Mutant Bik expression mediated by the enhanced minimal topoisomerase IIα promoter selectively suppressed breast tumors in an animal model

Abstract

To ensure the success of systemic gene therapy, it is critical to enhance the tumor specificity and activity of the promoter. In the current study, we determined that topoisomerase IIα promoter is selectively activated in breast cancer cells. An element containing an inverted CCAAT box (ICB) was shown to be responsible for the breast cancer specificity. When the ICB-harboring topoisomerase IIα minimal promoter was linked with an enhancer sequence from the cytomegalovirus immediate early gene promoter (CMV promoter), this composite promoter, CT90, exhibited activity comparable to or higher than the CMV promoter in breast cancer cells in vitro and in vivo, yet expresses much lower activity in normal cell lines and normal organs than the CMV promoter. A CT90-driven construct expressing BikDD, a potent proapoptotic gene, was shown to selectively kill breast cancer cells in vitro, and to suppress mammary tumor development in an animal model of intravenously administrated, liposome-delivered gene therapy. Expression of BikDD was readily detectable in the tumors but not in the normal organs (such as heart) of CT90-BikDD-treated animals. The results indicate that liposomal CT90-BikDD is an effective systemic breast cancer-targeting gene therapy.

Introduction

Breast cancer is the most common cancer and the second leading cause of cancer-related death for women in the United States.1 The 5-year survival rates for patients with local or regional diseases are about 80% or higher. For patients with metastasis, however, the survival rate drops to about 20%. Many patients experience disease recurrence even after adjuvant therapy such as hormone therapy, radiotherapy or chemotherapy. Therefore, the development of new therapies is a very important clinical issue in breast cancer. As micrometastasis occurs frequently in breast cancer, newly developed therapies must be delivered systemically.

Almost all types of cancers exhibit genomic instability,2, 3, 4 resulting in mutation, loss or gain of genes. Gene therapy suppresses tumors by transferring a gene into cancer cells to correct their genetic defects, block their abnormal signaling or induce their death. In recent years, advances in DNA delivery technology, especially cationic lipids, have made systemic delivery feasible for gene therapy. Cationic liposome is easy to produce and is relatively nontoxic. Its low immunogenicity allows its repeated administration to patients, increasing overall efficiency of gene delivery and therapeutic efficacy. Multiple preclinical studies and clinical trials have demonstrated the therapeutic efficacy and minimal side effects of liposomal gene therapy for cancers.5, 6, 7, 8

The efficacy of gene therapy depends on the promoter activity. As breast cancer frequently develops metastasis, the new therapy should be designed for systemic administration. Several studies have pointed out that, to ensure efficacy for systemic gene therapy, promoter activity in the targeted cells should be comparable to that of the promoter of cytomegalovirus immediate early gene (CMV promoter).10, 11, 12 In fact, the CMV promoter is the most widely used promoter in gene therapy settings,10, 13 especially those in clinical trials (according to The NIH/FDA Genetic Modification Clinical Research Information System (GeMCRIS; www4.od.nih.gov/oba/rac/GeMCRIS/GeMCRIS.htm) and The Journal of Gene Medicine Clinical Trial site (www.wiley.co.uk/genetherapy/clinical/)). However, the CMV promoter has ubiquitous activity; it drives high level of gene expression in both cancer and normal cells. When cationic liposome is used to deliver CMV promoter-driven reporter gene systemically, significant uptake of the DNA construct occurs in lung and heart, resulting in a high level of reporter activity in these organs.14, 15, 16, 17 Undesired gene expression in normal organs may cause side effects in cancer gene therapy.10, 11 Strategies to reduce such risk for liposomal gene therapy include the adoption of a promoter that induces a higher level of gene expression in tumor cells and a lower level in normal cells, thus decreasing undesired gene expression secondary to systemic treatment. For systemic treatment purposes, therefore, the promoter in gene therapy should possess (1) activity higher than or comparable to that of the CMV promoter in tumor cells and (2) a higher cancer specificity index (ratio of promoter activity in tumor to that in normal tissue (tumor:normal))18 than the CMV promoter. Several promoters have been used to drive breast cancer-targeting gene therapy in cell lines or animal models,13 such as the human alpha-lactalbumin promoter, ovine beta-lactoglobulin promoter,19 type II hexokinase promoter,20 HER2/neu promoter9 and hTERT promoter.21 However, the activities of these promoters are in general weaker than the CMV promoter. Identification of a promoter with these two properties improves the safety of systemic gene therapy protocols while achieving sufficient therapeutic efficacy.

