Cells Tissues Organs 2002;172:126132 DOI: 10.

1159/000065609

Apoptotic Genes in Cancer Therapy

Bertram Opalka a Alexandra Dickopp b Hans-Christoph Kirch a
a Department

of Internal Medicine (Cancer Research) and b Institute for Molecular Biology (Cancer Research), University of Essen, Germany

Key Words Apoptosis W p53 W E1A W Cancer gene therapy

Abstract Induction of apoptosis in malignant cells is a major goal of cancer therapy in general and of certain cancer gene therapy strategies in particular. Numerous apoptosisregulating genes have been evaluated for this purpose. Besides the most prominent p53 gene others include p16, p21, p27, E2F genes, FHIT, PTEN and CASPASE genes. Recently, the potential for therapy of an adenoviral gene, E1A, known for a long time for its apoptosisinducing activity, has been discovered. In experimental settings, these genes have proven their tumor-suppressive and apoptosis-inducing activity. Clinical trials are currently being performed with selected genes. By far the most studies transfer the p53 gene using retro- or adenoviral vectors. Disease stabilization or other benefits were observed in a limited number of patients when p53 was applied alone or in combination with cytotoxic drugs. A second proapoptotic gene that has entered clin-

ical trials is adenovirus E1A. Here, too, disease stabilization as well as/or local regression in one case have been demonstrated in selected patients. In all cases, side effects were tolerable. To further improve E1A as a therapeutic transgene, we have deleted transforming domains from the adenovirus 5 and 12 13S cDNAs. Mutants were derived which had completely lost their transforming activity in combination with the E1B oncogene but retained a pronounced tumor-suppressive activity. Cells transduced with these constructs showed a highly reduced ability to grow in soft agar, and tumor growth in nude mice could be substantially suppressed. Outgrowing tumors had lost E1A expression when analyzed in Western blots. These E1A constructs may represent valuable tools for cancer gene therapy in the future.
Copyright 2002 S. Karger AG, Basel

Abbreviations used in this paper

CR NSCLC PCR PFU RT-PCR

conserved region non-small-cell lung cancer polymerase chain reaction plaque forming unit reverse transcriptase polymerase chain reaction

This paper briefly summarizes selected preclinical and clinical data on apoptotic genes used in cancer gene therapy. There are good arguments why the use of apoptotic genes is a straightforward strategy for cancer gene therapy. First, it is now known that most, if not all, anticancer treatments induce apoptotic death of the malignant cells. Most mutations found in cancer affect genes involved in the regulation of apoptosis and this results in a deregulated cell cycle and resistance to undergo apoptosis induced by cytotoxic drugs or irradiation. Many genes involved in apoptosis have already shown therapeutic effects in experimental systems either in vitro or in vivo.

ABC
Fax + 41 61 306 12 34 E-Mail karger@karger.ch www.karger.com

2002 S. Karger AG, Basel 14226405/02/17220126$18.50/0 Accessible online at: www.karger.com/cto

Bertram Opalka, PhD Department of Internal Medicine (Cancer Research), University Clinics Essen Hufelandstrasse 55 D45122 Essen (Germany) Tel./Fax +49 201 723 2020, E-Mail bertram.opalka@uni-essen.de

Table 1. Apoptotic genes tested for applications in gene therapy

Gene p53 p16 p21 p27 E1A E2F-1 FHIT IB PTEN STAT3 E4orf4 CASPASE-3 CASPASE-8 BAX/BAK TSG

Function of gene product(s) TSG, transcription factor TSG, inhibitor of cyclin-dependent kinases inhibitor of cyclin-dependent kinases inhibitor of cyclin-dependent kinases viral regulatory gene transcription factor (Ap3A) hydrolase inhibitor of transcription factor TSG, signal tranducer? transcriptional regulator adenoviral regulatory protein proteolytic enzyme proteolytic enzyme regulators of apoptosis tumor suppressor gene

