigarette smoke (CS)-stimulated epidermal growth factor receptor-mediated FRA-1 induction in non-oncogenic HBE cells. Treatment of cells with LY294002, an inhibitor of the PI3K-Akt pathway, completely blocked CS-induced FRA-1 expression. Surprisingly pharmacological inhibition of Akt had no significant effect on CS-induced FRA-1 expression. Likewise the inhibition of protein kinase C ζ, which is a known downstream effector of PI3K, did not alter FRA-1 expression. We found that the PI3K through p21-activated kinase 1 regulates FRA-1 proto-oncogene induction by CS and the subsequent activation of the Elk1 and cAMP-response element-binding protein transcription factors that are bound to the promoter in HBE cells.

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.

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.

A JNK-Independent Signaling Pathway Regulates TNFα-Stimulated, c-Jun-Driven FRA-1 Protooncogene Transcription in Pulmonary Epithelial Cells

Among the several effectors that mediate TNF-α action is AP-1, which consists of transcription factors belonging to the JUN and FOS families. Although the effects of TNF-α in immune cells, such as the induction of NF-κΒ, are well known, the mechanisms by which it induces transcriptional activation of AP-1 in pulmonary epithelial cells are not well defined. In this study, we report that TNF-α stimulates the expression of the FRA-1 protooncogene in human pulmonary epithelial cells using c-Jun, acting via a 12-O-tetradecanoylphorbol-13 acetate response element located at −318. Although TNF-α stimulates phosphorylation of c-Jun, the inhibition of JNK activity had no significant effect on FRA-1 induction. Consistent with this result, ectopic expression of a c-Jun mutant lacking JNK phosphorylation sites had no effect on the TNF-α-induced expression of the promoter. In contrast, inhibition of the ERK pathway or ectopic expression of an ERK1 mutant strikingly reduced FRA-1 transcription. ERK inhibition not only blocked phosphorylation of Elk1, CREB, and ATF1, which constitutively bind to the FRA-1 promoter, but also suppressed the recruitment of c-Jun to the promoter. We found that short interfering RNA-mediated silencing of FRA-1 enhances TNF-α-induced IL-8 expression, whereas overexpression causes an opposite effect. Our findings collectively indicate that ERK signaling plays key roles in both Elk1, CREB, and ATF-1 activation and the subsequent recruitment of c-Jun to the FRA-1 promoter in response to TNF-α in pulmonary epithelial cells.