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Transition from SCLC to NSCLC Phenotype Is Accompanied by an Increased TRE-Binding Activity and Recruitment of Speci®C AP-1 Proteins

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Transition from SCLC to NSCLC Phenotype Is Accompanied by an Increased TRE-Binding Activity and Recruitment of Speci®C AP-1 Proteins Oncogene (1998) 16, 3057 ± 3068 1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc Transition from SCLC to NSCLC phenotype is accompanied by an increased TRE-binding activity and recruitment of speci®c AP-1 proteins Gundula Risse-Hackl1,JuÈrgen Adamkiewicz2, Anja Wimmel1 and Marcus Schuermann1 1Zentrum fuÈr Innere Medizin, Abteilung HaÈmatologie/Onkologie, Philipps-UniversitaÈt Marburg, Baldingerstrasse, D-35033 Marburg; 2Institut fuÈr Molekularbiologie und Tumorforschung (IMT), Philipps-UniversitaÈt Marburg, Emil-Mannkop-Str. 2, D-35037 Marburg, Germany Transitions from small cell (SCLC) to non-small cell lung lung cancer are supposed to originate from dierent cancer (NSCLC) cells have been documented both in vitro cell types of the bronchial epithelium and alveoli. In and in vivo and are thought to be an important step during clinical practise, however, lung cancer frequently tumor progression of human small cell lung cancer exhibits more than one histologic pattern (Kalemker- towards a treatment-resistant tumor state. We have ian and Mabry, 1993; Mabry et al., 1988, 1991; Roggli screened NSCLC and SCLC cell lines for dierences in et al., 1985). Thus, mixtures of cells with SCLC and the composition of nuclear transcription factors using NSCLC histologies appear in SCLC tumors and, consensus oligonucleotide sequences (SRE, Ets, TRE, moreover, both SCLC and NSCLC phenotypic CRE, B-motif, GAS, E-box). We found NSCLC cells to markers have been detected simultanously in indivi- exhibit signi®cantly higher AP-1 binding activity than dual cells. These observations have led to the SCLC cells consistent with the increased expression of hypothesis that in vivo transitions from an SCLC to CD44, an AP-1 target gene. To gain more insight into the an NSCLC phenotype might occur. Further evidence molecular mechanisms underlying these dierences, we in favour of this hypothesis came from in vitro analysed SCLC cell lines (NCI-N592 and NCI-H69) experiments in which overexpression of activated H- which were phenotypically transformed into NSCLC-type ras and c-myc oncogenes in SCLC cell lines resulted in cells by transfection with activated H-ras and c-myc the loss of SCLC-speci®c markers and gain of oncogenes. In these cells, ras-induced transition is phenotypic characteristics resembling those of NSCLC accompanied by a strong induction of AP-1-binding cells (Mabry et al., 1988, 1991 BuÈ rger et al., 1994). activity along with increased expression of CD44 mRNA Such cell type transitions may be crucial for the and protein. When analysing the composition of the AP-1 progression of each lung cancer type and its resistance complex in more detail and comparing ras-induced versus to current modes of therapy. phorbol ester-induced changes, we found Fra-1 to be the Although the molecular events underlying these cell major component induced in ras-transfected but not in type transitions have not yet been elucidated, it is likely phorbol-ester treated or non-treated parental SCLC cells. that distinct transcription factors are involved in this This ®nding is paralleled by the observation that among process. This is exempli®ed by the aforementioned in the various members of the Fos and Jun family analysed vitro observation that the combined action of H-ras (c-Fos, FosB, Fra-1, Fra-2, c-Jun, JunD, JunB) fra-1 is and c-Myc is sucient to promote `transdierentiation' the only gene to be exclusively expressed in NSCLC cells into a dierent phenotype. c-Myc itself is a member of but not in cells of SCLC origin. Our data, thus, point to a a larger family of transcription factors binding to the histiotype-related mechanism of recruitment among AP-1 E-box element (for review see Amati and Land, 1994; proteins which may have bearings on the fate of lung Bernards, 1995) while H-ras as a signal transduction cancer development. molecule transmits its activity via the Raf-MAP kinase pathway (reviewed in Karin and Hunter, 1995; Keywords: AP-1; Jun; Fos; Fra-1, Ras; lung cancer; Marshall, 1995) and modulates the activity of distinct transdierentiation nuclear transcription factors, such as the activity of AP-1, Ets/TCF and NF-kB proteins. Alteration of AP- 1 activity has been particularly associated with increased Ras activity and therefore has been Introduction extensively characterized in the past (for review see Angel et al., 1987; Hill and Treisman, 1995; Marshall, Lung cancer is the leading cause of tumor-related death 1995). AP-1 consists of proteins of the Jun and Fos in both men and women. Tumors arising in the lung families which associate as homo-(Jun/Jun) or hetero- form a heterogenous group by histology