A list of important apoptotic genes that are currently used for experimental cancer therapy is shown in table 1. By far the most frequently used gene in therapeutic approaches is the p53 tumor suppressor gene, which plays a central role in the regulation of apoptosis [for a review, see Balint and Vousden, 2001]. Other genes are the p16 tumor suppressor gene, which encodes an inhibitor of cyclin-dependent kinases [Sandig et al., 1997] or p21 [Shibata et al., 2001] and p27 [Craig et al., 1997], whose products are also inhibitors of cyclin-dependent kinases, or the gene encoding the transcription factor E2F-1 [Dong et al., 1999]. Adenoviral gene products have been shown to induce apoptosis and are also used for anticancer treatment. The E1A gene which encodes regulatory proteins for transcriptional modulation of several viral and cellular genes [Mymryk, 1996] and a gene from the E4 region, the so-called open reading frame No. 4 [Shtrichman et al., 1999] are of great importance. Other genes include the recently discovered FHIT gene [Ji et al., 1999], which encodes a nuclear type (Ap3A) hydrolase, the PTEN gene [Cheney et al., 1998], encoding a phosphatase active in signal transduction, the IB gene encoding an inhibitor of the NFB transcription factor [Batra et al., 1999], or the CASPASE-3 or CASPASE-8 genes encoding major proteases involved in various apoptotic pathways [Yamabe et al., 1999; Shinoura et al., 2000]. Another important molecule is the Fas ligand [Arai et al., 1997]. It is noteworthy that most of these genes are currently used in experimental systems and only p53 and E1A are already evaluated in clinical trials that are summarized at the end of this review.

As an example of therapeutic apoptosis, ovarian cancer cells (SK-OV3 cells), overexpressing the HER2/neu oncogene, were transfected in vitro with a construct expressing the E1A gene [Zhang et al., 1995]. Uninfected cells as well as cells infected with an empty vector showed high HER2/ neu gene expression, whereas cells transduced with a vector expressing the E1A gene have apparently lost the HER2/neu protein. It has shown earlier that this loss of HER2/neu expression was due to direct transcriptional downregulation of the HER2/neu gene by E1A [Yu et al., 1990]. The survival curve of mice that were inoculated intraperitoneally with SK-OV3 tumor cells and injected with an E1A construct at the same site showed a significantly prolonged survival compared with untreated or sham-treated mice [Zhang et al., 1995]. In another experimental example the FHIT gene was evaluated as a therapeutic transgene in two lung cancer cell lines H1299 and A549 [Ji et al., 1999]. Tumor cells were inoculated subcutaneously into nude mice, grown to a diameter of 510 mm and then treated intratumorally with adenoviral vectors expressing the FHIT cDNA. Tumor treatment was repeated 45 times. While tumors that were untreated or treated with the empty control vector grew, tumors treated with the FHIT construct had a substantially reduced growth rate and remained 1/10 in size of the control tumors. These two examples show that treatment of tumor cells with apoptosis-inducing gene constructs is effective in vitro and in vivo. Apart from these experimental systems, clinical studies with apoptotic genes listed in table 2 have been approved until spring of 2000 [Rosenberg et al., 1999]. This summary gives only some examples of active and completed clinical trials. However, published in 2000, the contents of this table still reflects quite well those apoptotic genes that are currently evaluated in clinical trials [human gene therapy protocols, 2001]. As can be seen in table 2, the most widely used gene is p53. It is proposed to treat various cancers like non-small-cell lung cancer (NSCLC), breast cancer, squamous cell carcinoma of the head and neck, hepatocellular carcinoma and prostate cancer. The E1A gene is used for treatment of cancers overexpressing HER2/neu for the mechanisms mentioned above in breast and ovarian cancer as well as head and neck squamous cell carcinoma. Data from a clinical study performed at the MD Anderson Cancer Center were published in 1999 and are summarized in table 3 [Swisher et al., 1999]. In this trial an adenoviral vector expressing the p53 cDNA (Adp53) was used to treat 28 patients with advanced NSCLC who had failed on chemo- and radiotherapy. These patients

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Table 2. Examples of clinical trials with proapoptotic genes [selected from Rosenberg et al., 1999]

Gene p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 p53 E1A

Principal investigator J.A. Roth G. Clayman C.P. Belani A. Belldegrun R. Figlin R.L. Breau M. v. Mehren L.G. Pagliario S. Swisher C.Y. Muller F.F. Lang W.K.A. Yung N.A. Habib RG.N. Hortobagyi G.L. Berstein M.C. Hung S. LaFollette J.L. Murray G.H. Yoo

Institution MD Anderson CC MD Anderson CC University of Pittsburgh UCLA Various Fox Chase CC MD Anderson CC MD Anderson CC University of Texas MD Anderson CC Hammersmith Hospital MD Anderson CC

Disease NSCLC HNSCC HCC prostate cancer SCCHN breast cancer bladder cancer NSCLC ovarian cancer malignant glioma metastatic liver tumors breast/ovarian cancer

Remarks +cisplatin

HER2/neu+++

E1A E1A

St. Lukes Chicago, Ill. Various

ovarian cancer HNSCC

HER2/neu+++

CC = Cancer Center; NSCLC = non-small-cell lung cancer; HNSCC = head and neck squamous cell carcinoma; HCC = hepatocellular carcinoma; SCCHN = squamous cell carcinoma of the head and neck.