In the current study, we identified a promoter with these two properties, and used it to develop a liposome-delivered breast cancer-targeting gene therapy for systemic administration. To accelerate the clinical use of this system, we adopted materials that have already been tested in clinical trials, including backbone DNA vector pUK215, 22 and extruded DOTAP:Chol liposome (Human Gene Transfer Protocol 0201-513, NIH Genetic Modification Clinical Research Information System).14, 15 As a first step, we determined that the topoisomerase IIα (topoIIα) promoter is specifically activated in breast cancer cells, and that its cis-element, the inverted CCAAT box (ICB),23, 24, 25 mediates this specific activity. To enhance its basal activity level, we linked the minimal topoIIα promoter harboring ICB1 with the enhancer sequence from the CMV promoter. We demonstrated that the activity of this composite promoter, CT90, is comparable to that of the CMV promoter in breast cancer cells, but much lower in normal cells in vitro and in vivo. The CT90 promoter was then used to drive a potent proapoptotic gene, BikDD,26 in the pUK21 vector (CT90-BikDD). We further showed that the intravenously injected DOTAP:Chol liposome–CT90-BikDD complex effectively suppressed tumor growth and extended animal lifespan in orthotopic breast cancer mouse models. These results demonstrated the tumor-targeting and therapeutic efficacy of the DOTAP:Chol–CT90-BikDD complex when administered systemically, which has important clinical implications. Moreover, as most of its materials have been tested in clinical trials, this cancer gene therapy regimen has great potential for transfer to clinical use.

Materials and methods

Cell lines

Human cancer cell lines (breast cancer MCF-7, MDA-MB-231, MDA-MB-468, MDA-MB-453, SK-BR-3 and T47D; ovarian cancer SKOV-3.ip-1; non-small-cell lung cancer A549; pancreatic cancer Capan-1) and normal human cell lines (breast epithelial cell line 184A1, normal fibroblast line WI-38 and nonmalignant liver cell line Chang liver) were all purchased from American Type Culture Collection (Manassas, VA), and cultured according to the vendor's instructions.

