Gene targeting approach to selectively kill colon cancer cells, with hyperactive K-Ras pathway

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Biomedicine & Pharmacotherapy 59 (2005) $370-$374 http://france.elsevier.com/directlBIOPHA/

Gene targeting approach to selectively kill colon cancer cells, with hyperactive K-Ras pathway H. Dvory-Sobol a,b, D. Kazanov a, N. Arber a,b,. a Department of Cancer Prevention, Integrated Cancer Prevention Center, Tel Aviv Medical Center, 6 Weizmann Street, Tel Aviv 64239, Israel b Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel

Abstract Background. - Ras mutations are present in approximately 50% of human colorectal tumors. We have previously shown that transfection of a non-tumorigenic rat intestinal epithelial cell line, IECI 8, by the K-Ras oncogene (RI cells), resulted in malignant cell transformation. Utilizing the constantly active Ras signaling pathway to selectively target transformed but not normal cells is a plausible goal. Aim. - To selectively kill Ras transformed cells by over expressing a lethal gene using a Ras-responsive promoter. Material a n d methods. - IEC18, R I and a number of colon cancer cell lines were transfected with luciferase (Luc) reporter gene under the control of different Ras-responsive elements. The Ras-responsive promoter Py2 contains two copies of adjacent Ets and AP I binding sites followed by a minimal promoter. Apoptotic genes (bax, caspase-8 and PKG) were cloned into the Py2 plasmids. R I cells co-transfected with expression constructs and a selected vector and then grown for 3 weeks under selection. Results. - R1, SW480 and HCT116 with mutated c-K-Ras expressed high level of Luc activity following transfection with the Py2 element. IECI8 cell lines that do not contain this mutation expressed negligible low Luc activity. Following transfection of SW480 and R1 cells with Py2-bax, caspase-8 and PKG, there was a significant reduction in the number of colony formation. Conclusions. - 1. Selective over-expression of pro-apoptotic genes, inhibits the growth of Ras transformed cells, and not normal cells. 2. This gene approach therapy may become a useful, effective and safe to target Ras mutated tumor cells with sparing of the normal cells. © 2005 Elsevier SAS. All rights reserved. Keywords: Colon cancer; K-Ras; Responsive element

1. Introduction Colorectal cancer (CRC) is a multi-step process that develops through progressive accumulation of genetic and epigenetic changes that lead to the conversion of normal colonic epithelium to dysplasia, followed by adenomas and adenocarcinomas [ 11,15]. The R a s proteins are encoded by a closely related set of genes, initially identified in acutely transforming sarcoma viruses and subsequently in human tumor cell lines [11,8,32]. There are three mammalian R a s isoforms; K - R a s (which is the most common mutation in human cancer) H a - R a s and N - R a s , as well as two R a s related proteins ( R - R a s and TC21). R a s mutations appear early and persistently in the multistage process of

* Corresponding author. E-mail addresses: [email protected], [email protected] (N. Arber). © 2005 Elsevier SAS. All rights reserved.

tumorigenesis [11]. Mutations in R a s can activate genes causing them to become oncogenic [21,10]. K - R a s mutations occur early during the transformation process in 40-50% of colon carcinomas [7,4]. These mutations, which invariably occur at codons 12, 13, or 61, prevent efficient GTP hydrolysis and thus render the proteins in an activated state. As a result, multiple Ras effector pathways which control fundamental biological processes such as proliferation, apoptosis, and cell motility become constitutively activated and/or deregulated. Increased proliferation may result from up-regulation of cyclin D 1 expression, and decreased apoptosis due to downregulation of Bak levels in R a s - t r a n s f o r m e d cells [20]. Oncogenic Ras activates the transcription of a variety of cellular genes through promoter elements consisting of binding sites for transcription factors from several distinct families. The first promoter oncogene responsive element (ORE) to be characterized was from the polyoma virus (Py) enhancer, and it consists of overlapping binding sites for the