Table 3. Clinical phase I trial with Adp53

Patients Treatment Results

28 patients with advanced NSCLC 1061011 PFU Adp53 percutaneously (23 patients) or by bronchoscopy (5 patients) presence of vector in 18/21 patients (86%) by PCR analysis, mRNA detected in 12/26 patients (46%); apoptosis in 11/21 patients (52%); partial response: 2 patients (8%); disease stabilization: 16 patients (64%); progressive disease: 7 patients (28%)

Data from Swisher et al. [1999]. Adp53 = Adenovirus vector expressing p53.

were treated with escalating doses from 1061011 PFU of an adenoviral construct administered either percutaneously or by bronchoscopy. The presence of the vector was monitored by DNA PCR and was found in 18 of 21 (86%) patients. The mRNA was detected in 46% of patients by RT-PCR and increased apoptosis was observed in 52% of patients as assessed by immunohistochemistry. The clinical responses were as follows. A partial response, defined as a significant reduction in tumor size, was observed in 8% of the patients, whereas 64% patients showed stable disease. Progressive disease was found in 7 patients. The side effects were only moderate with flu-like symptoms and fever and occasional pain at the injection site. A CAT scan of one of the patients demonstrated an ongoing regression. After 18 months and 6 courses of treatment with the p53 (Adp53), no evidence of tumor cells was detected in this patient. In a second study, p53 adenovirus therapy was combined with cisplatin treatment [Nemunaitis et al., 2000].

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Table 4. Clinical phase I trial with Adp53 plus cisplatin

Patients Treatment Results

24 patients with advanced NSCLC 80 mg/m2 cisplatin followed by 1061011 PFU Adp53 mRNA detected in 6/14 patients (43%), increase in apoptosis in 11/14 patients (79%); disease stabilization: 17 patients (71%); partial response: 2 patients (10%); progressive disease: 4 patients (19%); not assessable: 1 patient (5%)

Data from Nemunaitis et al. [2000]. Adp53 = Adenovirus vector expressing p53.

Table 5. Problems with the use of apoptotic genes for cancer gene

therapy For an efficient therapeutic intervention ideally all tumor cells have to express the transgene This is unachievable in practice, e.g. due to inefficient transfer vectors Bystander effects and/or immunostimulation by apoptotic cells may circumvent these problems

This study enrolled 24 patients with advanced NSCLC who had failed chemotherapy alone or many other kinds of treatment (table 4). 80 mg/m2 of cisplatin were administered, followed by an escalating dose of 1061011 PFU of p53 (Adp53). In these patients mRNA was detected in 43% and an increase of the apoptotic index was found in 79% of the patients. This combination therapy led to disease stabilization in 17 patients (71%) and a partial response in 2 patients (10%). Progressive disease was seen in 4 patients only; 1 patient was lost to follow-up. The evidence of a clinical response was confirmed by bronchoscopy. More recently, additional data on the use of p53 for the treatment of NSCLC have been reported [Schuler et al., 2001; Weill et al., 2000; Yen et al., 2000]. Application of p53 in an adenoviral vector was shown to be safe and an improvement of airway obstruction was observed in 6 of 12 patients [Weill et al., 2000]. However, data on the benefit of p53-treated patients were inconsistent among the different studies [Schuler et al., 2001; Yen et al., 2000]. The biological safety, feasibility and effective transduction with an adenoviral p53 vector was also demonstrated in melanoma and breast cancer patients [Dummer et al., 2000]. Initial data are also available from trials using the adenoviral E1A gene for treatment of HER2/neu-overex-