Liposome and DNA constructs

Extruded DOTAP:Chol liposome (20 mM) from the nonviral core facility at Baylor Collage of Medicine was used. It was prepared following the protocol described previously.15 The defined full-length −572/+90 sequence of the topoIIα promoter was amplified via PCR from genomic DNA of HeLa cells by using primers T2A-F (5′-IndexTermGGGGCGGGGTTGAGGCAGATG-3′) and T2A-R (5′-IndexTermCCATGGTGACGGTCGTGAAGGGGC-3′), and then ligated into the pCR2.1-TOPO TA cloning vector (Invitrogen, Carlsbad, CA). The −572/+90 sequence was released by digesting the TA vector at HindIII/XhoI and ligated into pGL3-basic (Promega, Madison, WI) at the corresponding sites to construct the topoIIα promoter-driven luciferase reporter topoIIα-pGL3. The deletion mutants of the topoIIα promoter were amplified by PCR from topoIIα-pGL3 and subcloned into the pGL3-basic vector to generate T2A354-pGL3, T2A182-pGL3, T2A142-pGL3, T2A90-pGL3 and T2A60-pGL3. To generate the ICB1-mutated topoIIα promoter, two fragments were PCR amplified by using two sets of primers: T2A-F and 5′-IndexTermCAGACCAGCCTTTCCCTGAC-3′, as well as 5′-IndexTermGTCAGGGAAAGGCTGGTCTG-3′ and T2A-R. The full-length mutant fragment, mICB1-T2A, was obtained in secondary PCR by using the mixture of the primary PCR products as template, and T2A-F and T2A-R as primers. The sequence of 214–645 bp in the pCDNA3.1 vector (Invitrogen), which contained the CMV enhancer sequence (−604/−173 in the CMV promoter),27 was PCR amplified with primers 5′-IndexTermTTGCTAGCTACGGGCCAGATATACGCGTT-3′ and 5′-IndexTermAACTCGAGTGAGTCAAACCGCTATCCACG-3′. The PCR product was digested with NheI/XhoI and subcloned at corresponding sites into topoIIα-pGL3, T2A182-pGL3 and T2A90-pGL3 to obtain CT572-pGL3, CT182-pGL3 and CT90-pGL3, respectively. The BikDD sequence was PCR amplified from the CMV-BikDD expression vector26 and subcloned into a HindIII/XbaI-digested CT90-pGL3 vector to obtain CT90-BikDD. For antitumor activity assay, CT90-BikDD and CMV-BikDD were PCR amplified and subcloned into pUK21 vector (GenBank accession no. AF223640). Expression vectors of cdk2 and dominant-negative cdk2 mutant (cdk2-dn) were kindly provided by Dr Sander van den Heuvel (Massachusetts General Hospital, Boston, MA). Cyclin A (CCNA) expression vector was a kind gift from Dr Brenda Schulman (St Jude Children Research Hospital, Memphis, TN). Cyclin E expression vector was a generous gift from Dr Khandan Keyomarsi of our institute.

In vitro promoter activity assay

The promoter activity in the cell lines was determined by transient transfection and reporter assay. Cell lines were cultured in six-well tissue clusters to 80% confluency. Tested plasmid (1.5 μg) and 0.075 μg of pRL-TK (Promega), which was used to normalize transfection efficiency, were co-transfected into cells by the liposome method described previously.6 Twenty-four hours after transfection, cells were harvested and cell lysates were prepared. The reporter activity in the cell lysates was determined by using the dual luciferase reporter assay system (Promega) according to the manufacturer's instructions. Three independent experiments were performed for each data point reported.

In vitro luciferase viability assay

The cell-killing effect of the gene therapy construct was determined by in vitro luciferase viability as described previously.26 Briefly, cell lines in six-well plates were co-transfected with gene therapy constructs (CT90-BikDD or CMV-BikDD) in different doses (0, 1.0 or 3.0 μg), and with CMV-luc (50 μg), the CMV promoter-driven luciferase reporter construct). At each dose, the pCDNA3.1 vector was replenished to keep the total amount of transfected DNA equal (3.05 μg). Twenty-four hours after transfection, cells were harvested and cell lysates were prepared. Luciferase activities were measured by using the single luciferase assay system (Promega) according to the manufacturer's instructions. The proportions of surviving cells were calculated by dividing the reporter reading from the experiment group (therapeutic construct 1.0 or 3.0 μg) by that from control group (no therapeutic construct) and calculating the percentage. Values are means±s.d. of three independent experiments.

Tissue distribution of the liposome-delivered reporter genes

The human breast cancer cells (MDA-MB-231 or MDA-MB-468) were trypsinized, harvested, and 106 cells were inoculated into the mammary fat pads of nude mice (nu/nu, 7–8 weeks old, 18–22 g; Charles River Laboratories, Wilmington, MA) to induce tumors as described previously.18 Five weeks later, the tumor-bearing mice were divided into two groups (five mice per group). CT90-pGL3 or CMV-pGL3 plasmid (50 μg) was mixed with DOTAP:Chol as described elsewhere15 for each mouse. The DNA–liposome complex was injected into each mouse through the tail vein. Forty-eight hours after injection, tumors and normal organs, including heart, lung, liver, spleen, kidney and muscle, were resected and immediately frozen on dry ice. The collected tissues were homogenized with 1 × passive lysis buffer (Promega) and subjected to centrifugation, and the supernatants were collected for measurement of luciferase activity and protein concentration. Protein concentrations were determined by using the Bio-Rad protein assay reagent (Bio-Rad, Fremont, CA). The luciferase activity per microgram of protein was used to compare gene expression in different tissues. The background levels of luciferase activity in tumors and organs were measured in tumor-bearing mice intravenously injected with the empty pGL3–liposome complex.