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Ets and AP-1 transcription factors [23]. Analysis of the Py ORE, showed that both the Ets and AP-1 binding sites are essential for the transcriptional activation by non-nuclear oncogenes [30,16,23]. Similar elements have since been found in the promoters of many genes, and where it has been tested, Ets can cooperate with AP-1 family members and Ras to activate their transcription. The products of these Ras-induced genes have a wide spectrum of functions, including roles in cellular metabolism, growth control, and metastasis [6,3,9]. A variety of other types of promoter elements distinct from the adjacent Ets and AP-1 binding sites, were reported to mediate transactivation by oncogenic Ras. Multiple tandem binding sites for members of the AP1 [25], NF-~:B [12] or SP-1 [26] families of transcription factors were found to be sufficient to function as OREs, and tandem Ets binding sites were found to be necessary for maximal Ras-responsiveness [31]. We had previously shown that transfection of nontumorigenic rat intestinal epithelial cell line, IEC18, by the K-Ras oncogene resulted in malignant cell transformation (R1 cells). The R1 cells proliferate faster, form colonies in soft agar, have higher saturation density and plating efficiency, and are tumorigenic when injected into nude mice [5]. Herein, a novel strategy to exploit and use the differential transcription potential of Ras to selectively target and kill tumor cells is described. The introduction of cell death genes under the control of the promoter containing wildtype Ets and AP-1 binding sites resulted in preferential killing of cells with hyperactive Ras pathway. Three proapoptotic genes were evaluated; bax, caspase-8 and mutant PKG (PKG 113) which elicit cell death through induction of different apoptotic pathways. Bax, a member of the Bcl-2 family protein, that resides in the outer mitochondrial membrane. Bax has been shown to accelerate apoptosis by directly inducing the release of Cytochrome c from the mitochondria [17]. Caspase-8, a member of the cystein protease family, is activated at the initial step in the Fas-induced apoptosis cascade [22]. Activated caspase-8 directly activates other caspases, including executioner caspase (for example, caspase-3) [28]. PKG activates JNK1 through a novel PKG-MEKK1-SEK1-JNK1 pathway, leading to induction of apoptosis. Mutant PKG I[3 has a deletion of the N-terminal 93 amino acids [27]. This deletion renders PKG independently of cGMP, and therefore, it is constitutively active.

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95% oxygen and 5% CO 2. The medium for R1 cells was supplemented with 200 ~tg/ml G418. Human colon cancer cells, SW480, DLD-1, HCT116 and HT29 were purchased from American Type CultureCollection (Manassas, VA, USA) and grown in DMEM supplemented with 10% FCS, 1% penicillin and 1% streptomycin. 2.2. Construction o f plasmids

The AP1-A56Fos-Luc, Ets-A56Fos-Luc, A56Fos-Luc and Py2-A56Fos-Luc were generous gifts from Dr. D. Engelberg (The Hebrew University, Jerusalem, Israel). These reporter plasmids are identical to the minimal fos promoter-containing CAT reporter that were previously described [13]. The coding sequence of CAT was replaced by that of luciferase [33]. The Py2 contains two adjacent Ets and AP-1 binding sites from the polyoma enhancer [23]. The A56Fos-Luc and Py2-A56Fos-Luc were used as templates for the PCR fragments containing the cFos basal promoter alone or plus wild-type Py2 binding sites. These PCR fragments were then cloned into the NheIBglII sites in the pGL3-basic vector (Promega, USA). The resultant plasmids were designated as pGL3-Fos-Luc pGL3-Py2Fos-Luc. To construct the Py2-SV40-Luc plasmid, the BglII-NheI Py2 fragment (Sigma, Israel), was cloned into NheI and BglII sites upstream to SV40 minimal promoter in the pGL3-promoter plasmid (Promega). In order to construct the Py2-SV40-bax, Py2-SV40-caspase-8, Py2-SV40-PKG and their control plasmids (pSV40-bax, pSV40-caspase-8, pSV40-PKG) a polylinker sequence, HindIII-XbaI, which contains the HindIII, EcoRV, EcoRI, PstI, SnabI and XbaI sites was used to replace the HindIII-XbaI Luc fragments in the pGL3-promoter and Py2-SV40-Luc vectors. The resultant plasmids were designated as pGL3-promoter-HX, Py2-SV40-HX (HX-plasmids). A blunted EcoRV fragment from pCDNA3-caspase-8 plasmid (a gift from Dr. Etan Gross, Weizmann Institute of Science, Rehovot, Israel) cloned into the EcoRV site in the pGL3-promoter-HX and Py2-SV40-HX plasmids. To create the Py2-SV40-PKG and the control plasmid pSV40-PKG, the blunted XhoI fragment from pRc/CMV-A93GK plasmid (A gift from Dr. I. Bernard Weinstein, Columbia University, NY), which encodes a mutant PKG I[3 sequence with an N-terminal truncation, was also cloned into the EcoRV site in the HXplasmids. The NcoI-XbaI Bax sequence was amplified by PCR and was used to replace the NcoI-XbaI Luc fragment in the pGL3-promoter and Py2-SV40=Luc vectors.

