Gene and Its Relationship with P53 in Non Small Cell Lung Cancer

Alterations of Fas (APO-1/CD 95) gene and its relationship with p53 in non small cell lung cancer

Laura Boldrini1, Pinuccia Faviana1, Francesca Pistolesi1, Silvia Gisfredi1, Dagmar Di Quirico1, Marco Lucchi2, Alfredo Mussi2, Carlo Alberto Angeletti2, Fulvia Baldinotti3, Antonella Fogli3, Paolo Simi3, Fulvio Basolo1 and Gabriella Fontanini*,1

1Department of Oncology, Transplants and Advanced Technologies in Medicine, University of Pisa, Italy; 2Department of Cardio-Thoracic Surgery, University of Pisa, Italy; 3Department of Oncology, U.O. of Cytogenetic and Molecular Genetic, 56100 Pisa, Italy

The Fas (APO-1/CD95) system regulates a number of The Fas receptor protein, also known as APO-1 or physiological and pathological processes of cell death. CD95, was ®rst identi®ed in 1989 (Yonehara et al., The ligand for Fas induces apoptosis by interacting with 1989) and subsequently shown to be a member of the a transmembrane cell surface Fas receptor. The key role tumor necrosis factor (TNF)-receptor superfamily, of the Fas system has been studied mostly in the immune mapping to the chromosome 10q23 (Lichter et al., system, but Fas mutations, one of the possible 1992). The majority of human cells express Fas mechanisms for resistance to apoptosis signaling, may receptor, although at dierent levels (Leithauser et be involved in the pathogenesis of non-lymphoid al., 1993; Midis et al., 1996; O'Connell et al., 1997), malignancies as well. To better understand the potential and its presence on several neoplastic cells may open involvement of Fas system in non-small cell lung cancer new approaches in cancer research. (NSCLC) we evaluated Fas and Fas-ligand mRNA The Fas ligand is a membrane protein predominantly expression by polymerase chain reaction in 102 tumor expressed on natural killer cells and activated T-cells. samples and in 44 normal surrounding tissues. Although The link of Fas ligand to Fas receptor induces apoptosis, over 60% of the human NSCLC analysed expressed particularly in cultured human T (Jurkat) cells but also in both genes, they seem to be unable to induce apoptosis in human B cells, cultured human rhabdomyosarcoma cells vivo by autocrine suicide. In this regard, we investigated (Yonehara et al., 1989), and murine ®broblast L929 cells in 79 cases, the promoter and the entire coding region of and T-cell lymphoma WR19L cells (Itoh and Nagata, the Fas gene by polymerase chain reaction, single strand 1993; Firestein et al., 1995). Therefore, the apoptosis conformation polymorphism and DNA sequencing for induced by the Fas-ligand seems to be predominantly detecting putative alterations. Sixteen tumors (20.25 %) involved in anti-viral immune responses, elimination of were found to