which can be dimers (Fos/Jun; reviewed in Angel and Karin, 1991; classi®ed into four major subgroups: small cell Cohen and Curran, 1990). To date, three Jun proteins carcinoma (SCLC), accounting for 25% of human (c-Jun, JunB and JunD) and four Fos family members lung tumors, squamous cell carcinoma, large cell (c-Fos, FosB, Fra-1 and Fra-2) have been identi®ed as carcinoma and adenocarcinoma, collectively termed components of the AP-1 factor (Bohmann et al., 1987; non-small cell lung carcinoma (NSCLC). Due to their Cohen and Curran, 1988; Hirai et al., 1989; Matsui et dierent phenotypic properties, the various types of al., 1990; Nakabeppu et al., 1988; Nishina et al., 1990; Ryder et al., 1989; Zerial et al., 1989). Fos/Jun hetero- or Jun/Jun homodimers recognize a cis-acting element, the phorbol 12-myristate 13-acetate (TPA)-responsive Correspondence: M Schuermann Received 22 October 1997; revised 19 January 1998; accepted 20 element (TRE: TGACTCA), which is found in a January 1998 number of cellular promoters such as the genes coding Histiotype-dependent recruitment of Fra-1 in lung cancer GRisse-Hacklet al 3058 for interstitial collagenase, transin, typeIV collagenase, component in various dierentiation processes invol- procathepsin L or CD44 (Angel et al., 1987; Hofmann ving growth arrest (Dixit et al., 1993; Melamed et al., et al., 1993; Kerr et al., 1988; Sato and Seiki, 1993; 1993) or apoptosis (Estus et al., 1994; Goldstone and Troen et al., 1991). Lavin, 1994; Smeyne et al., 1993). Most of these eects While AP-1 activity is generally associated with have been reported in cell systems of diverge tissue proliferation (Angel and Karin, 1991; Kovary and origin and thus, the function of AP-1 seems to be Bravo, 1992) it is also an important signaling dependent not only on the pathway of signal a NSCLC b SCLC NHBE 32M1 U1752 97TM1 103H U1810 H23 H69 H82 H841 H146 H187 86M1 DMS79 – + ––––––++++++ PMA – + ––––––++++++ PMA fp — fp — 12345678910 11 12 13 14 12345678910 11 12 13 14 H125 H820 H1573 H2009 H2077 H2126 H526 H1092 H510 SW210 ––––––++++++ PMA PMA ––––++++ fp — fp — 15 16 17 18 19 20 21 22 23 24 25 26 15 16 17 18 19 20 21 22 c SCLC NSCLC H69 H82 H841 H146 32M1 103H H1573 H2077 PMA ––––––––+ + ++++ + + Figure 1 AP-1 DNA binding activity in NSCLC (a) and SCLC cell lines (b). Whole cell extracts (10 mg) were prepared from NHBE cells and NSCLC and SCLC cell lines as described under Materials and methods either in the presence (+) or absence (7) 78 of 5610 M PMA added for 24 h (indicated above the lanes). Aliquots were incubated with 32P-labeled, double-stranded consensus AP-1 DNA binding sequence. The bound complexes were resolved on a 4% polyacrylamide gel (fp=free probe). (c) Same experimental approach using a double-stranded consensus fp — sequence for SP-1 as control for equal quality of extracts (shown only for eight cell lines) Histiotype-dependent recruitment of Fra-1 in lung cancer G Risse-Hackl et al 3059 transduction itself but also on the context of cellular dierentiation. a NSCLC In this study, we have screened a number of transcription factors with respect to their binding 97TM1 32M1 H125 H820 103H H23 U1752 U1810 potential in lung cancer cell lines of dierent NHBE H1573 H2009 H2126 H2077 histological origin. Among these, we found AP-1 to be a protein complex which is abundantly present in CD44 — NSCLC but not in SCLC cells. Using H-ras/c-myc transfected SCLC cells, we can further show that upon transition from SCLC- to NSCLC phenotype these cells also gain strong AP-1 DNA binding capacity along with GAPDH — elevated AP-1 dependent gene expression. The strong TRE-binding activity in these NSCLC-like cells is largely achieved by the recruitment of Fra-1 into the complex the expression of which also is a hallmark of b SCLC NSCLC cells. This ®nding, thus, may point to a histiotype-related mechanism of regulation among 24H SW210 86M1 H841 H146 H187 H526 H510 H69 H82 members of the AP-1 family in lung cancer cells. DMS79 H1092 CD44 — Results Comparison of sequence-speci®c DNA-binding activities GAPDH — in NSCLC and SCLC cell lines A collection of 12 NSCLC and 11 SCLC cell lines was Figure 2 Expression of CD44 mRNA in lung cancer cell lines as examined in electrophoretic mobility shift assays analysed by RT ± PCR. Total RNA was isolated from the (EMSA) for the presence of proteins binding to indicated cell lines and reverse transcribed. Aliquots of the resulting cDNA were then used as a template for PCR dierent synthetic consensus sequences. The latter ampli®cation using gene-speci®c primers. [33P]dCTP was added either corresponded to consensus sites for nuclear to the PCR reaction to allow the sensitive detection of ampli®ed factors involved in the Ras-MAP kinase signaling DNA fragments using only a limited number of PCR cycles. The pathway such as the TRE (TPA-responsive element, reaction products were electrophoresed on 6% polyacrylamide gels and visualized by autoradiography.
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