2. Material and methods 2.3. Transfection and Luc assay 2.1. Cell culture

IEC 18 cells were grown in Dulbecco's minimal essential medium (DMEM) (Biological Industries, Kibbutz Beit Haemek, Israel), containing 10% fetal calf serum (FCS), 1% penicillin and 1% streptomycin at 37°C, in an atmosphere of

Transfections were performed using LipofectAMINE (Roche Molecular Biochemicals, Indianapolis, IN) according to the manufacturer's instructions. 5 x 105 cells were seeded in 6-well plates for Luc assays. The next day, when the cells were about 50% confluent, they were co-

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transfection with l~tg of either vector plus 100 ng of pRLT K or 30 ng p R L - C M V (Promega) was performed. Luc assay was performed 24 h post transfection. Briefly, cells were washed once with PBS and then lyzed in 400 ~tl of lysis buffer for 15 min at room temperature. The lysates were centrifuged at 14,000 rpm for 5 min, and 20 lal of each lysate were used to measure Luc reporter gene expression. The Luc activity was normalized to Renilla Luc activity from a parallel co-transfection of p R L - T K or p R L - C M V (Dual Luc system, Promega). All experiments were performed in triplicate at least twice and gave similar results. 2.4. Colony formation assays IEC18 and R1 cell lines were co-transfected with p M V 12 and one of the expressions constructs as indicated. Cells were harvested 24 h after transfection, and equal cell numbers were serially diluted in 10 cm dishes and grown under selection in hygromycin B for 3 weeks then fixed and stained with Comassie blue (0.2% Comassie blue, 50% methanol, 10% acetic acid, 40% H20).

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Fig. 2. Ras-activated promoters containing Py2 sequence. IEC18 and R I cells were transfected with two sets of ras-responsive promoters Py2SV40 and Py2Fos. Transfectionexperiments were normalizedby the Dual Luciferase system, pRL-CMV was used for normalization. Luciferase activities were measured 24 h after transfection. Py2 activity was significantly higher in R1 than in IEC18 cells. Py2-SV40 construct exhibited several folds higher activity than Py2-A56Fos. The apoptotic genes were cloned downstream to the SV40 minimal promoter (Fig. 2). 3.2. Elevated Ras dependent transactivation in colon cancer cell lines

3. Results

3.1. Ras-mediated promoter activities In order to create the most sensitive response elements, the activities of three OREs were tested: Ets, AP1 and their combination (Py2). These constructs were connected to a minimal A56Fos promoter upstream to the Luc gene. Fig. 1 demonstrates Luc activity in RI cells using the different constructs. Transfection with Py2-A56Fos-Luc activity was six and threefold higher as c o m p a r e d to the activity of A P I - A 5 6 F o s - L u c and Ets-A56Fos-Luc constructs, respectively. Hence, Py2 element was chosen in the rest of the studies. Next, the activities of two different sets of Rasresponsive promoters were tested in IEC18 and R1 cells.