have Fas alterations, in promoter and/or tumor cells, and regulation of lymphocyte development. exon region. In all cases samples carried heterozygous A 150 amino acid sequence at the C-terminus of Fas- alterations and mostly showed simultaneous mutations of ligand, which is located at the outside of the cell, shares p53 gene. Moreover, the quantitative analysis of Fas signi®cant homology with members of the TNF family, mRNA expression showed high levels of Fas messenger such as TNF or lymphotoxin (Boussaud et al., 1998; associated with p53 wild-type status alone. Taken Kashii et al., 1999). Binding of soluble Fas-ligand (a together, these ®ndings point to an involvement of Fas/ trimer) or cell-surface-expressed Fas-ligand (whose Fas-ligand system in the development of NSCLC, quaternary structure is unknown) to Fas receptor leads suggesting that the loss of its apoptotic function might to oligomerization (probably trimerization) of the be linked to p53 alterations which contribute to the self- receptor (Matsumura et al., 2000; Siegel et al., 2000). maintenance of cancer cells. Oncogene (2001) 20, This provides for the transmission of the apoptotic 6632 ± 6638. signal. Interestingly, it appears that soluble human Fas- ligand can also antagonize the action of the membrane- Keywords: NSCLC; Fas (CD95/APO-1); p53; muta- bound form. tions Recently, Fas-ligand has also been described on a variety of tumors, including melanoma (Hahne et al., 1996), colon carcinoma (O'Connell et al., 1996), astrocytoma (Gratas et al., 1997; Saas et al., 1997), and lung carcinoma (Niehans et al., 1997). Moreover, recent evidence suggests a role of Fas/Fas-ligand *Correspondence: G Fontanini, Department of Oncology, Division system in chemotherapy-induced apoptosis. Cytotoxic of Pathology, via Roma, 57 56126 Pisa, Italy; drugs commonly used in eective chemotherapy of E-mail: [email protected] Received 25 January 2001; revised 13 June 2001; accepted 14 June leukemias induce a strong Fas-ligand and Fas expres- 2001 sion in tumor cells following drug treatment (MuÈ ller et Alterations of Fas and p53 genes in non small cell lung cancer (NSCLC) L Boldrini et al 6633 al., 1997). Up-regulation of Fas by cytotoxic drugs seems to depend on wild-type p53 status (Munker et al., 1995, 1996; MuÈ ller et al., 1998). Events which initiate or trigger the apoptotic phenomenon may take dierent pathways, depending upon environmental context and apoptotic agents. In order to clarify the involvement of Fas and p53 in apoptotic process, we analysed the Fas/Fas-ligand system and p53-status in a series of non-small cell lung carcinomas and normal surrounding lung tissue, analysing their expression and their genetic assessment, with particular attention given to the simultaneous presence of mutations.