In vivo antitumor activity

The antitumor activity assay was performed following the principles of a previous study.18 The human breast cancer cells were inoculated into nude mice to induce tumors, as described in the above section. Mice were divided into two groups to receive injections of 15 μg of DNA–liposome complex once per week. In each group, mice were further divided into four subgroups of eight mice, and each subgroup received a different treatment (CT90-BikDD, CMV-BikDD, PGL3 vector or 5% dextrose). Tumor volume was measured on days 1 and 4 in each week. The tumor growth ratio, as described previously,18 was determined by comparing the size of the treated tumors at various times with the size of the original tumor before any treatment. The tumor volume (V) was calculated using the formula V=SSL/2, where S is the short length of the tumor in mm and L is the long length of the tumor in mm. The tumor volume ratio (R) was calculated as R=VN/V0, where VN is tumor volume at day N and V0 is tumor volume at day 0. The tumor volume ratio was introduced to normalize the individual difference of tested animals as described previously.18 The mice received a total of eight injections in the QW group and 12 injections in the BIW group. The tumor volume and survival data were analyzed with two-sided log-rank statistical tests and Kaplan–Meier analysis with log-rank test, respectively, using statistical analysis software SPSS (SPSS Inc., Chicago, IL).

In situ hybridization

Mice were prepared and inoculated as described previously. After the tumors grew to an average of 500 mm3, CT90-BikDD or CMV-BikDD was mixed with DOTAP:Chol and injected intravenously at a dosage of 50 μg of DNA per mouse in each group. Forty-eight hours after injection, the mice were killed. Tumors and several organs were resected, fixed in 10% buffered formalin for 48 h, and then processed and embedded in paraffin. The BikDD coding region sequence was obtained by digesting the CMV-BikDD vector at Hind III/Xba I and subcloned into the corresponding sites in the pSPT18 vector (Roche Diagnostics, Indianapolis, IN). The antisense and sense BikDD hybridization probes were synthesized by in vitro transcription using SP6 and T7 polymerase, respectively, in the presence of digoxygenin-UTP from the DIG RNA labeling kit (Roche Diagnostics, Indianapolis, IN) following the manufacturer's instructions. Hybridization was performed as described.28, 29 Signals were detected using the DIG nucleic acid detection kit (Roche Diagnostics, Indianapolis, IN), following the instruction manual.

Results

TopoIIα promoter activity is specifically activated in breast cancer cells

As the first step in identifying a promoter with higher specificity for breast cancer cells, we searched for genes known to be overexpressed in breast tumor tissues. Although mechanisms of overexpression include gene amplification, transcriptional and translational upregulation, or increased stability of mRNA and protein, we reasoned that, for certain genes, overexpression might result from transcription upregulation, and the activities of their promoters could be much stronger in breast cancer cells than in other types of cells. With this notion in mind, we sought genes overexpressed in breast cancer cells from published data of gene expression profile30, 31, 32, 33 and serial analysis of gene expression (SAGE).34, 35 We selected those overexpressed genes commonly shown in these studies and identified four candidate breast cancer-specific genes: topoisomerase IIα (topoIIα), B-myb, ceruloplasmin and transferrin receptor. We obtained or cloned their promoters and examined their activities in normal and breast cancer cell lines with a reporter assay (Table 1). As an initial screening, we selected three breast cancer cell lines (MDA-MB-468, MDA-MB-453 and MDA-MB-231), a normal lung fibroblast cell line (WI-38) and a nonmalignant hepatocyte (Chang liver).36 We selected the lung fibroblast cell line as a representative normal cell line for comparison of promoter activity in this quick screening because lung is the major nonspecific targeted organ when a gene is delivered intravenously by cationic liposome.14, 15, 16, 17 On average, the topoIIα promoter possessed the highest level of activity in breast cancer cells and the lowest level of activity in normal or nonmalignant cell lines, suggesting that it has the highest degree of breast cancer specificity. The other promoters had either a lower level of activity in breast cancer cells (ceruloplasmin and transferrin receptor) or a higher level of activity in normal cells (B-myb). Therefore, we used the topoIIα promoter to develop a breast cancer-targeting gene therapy setting in the following experiments.