A number of human colorectal cancer cell lines (SW480, HCT116, D L D - ! and HT29), harboring mutations at codon 12 or 13 of the c-K-Ras gene were further tested. As shown in (Fig. 3) upon transfection o f Py2-SV40-Luc, all the four cell lines expressed high levels of Luc activity. In contrast, transfection of pGL3-promoter yielded only basal level of Luc activity, indicating that the elevated activity of Py2SV40-1uc in these cell lines is Ras dependent. 3.3. Induction o f cell death in R1 cells Cotransfection of Py2-SV40-bax, Py2-SV40-caspase-8, Py2-SV40-PKG into R1 cells resulted in an impressive cell growth inhibition, manifested by reduced colony formation (Fig. 4). 70

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Fig. I. Ras-mediated promoter activities. A56Fos-Luc or Py2-A56Fos-Luc or AP1-A56Fos-Lucor Ets-A56Fos-Lucwas transfected into IEC18 and R1 ceils. Transfectionexperiments were normalizedby the Dual Luciferase system (Promega). Thirty nanograms of control plasmid pRL-CMV (Promega) was used for normalization. Luciferase activities were measured 24 h after transfection. The results are the mean values from three different experiments performed in triplicate.

SW480

DLD-1

HCT116

HT29

Fig. 3. Ras-responsive promoter mediated Luc reporter activity in colon cancer cell lines. Py2-SV40-Luc or pGL3-promoter was transfected into colorectal cancer cell lines SW480, HCT116, DLD-1 HT29. Transfection experiments were normalized by the Dual Luciferase system. Hundred nanograms of control plasmid pRL-TK (Promega) was used for normalization. Luciferase activities were measured 24 h after transfection.The results are the mean values from three different experiments performedin triplicate.

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Py2-SV40-PKG

pSV40-PKG

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tumor cells only. Indeed, when Bax, caspase-8 or P K G are under the control of a defined promoter containing the Py2responsive element, a selective cell death was induced by pSV40-PKG, Py2-SV40-bax, Py2-SV40-caspase-8 in cancer cells harboring mutant c - K - R a s . To further develop this gene transfer strategy, an adenoviral vectors system should be developed. The adenovirus has a highly effective nuclear entry mechanism and a very low pathogenicity in humans [29]. The adenoviral vectors can be produced in high titers, they can transduce cells in vivo and they do not integrate into the host cell genome. The current studies provide the proof of concept that has to be confirmed in animal models. It does provide a new approach to use the mutated pathway rather than to inhibit it. This approach may have a very promising potential in the treatment of cancer, depending on its molecular profile.

Acknowledgments This work was part of the requirements of Hadas DvorySobol for her PhD degree at the Sackler School of Medicine at Tel Aviv University, Israel. We wish to thank Ludmila Strier for her technical assistance and Talya Kunink for her assistance with the plasmids construction. This study was supported by the Israel Cancer Association.

References [1] Fig. 4. Bax, caspase-8 and PKG suppresses the growth of colon cancercells. RI cell line were transfectedwith constructsencodingBax, caspase-8,PKG, as well as Py2-Bax,Py2-caspase-8and Py2-PKG.Cells were harvested 24 h after transfection,and equal cell numbers were diluted in duplicates (1:10 and 1:25) in 10 cm dishes and grown under selection in hygromycinB for 3 weeks, then fixed and stained with Comassie blue. Colony formationin representativedishes (1:10 dilution)of transfected R 1 cells is shown.

4. Discussion and conclusion R a s is the most important oncogene being mutated in about 30% of all tumors [1]. Herein, we showed a novel approach that allows to selectively express lethal genes by targeting an active Ras signaling pathway in transformed cells, but not in normal cells. A proof of concept is shown, that mutant K - R a s pathways may be used as a promising target in gene therapy of cancer, and colon cancer in particular. So far, attempts were mostly done to inhibit and suppress Ras activity in cancer. Suppression of H - R a s expression by antisense oligonucleotide, antisense RNA, or ribozyme led to an inhibition of the neoplastic phenotype of bladder carcinoma cells and N1H3T3 cells transformed by H - R a s oncogene [18,19,24]. The anti-sense K - R a s retroviral [14] and adenoviral [2] vector infections were useful in suppressing tumorigenicity in lung cancer models in nude mice. The current approach uses a completely opposite approach by targeting the existence active Ras signaling in

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