Reverse Transcription-Polymerase Chain Reaction analysis of Fas and Fas-ligand mRNA expression Fas mRNA expression was similar in tumors and normal tissue, with PCR signals in 68 of 102 cancers (66.6%) and 25 of 44 surrounding tissue (57%). Representative results of normal PCR for Fas mRNA Figure 1 Electrophoretic analysis of PCR products for Fas expression are shown in Figure 1 (upper panel). (upper panel) and of nested-PCR products for Fas ligand (lower As regards Fas-ligand mRNA expression, the panel) on a 1.5% agarose gel containing 0.5 mg/ml ethidium percentage of positivity was 69.6% in tumors (71 of bromide. Lanes L: marker (100-bp ladder, Pharmacia); lanes 1 ± 6: 102) and 45.4% in normal tissues (20 of 44). Nested human lung tumor samples. Total RNA was extracted from frozen tissue, primed with random hexamers to synthesize PCR was performed due to the higher sensitivity of complementary DNA (cDNA) using AMV reverse transcriptase. this technique than normal PCR. Figure 1 (lower PCR was carried out in a Perkin-Elmer Thermal Cycler following panel) shows some representative results of nested PCR these conditions: for FAS, cDNAs were ampli®ed for 35 cycles for Fas-ligand. (30 s at 948C, 60 s at 558C, and 60 s at 728C, with a ®nal A signi®cant correlation was found between Fas and additional extension step at 728C for 5 min) using primers capable of amplifying a segment from nucleotides 271 to 820 with: FAS 1 2 Fas-ligand mRNA expression in tumor samples (w 5'-CAAGTGACTGACATCAACTCC-3' and FAS 2 5'- test; P=0.0005). CCTTGGTTTTCCTTTCTGTGC-3' (Mitra et al., 1996); for FAS-Ligand Nested PCR, cDNAs were ampli®ed for 20 cycles (30 s at 948C, 60 s at 558C, and 90 s at 728C, with a ®nal Quantitation of Fas mRNA expression additional extension step at 728C for 5 min) using these primers: forward 5'-CAGCTCTTCCACCTACAG-3' (position 495) and On the basis of Fas PCR band-intensity for Fas, we reverse 5'-TCATGCTTCTCCGTCTTGACATGG-3' (position measured its expression using a competitive approach 1262) (Mitra et al., 1996). After ampli®cation with this ®rst pair in 77 tumor samples. In order to determine the of primers, 8 ml of the product was reampli®ed in 50 ml of fresh appropriate amount of Fas competitor to be used in reaction mixture, for 30 cycles under the same conditions using a second pair of primers: forward 5'-CTGGGGATGTTT- the PCR ampli®cation, we performed a preliminary GAGCTCTTC-3' (position 483) and reverse 5'-CTTCACTCCA- experiment in which Fas cDNA, obtained by retro- GAAAGCAGGAC-3' (position 693) (Shiraki et al., 1997) transcription of a constant amount of total RNA, was ampli®ed in the presence of serial dilutions of competitor. The results of these experiments enabled ampli®ed DNA fragments, indicating a structural us to perform competitive PCR reactions with this aberration of the Fas gene, were observed in 16 of known amount of competitor and cDNA derived from the 79 (20.25%) tumor samples; on the contrary, the 8 mg of total RNA. A representative electrophoretic percentage of p53 mutation was higher (25/40, 62.5%). analysis of competitive PCR products for Fas appears Figure 3 shows SSCP analysis in samples showing an in Figure 2. aberrant electrophoretic pattern of Fas and p53 genes. The mean value of Fas cDNA quantitation (57.93 Within the Fas gene we identi®ed nineteen aberrant molecules) allowed us to separate tumors with high bands: seven in promoter region, seven in exon 3, three (457.93 molecules of Fas cDNA) from tumors with in exon 5 and two in exon 7. Three samples low (457.93 molecules) Fas expression. Seven cases concomitantly showed two alterations of the gene. alone showed high Fas mRNA expression and these Sequencing analysis of these aberrant bands demon- cases were all Fas ligand positive (w2 test; P=0.036). strated that alterations were mostly missense or involved non-coding regions; only one sense mutation was present in exon 7, resulting in aminoacidic PCR ± SSCP analysis for Fas and p53 substitution. Interestingly, all the samples showing The highly-preserved regions of the p53 gene (exons alterations in the promoter region had the same 4 ± 9), the promoter and the coding regions of the Fas heterozygosity in position 90. By computer analysis gene were ampli®ed by PCR. Altered mobilities of we compared consensus p53-binding site, de®ned by