Table 1 Specificity of the candidate breast cancer-specific promotersa

To test its specificity for breast cancer cells, we then examined the activity of a defined full-length (−572 to +90) topoIIα promoter25 in an expanded panel of cell lines by reporter assay. The results confirmed that, on average, topoIIα promoter possessed a higher level of activity in breast cancer cells and a lower level in other cell types (Figure 1a), including immortalized normal cell lines (184A1, WI38 and Chang liver) and other cancer cells (CAPAN-1, SKOV3-ip1 and A549), fulfilling one property of the promoter for systemic gene therapy. We then measured the ratio of topoIIα promoter activity to CMV promoter activity (Figure 1b, topoIIα/CMV). We observed that topoIIα/CMV ratios in most breast cancer cell lines are higher than those in other types of cell lines. It is interesting to note that the topoIIα/CMV ratio is also relatively high in SKOV-ip1 cell line, which is an ovarian cancer cell line harboring overexpression of HER2. The activity of topoIIα promoter in breast cancer cells was much weaker than that of the CMV promoter (<2.5%), however, and thus was expected to be insufficient for therapeutic efficacy, especially in the systemic treatment.

Figure 1
figure1

TopoIIα promoter is specifically activated in breast cancer cells. (a) TopoIIα promoter activity in various cell lines. The topoIIα-pGL3 vector (see Materials and methods) was transfected into the various cell lines, and then the promoter activity was determined by the dual luciferase assay and is given in arbitrary units. The cell lines included normal breast epithelial cells (184A1), normal lung fibroblast cells (WI-38), nonmalignant hepatocytes (Chang liver), pancreatic cancer cells (CAPAN-1), ovarian cancer cells (SKOV3-ip1, referred as ip-1) and breast cancer cells (BC): MDA-MB-231 (231), MDA-MB-468 (468), MDA-MB-453 (453), MCF7, SKBR-3 and T47D. (b) TopoIIα promoter activity relative to that of the cytomegalovirus (CMV) promoter in the various cell lines. The activities of topoIIα and CMV promoters were determined as described in (a). Y axis represents the ratio between promoter activity of topoIIα and that of CMV defining as 100% (topoIIα/CMV) in each cell line.

ICB1 can mediate activation of topoIIα promoter by Cdk2/cyclin A specifically in breast cancer cells

One straightforward approach to enhance the basal activity level of topoIIα promoter in breast cancer cells is to link a strong enhancer to it. Owing to possible interference from some of the regulatory elements in the topoIIα promoter, however, addition of a strong enhancer might not achieve a high level of basal activity, and could even negate breast cancer specificity (see Figure 4). To obtain a promoter possessing both specificity and a high level of basal activity in breast cancer cells, we reasoned that a minimal breast cancer-specific element linking to an enhancer would have a better chance of success. Thus, we set out to identify a breast cancer-specific element in the topoIIα promoter. To this end, we first explored the possible breast cancer-specific signal to activate topoIIα promoter, then looked for the cis-element that mediated this signaling. TopoIIα expression is upregulated during S phase and peaks during G2/M.37 As the essential cell-cycle regulator Cdk2 is activated during S and G2 phases,38 we asked whether topoIIα promoter activity might be upregulated by Cdk2. We examined the effect of Cdk2 on topoIIα promoter activity by co-transfecting a dominant-negative cdk2 mutant (cdk2-dn)39 and topoIIα promoter reporter construct in a reporter assay. Cdk2-dn suppressed topoIIα promoter activity in three different breast cancer cells but not in normal breast epithelial cells (184A1), lung cancer cells (A549) or nonmalignant hepatocytes (Chang liver) (Figure 2a). As a control, co-transfection of dominant-negative E2F1 mutant had no obvious effect on the topoIIα promoter activity (data not shown). As Cdk2 associates with cyclin E and cyclin A in late G1 and S phases, respectively, we investigated which of these cyclins could activate topoIIα promoter. Expression of cyclin A significantly induced activation of the topoIIα promoter in breast cancer cells, but not in other cell types (Figure 2b). On the other hand, cyclin E had minimal effect on the topoIIα promoter in breast cancer cells (data not shown). These data suggest that cyclin A/Cdk2 activates the topoIIα promoter in a breast cancer-specific manner.