pressing breast, ovary and head and neck cancer [Hortobagyi et al., 2001; Yoo et al., 2001]. In contrast to p53, which is most frequently applied in viral vectors, E1A was given as a nonviral preparation consisting of plasmid DNA/lipid complexes. The application was found to be safe and side effects such as fever, nausea, vomiting, discomfort or bleeding at the injection site were tolerable. Downregulation of HER2/neu was observed along with reduced proliferation and increased apoptosis [Hortobagyi et al., 2001]. In 1 breast cancer patient, the tumor was undetectable in biopsies of the treated tumor site; in 2 of 16 evaluable patients the tumor showed a minor response, while in 8 stable disease occurred [Yoo et al., 2001]. Thus, it can be concluded that apoptosis-inducing regulatory genes yield promising antitumor activity in vitro and in vivo in several animal models and even in some clinical studies. In the future, these genes may be useful for clinical tumor treatment, either alone or, more probably, in combination with cytotoxic drugs, radiotherapy or immunostimulatory protocols. Major problems with the use of apoptosis-inducing genes as opposed to immunotherapy arise due to the necessity to hit every tumor cell with these genes to induce apoptosis, unless bystander effects were to occur (table 5). In patients with large tumor masses or multiple metastases this goal is hard to achieve. Therefore, it would be advantageous to perform systemic treatment with the apoptotic transgenes in vectors suitable to transduce as many cells as possible. Towards this goal it becomes important to develop efficient gene delivery and/or tumor-targeting systems. Apart from p53, the second proapoptotic gene which has entered clinical trials is adenovirus E1A. A theoretical disadvantage of E1A is the fact that it may behave as an oncogene. It has been shown that E1A proteins are able to immortalize primary cells and fibroblasts, and in combination with the viral E1B gene or the RAS protooncogene it can fully transform fibroblasts. Despite these facts, this gene has been approved for clinical trials (see above); up to now, no correlation of malignancy of human cells and E1A expression has been confirmed. Nevertheless, our laboratory focused on the development of safe E1A constructs and asked the question of whether it would be possible to eliminate transforming functions and still maintain tumor suppressor effects. For this purpose, we constructed a series of derivative E1A cDNAs derived from the major transcript of E1A, the socalled 13S cDNA [Dickopp et al., 2000]. The 13S mRNA is characterized by 3 conserved regions (CR), which are highly conserved among different adenoviral serotypes. In one of our mutants the complete CR2, which is known

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Fig. 1. E1A constructs used in our studies. From the top: 13S wild-type E1A cDNA, the CR13, respectively, are

shaded and marked; mutant lacking CR2 (E1A-delCR2); mutant expressing CR3 and exon 2 (E1A-CR3Ex2); mutant expressing exon 2 solely (E1A-Ex2).

Fig. 2. Growth of soft agar colonies of

H1299 lung cancer control cells and cells transduced with different E1A constructs. * Significant differences (p ! 0.05) [Dickopp, PhD thesis, Essen, 2000].

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20 18 16 Tumor volume ( 10 2 mm3) 14 12 10 8 E1AEx2110 6 4 2 0 2 0 1 2 3 4 5 6 7 8 9 10 11 12 Weeks after injection pRc-RSV H1299 E1AWT19 E1AdelCR239 E1ACR3Ex2123 E1ACR3Ex2316 E1AEx239

Fig. 3. Tumorigenicity of E1A-expressing and control H1299 cell lines in nude mice.

to be necessary for transforming functions and which is also known to bind RB and other related pocket proteins, was eliminated. Another construct consists exclusively of the CR3, a transactivation domain, and the exon 2, which binds a cellular protein, the so-called CtBP, that is involved in the tumor suppression by E1A. Another mutant expresses only the exon 2 of E1A (fig. 1). We have established E1A-expressing cell lines from the lung cancer H1299 cell line as well as from a malignant metastatic melanoma cell line, BLM, by stable transfection. In this paper, we show the results of the H1299 lung cancer cells [Dickopp et al., unpubl. data]; the results with the BLM cells were similar [Dickopp et al., 2000]. First, we tested the growth of these cell lines in soft agar medium. Parental cells and cells containing the empty vector formed colonies in soft agar, as did, to our surprise, cells expressing the 13S wild-type E1A construct. However, deletion mutants lacking the CR2 (E1A-delCR2) and those expressing only the CR3 (E1A-CR3Ex2) in conjunction with exon 2 showed significant reduction of colony formation in soft agar. In contrast, cells expressing exon 2 alone showed no suppression (fig. 2). Next, E1A-expressing cell lines and control cells were injected subcutaneously into nude mice. Tumors of un-

transduced cells grew continuously after 3 ! 106 cells were administered, and the cells expressing the empty vector or the exon 2 construct also formed similar tumors. However, cells expressing E1A-delCR2 or E1A-CR3Ex2 showed a significant delay in tumor formation, and, in one case, no tumors were found in nude mice (fig. 3). Thus, we conclude that it is possible to dissect the transforming or immortalizing functions of E1A from the tumor suppressor functions and that constructs, either lacking CR2 or consisting only of CR3 plus exon 2, are interesting transgene candidates for tumor gene therapy in the future.

Acknowledgments
We thank J. Sternberg for technical assistance, H. Esche for ongoing support, and the Wilhelm Sander Foundation for generous funding. Note: This brief article is for the most part the summary of a presentation held at the meeting International Conference on Gene Technology and Skin Gene Therapy, April 2000, Essen, Germany, and thus, does not give a detailed overview over the entire field or provides an indepth review. Moreover, for the sake of brevity, only selected articles are cited, for which the authors apologize.

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