Oncogene Alterations of Fas and p53 genes in non small cell lung cancer (NSCLC) L Boldrini et al 6634 demonstrated low levels of Fas mRNA in the majority of NSCLC tissues. Recent observations suggest that tumor cells can exploit the Fas system by down-regulating the expression of functional Fas to resist Fas-ligand- mediated killing by cytotoxic T-cells, or by using the Fas system in mounting a `counterattack' for deletion of tumor-in®ltrating T-cells via up-regulation of tumor cell-derived Fas-ligand (O' Connell et al., 1997; Ungefroren et al., 1998; Bennett et al., 1998a). The result of this `tumor counterattack' could be the depletion of the attacking antitumor lymphocytes by the tumor, immunosuppression and immune escape. This pattern may have relevance in vivo, and the aim of Figure 2 Quantitation of Fas mRNA expression. Lanes 1 ± 4 future therapy will be to cause tumor rejection by and 6 ± 9: human lung tumor samples. Lane 5: marker (100-bp temporarily-induced T-cell resistance toward apoptosis ladder, Pharmacia). Lane 10: Fas Competitor alone. In order to obtain the quantitation of Fas mRNA levels we used a technique while maintaining sensitivity in the tumor cells. based on a competitive PCR approach using non-homologous To date it is not clear whether NSCLC is resistant to internal standard called Fas competitor. We constructed a 600 bp Fas-mediated apoptosis in vivo, but some data have Fas competitor as previously described (Boldrini et al., 1999): a supported this idea; in fact, cell lines from adenocarci- tritation assay was made to valuate the opportune competitor dilutions to be used. Competitive PCR reactions were then noma showed resistance to Fas-mediated apoptosis, performed adding dilutions of Fas competitor to aliquots of despite expressing Fas (Nambu et al., 1998). Moreover, cDNA derived from 8 mg of total RNA. The relative densito- in patients with completely resected non-small cell lung metric measure of the electrophoretic bands was then plotted and stage tumors, those with Fas expression were found to the point of equal intensity between the bands of competitor PCR survive longer than those without Fas expression MIMIC and target gene was taken as concentration of the cDNA sample (Uramoto et al., 1999). The expression of Fas in tumor samples does not necessarily predict susceptibility to death (Beltinger et El-Deiry et al. (1992), with Fas gene-promoter al., 1998); Fas-mediated apoptosis can be blocked by sequence, identifying a putative p53-binding element several mechanisms, including the mutation of the exactly in the promoter region containing heterozygous primary structure of Fas (Lee et al., 1999a) or the lack alteration in our samples. of p53-mediated activation of Fas/Fas-ligand system On the other hand, sequencing of SSCP-altered band (Bennett et al., 1998b). Epigenetic events, including for p53 gene identi®ed mutations resulting in aminoa- promoter region methylation, were supposed to be cidic substitution or deletions with frame-shift. Two contributing factors to the reduction in apoptotic samples concomitantly showed two alterations of p53, capability of tumors, even if early data do not con®rm and exon 4 of this gene was always mutated in various this hypothesis (Butler et al., 2000). spots, often identical in dierent cases. The key role of the Fas system has been studied None of the mutations were present in normal DNA mostly in the immune system (Nagata, 1997), and from the same patients, suggesting that aberrant bands mutations of Fas gene in cancer patients have been did not originate in germline alteration. Detection of described in several lymphoid-lineage malignancies. Fas and p53 gene mutation were reproducibile through More recently, somatic mutations of the Fas gene duplicate experiments. There was no correlation have also been found in solid tumors, such as bladder between presence of Fas mutations and its mRNA cancer (Lee et al., 1999b), malignant melanoma (Rhin expression, but when alterations of Fas were detected, et al., 2000) and skin squamous-cell carcinoma (Shin et p53 gene was also mutated (Figure 4, upper panel; al., 1999). Mutations appear to be clonal in the tumor P=0.01). Quantitative PCR showed that high Fas cells, but their functional relevance remains to be mRNA expression was signi®cantly associated with established. wild-type p53 (Figure 4, lower panel; P=0.0085). Although functional studies have not yet been Fas is a cell-surface receptor involved in cell-death performed, all the mutations identi®ed in Fas gene in signaling. The death signal cascade is initiated in the our study are either missense or interest non-coding cross-linking of Fas by its natural ligand. There is now regions, with no alteration of the normal function of broad evidence demonstrating that malignant cells Fas. Only one sense mutation in exon 7 was found, draw advantage from aberrant loss of Fas and increase causing aminoacidic substitution. Moreover, our Fas-ligand expression, as compared to their normal patients carry heterozygous alterations in Fas gene; counterparts. The analysis of Fas and Fas-ligand therefore hemizygously-normal Fas could explain Fas expression in our series of NSCLC samples points to mRNA expression we found in mutated samples. a higher percentage of Fas-ligand positive cells in The low percentage of tumor cells harboring a tumors than in surrounding normal tissue. On the somatically-mutated Fas gene (26.5% in our work, in other hand, although Fas positivity was similar in accordance with the range 4 ± 28% reported in other neoplastic and normal samples, quantitative analysis studies) suggests that somatic mutation of Fas gene