Figure 4
figure4

Enhancer sequence of cytomegalovirus (CMV) promoter potentiates −90 topoIIα promoter activity specifically in breast cancer cells. (a) Design of CT572, CT182 and CT90. The CMV promoter enhancer sequence from the pCDNA3.1 vector was PCR amplified and cloned upstream to the topoIIα, −182 or −90 promoters in pGL3 reporter construct, forming the fusion promoters CT572, CT182 and CT90, respectively. (b) Activity of CT572, CT182 and CT90 in cell lines. The Y axis represents log scale. The promoter activity in each cell line was determined as described for Figure 1a. (c) Activity of CT572, CT182 and CT90 relative to the CMV promoter in cell lines. The promoter activity ratios of CT572 to CMV (CT572/CMV), CT182 to CMV (CT182/CMV) and CT90 to CMV (CT90/CMV) in each cell line were determined as described in Figure 1b. (d) The biodistribution of reporter activities from the CMV and CT90 promoters in the orthotopic MDA-MB-231 breast cancer xenograft mouse model. The Y axis represents log scale. The liposome-complexed luciferase reporter constructs controlled by CT90 (CT90-luc) or CMV promoter (CMV-luc) were injected into tail veins of tumor-bearing mice. Mice were killed 48 h after treatment, and major organs were removed for luciferase assays. Left panel: Reporter activities from the CMV and CT90 promoters in tumor and normal organs. The luciferase activity per micrgram of protein is shown. Right panel: The tumor:tissue ratio of the luciferase activity of CMV-luc and CT90-luc (tumor:normal; T, tumor; H, heart; LU, lung; LI, liver; S, spleen; K, kidney; M, muscle). (e) Reporter activities in MDA-MB-231 xenograft-bearing mice treated with intratumoral injection of CT90-luc and CMV-luc. The CT90-luc or CMV-luc construct complexed with DOTAP:Chol liposome was injected into MDA-MB-231 tumors on the mice. Mice were killed 48 h after injection, and tumor and major organs were removed for luciferase assay. Left panel: Reporter activities from the CMV and CT90 promoters in tumor and normal organs. The luciferase activity per microgram of protein is shown. The CT90 and CMV promoter activities in MDA-MB-231 tumors are significantly different in t-test (P<0.05). Right panel: The tumor:tissue ratio of the luciferase activity of CMV-luc and CT90-luc (tumor:normal; T, tumor; H, heart; LU, lung).

Figure 2
figure2

Inverted CCAAT boxes mediate breast cancer-specific signaling. (a) Dominant-negative cdk2 mutant (cdk2-dn) blocked topoIIα promoter activity in breast cancer cells. The control vector or cdk2-dn expression vector was co-transfected into cells with the topoIIα-pGL3 reporter vector, and then luciferase assay was performed to determine topoIIα promoter activity. The ratio of topoIIα promoter activity in the cdk2-dn group to the control group defined as 1 is shown as ‘suppression ratio by cdk2-dn’ in the Y axis. The cell lines used are described in Figure 1 (BC, breast cancer cells). (b) Cyclin A activates the topoIIα promoter in breast cancer cells. The control or cyclin A expression vector was cotransfected into SKBR3 cells with the topoIIα-pGL3 reporter vector, and then the luciferase assay was performed to determine topoIIα promoter activity. The ratio of topoIIα promoter activity in the cdk2-dn group to that in the control group is shown. (c) The design of topoIIα promoter deletion mutants (−572, −182, −90, −60) and ICB1-mutated topoIIα promoter (mICB1). Circled x, mutation at the ICB1 site. (d) ICB1 can mediate activation of the topoIIα promoter by cyclin A/Cdk2 signal in breast cancer cells. Left panel: The topoIIα promoter deletion mutants were co-transfected with the cyclin A expression vector or the control vector into the various cell lines, and their activity was determined by luciferase assay. The ratio of promoter activity in the cyclin A group to that in the control group (CCNA/Ctrl) is shown. Right panel: ICB1 site of the topoIIα promoter was mutated as described in Materials and methods. The ICB1-mutated promoter (mICB1-T2A) was co-transfected with the cyclin A expression vector or the control vector into the various cell lines, and the fold of activation was determined as described above.