Oncogene Alterations of Fas and p53 genes in non small cell lung cancer (NSCLC) L Boldrini et al 6635 does not constitute a unifying event of malignant transformation in lung cancer. Other factors could be involved in the impairment of Fas signaling, justifying the absence of Fas messenger also in samples without alteration of this gene. Recent evidence suggests an intriguing link between p53 and Fas pathway: infection of p53-resistant cancer cells with adenovirus vector capable of transferring functional human Fas sensitized them to p53-mediated apoptosis (Rakkar et al., 1999). Moreover, Munsch et al. (2000) reported that intron 1 and promoter of the human Fas death- receptor gene contain a p53-

Figure 4 Upper panel: relationship between presence of Fas Figure 3 PCR ± SSCP detection of Fas and p53 mutations in mutation and p53 status in our 40 NSCLC samples (P=0.01). human lung tumors (T) in comparison with surrounding normal Lower panel: relationship between Fas expression (expressed in tissues (P). Tissue samples were mechanically disrupted in liquid terms of cDNA molecules quanti®ed by competitive PCR assay) nitrogen, lysed by proteinase-K. DNA extraction was then and p53 status in our 40 NSCLC samples (P=0.0085) performed using the spin column procedure (QIAamp Tissue Kit, Qiagen). PCR ± SSCP screening for p53 gene and Fas gene mutations: the eluted DNA was used as template in a standard 20 ml-PCR reaction mixture consisting of 10 mM Tris-HCl, 50 mM KCl, 1.5 mM MgCl2 (pH 8.3), 0.2 mM dNTPs, 8 pmol of each sense and antisense primer, and 1 U of Amplitaq DNA DNA template was omitted in the reaction. The ampli®cation Polymerase (Perkin-Elmer Cetus). PCR analysis for p53 gene products were separated on 1.5% agarose gels and visualized by was performed using Human p53 Amplimer Panels (CLON- ethydium bromide staining. The PCR products were diluted 1 : 1 TECH); PCR product sizes for p53 exons 4, 5, 6, 7 and 8 were with denaturing solution (1% xylene cyanol, 1% bromophenol 307, 211, 185, 139, and 200 bp, respectively. Since all the primers blue, 0.1 mM EDTA, 99% formamide), boiled for 5 min and in the Human Amplimer Panels had similar melting temperatures, thereafter directly placed on ice to prevent reannealing of the the same PCR conditions could be used to simultaneously amplify single stranded product. SSCP screening for both genes was all the exons (in separated reaction tubes). Conditions of p53 carried out on the GenePhor Electrophoresis Unit using GeneGel exons 4 ± 8, after initial denaturation at 958C (5 min), were 35 Excel 12.5/24 (12.5% T, 2% C), according to the instructions cycles of denaturation at 948C for 2 min, annealing at 618C for supplied with the Kit (Pharmacia Biotech). Electrophoresis was 2 min, and synthesis at 728C for 3 min, followed by ®nal performed at 188C, at 600 V, 25 mA, 15 W for 80 min. Gels were extension for 10 min. PCR analysis for Fas gene was performed stained using PlusOne Silver Staining Kit (Pharmacia Biotech), as described by Lee et al. (1999). PCR product sizes for promoter according to the supplied instructions. Tumour samples demon- and exons 1, 2, 3, 4, 5, 7, 8 and 9 were 124, 171, 249, 192, 228, strating aberrantly migrating bands in two or more independent 180, 164, 117 and 191 bp respectively. Conditions of Fas PCR ± SSCP runs were considered to contain a mutation. For the promoter and exons 1 ± 9, after initial denaturation at 948C detection of mutations, PCR products showing mobility shifts (5 min), were 40 cycles of denaturation at 948C for 40 s, were puri®ed with QIAquick PCR Puri®cation Kit (Qiagen) and annealing at 49 ± 608C for 40 s, and synthesis at 728C for 40 s, sequenced using cyclic sequencing kit (Perkin-Elmer, CA, USA) followed by ®nal extension for 5 min. As negative control, the according to the manufacturer's recommendations

Oncogene Alterations of Fas and p53 genes in non small cell lung cancer (NSCLC) L Boldrini et al 6636 responsive element, speci®cally bound only by wild- Our results indicate that low or absent expression of type p53. Our precedent work (Fontanini et al., 1999) Fas in NSCLC is not due to alterations of its gene in analysed p53 status in some NSCLC samples. In the the coding region; probably a p53-dependent regula- present work, we investigated p53 status in 40 NSCLC, tion of Fas system exists with a putative consequent founding p53-mutations in all samples with Fas gene loss of apoptotic function. This loss of function may alterations; Fas negative samples showed mutated p53, contribute to the development and maintenance of this and high Fas mRNA expression, based on quantitative type of cancer, suggesting the possibility of therapeutic PCR analysis, was signi®cantly associated with wild- approaches which aim at reconstructing the function of type p53. Accumulation of mutations within p53 gene Fas/Fas-ligand system. and within Fas promoter region, containing putative p53-binding site, could progressively silence Fas expression in malignant progression. In addition to transcriptional regulation of Fas expression by p53, the function of the Fas protein is Acknowledgments further regulated by p53, since the transport of Fas This work has been supported by grants from the protein to the cell surface is also coordinated by p53. Associazione Italiana per 1a Ricerca sul Cancro (A.I.R.C.).

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