We concluded from these results that the cyclin A/Cdk2-responding element in the topoIIα promoter might be the potential breast cancer-specific element. To identify this element, we generated a series of deletion mutants of the topoIIα promoter, including −182, −90 and −60, which contain three, one and no ICB(s), respectively (Figure 2c).23, 24, 25 Their responses to CCNA activation were examined in the breast cancer cell line SK-BR3 (Figure 2d, left panel). Cyclin A activated the full-length promoter as well as its −182 and −90 deletion mutants, which harbor at least one ICB (−572, −182 and −90 in the left panel of Figure 2d, P<0.05), but not the −60 deletion mutant, which lacks any ICB (−60 in the left panel of Figure 2d). Similar results were obtained when these experiments were performed in two other breast cancer cell lines MDA-MB-468 and MDA-MB-231 (data not shown), indicating that ICBs could be important for mediating the cyclin A signal. This point was further supported by the fact that the mutation of ICB1 in full-length topoIIα promoter resulted in the greater reduction in the response to cyclin A (Figure 2d, right panel), suggesting that ICB1 in the topoIIα promoter is required for full response to cyclin A/Cdk2 activation, and may represent a minimal breast cancer-specific element.

To validate the role of ICB1 in mediating breast cancer specificity of topoIIα promoter, the activity of the −90 deletion mutant, which contained only one ICB site, was examined in a panel of cell lines. Like the full-length promoter (Figure 1a), the −90 promoter had a higher level of activity in most breast cancer cell lines than in other types of cells (Figure 3a), indicating that it retains breast cancer specificity. The ratio of −90 promoter activity to CMV promoter activity (−90/CMV) also had low values in breast cancer cells (<0.5% in Figure 3b). As most other regulatory elements had been deleted in the −90 promoter, we concluded that ICB1 is critical for the breast cancer-specific activity of topoIIα promoter.

Figure 3
figure3

The −90 deletion mutant of the topoIIα promoter retains breast cancer-specific activity. (a) T2A90-pGL3 was transfected into cell lines, and its promoter activity was determined by using the dual luciferase assay. BE, normal breast epithelial cells; BC, breast cancer cells; LF, normal lung fibroblasts; HC, normal hepatocytes; PC, pancreatic cancer cells; OC, ovarian cancer cells. (b) T2A-90 promoter activity relative to that of the CMV promoter in various cell lines. The promoter activity ratio of T2A-90 to CMV (topoIIα/CMV) in each cell line was calculated as described for Figure 2b.

Enhancer sequence of the CMV promoter potentiated breast cancer-specific activity of −90 topoIIα promoter in vitro and in vivo

To enhance the basal level of activity of the topoIIα promoter in breast cancer cells, we connected the enhancer sequence of the CMV promoter (CMV enhancer)40 to three ICB-harboring promoters: full-length topoIIα promoter and its −182 and −90 deletion mutants. The composite promoters were designated CT572, CT182 and CT90, respectively (Figure 4a). Interestingly, among these composite promoters, CT90 had the highest level of activity in breast cancer cells relative to that in normal 184A1 cells (Figure 4b, reporter activity shown by log scale). Moreover, the activity of CT90 was comparable to the CMV promoter in breast cancer cells (Figure 4c). As the Y axis represents log scale, Figure 4b showed that the promoter activity of CT90 is about 5- to 10-fold higher than that of CT572 and CT182 in breast cancer cell lines are. CT572 and CT182 had lower specificity for breast cancer cells than CT90 (Figure 4b), and their activities were enhanced to a very limited extent (Figure 4c), suggesting that some elements other than ICB1 in the topoIIα promoter may interfere with CMV enhancer activity.

To test whether CT90 possesses sufficient specificity and activity for systemic liposomal gene therapy, we further examined its activity in vivo. The luciferase constructs driven by the CT90 or CMV promoter (CT90-luc and CMV-luc, respectively) were complexed with DOTAP: Chol liposome, then intravenously injected into mice carrying either MDA-MB-231 breast cancer xenograft. Reporter activity in the normal and tumor tissues was determined by luciferase assay and was shown by the reading per microgram of proteins (Y axis is of log scale, left panels of Figure 4d). The biodistribution of reporter activity from CMV-luc was very similar to that in reported studies: highest in lung, second highest in heart and third highest in liver; it was lower in other tissues. CT90-luc, however, induced higher reporter activity in the tumors and lower activity in normal organs such as heart, lung and liver than CMV-luc did (Figure 4d, left panels), suggesting that CT90 induced less undesired gene expression in normal tissues. It should be mentioned that expression levels in spleen, kidney and muscle are so low and likely negligible. To further evaluate the cancer specificity of the promoters, we calculated the ratio of reporter activity in tumor to that in normal tissue (tumor:normal, right panel, Figure 4d).18 The tumor:normal ratio of the CT90-luc is 49-, 69-, 35-, 4.7-, 4.8- and 9.8-fold higher than that of the CMV-luc in the heart, lung, liver, spleen, kidney and muscle, respectively. These results indicated that the CT90 promoter is more tumor-specific than the CMV promoter. A similar tumor:normal profile of CMV-luc and CT90-luc activities was observed in mice bearing MDA-MB-468 breast cancer xenograft (data not shown). When the liposome–DNA complexes were intratumorally injected, the activity of CT90 promoter was still higher in tumor and lower in normal tissues than that of the CMV promoter (Figure 4e, left panel). In addition, in all the tested tissues, the tumor:normal ratio of CT90 is higher than that of the CMV promoter (Figure 4e, right panel), confirming the higher tumor specificity of CT90. Taken together, these results demonstrate that CT90 possesses higher activity and cancer specificity than the CMV promoter, and may be a better promoter for breast cancer-targeting gene therapy.

CT90-BikDD selectively kills tumor cells in vitro

As CT90 possesses strong breast cancer-specific activity in vitro and in vivo, we compared the therapeutic efficacy of CT90-BikDD and CMV-BikDD constructs, in which the BikDD gene was driven by CT90 and CMV promoter, respectively (Figure 5a). The in vitro luciferase viability assay (see Materials and methods) was performed to examine the cell-killing effects of these constructs in a normal breast epithelial cell line and two breast cancer cell lines (Figure 5b). CT90-BikDD had a more prominent cell-killing effect in breast cancer cells than in normal breast cells, indicating its selectivity for breast cancer cells. On the other hand, the CMV-BikDD treatment killed all cell lines without selectivity.

Figure 5
figure5

CT90-BikDD selectively killed breast cancer cells in vitro. (a) Design of the gene therapy construct CT90-BikDD. (b) Selective killing effect of CT90-BikDD on breast cancer cells. CT90-BikDD and a luciferase reporter vector were co-transfected into normal breast epithelial 184A1 cells and breast cancer MDA-MB-468 and MDA-MD-231 cells. The survival ratio of cells receiving the construct was determined by the luciferase viability assay.

Liposome-delivered CT90-BikDD selectively suppresses breast tumor growth and prolongs survival in an orthotopic mouse model

To test whether a systemically administered liposome–CT90-BikDD complex could direct selective BikDD expression in vivo, MDA-MB-231 cells were inoculated into the mammary fat pad of female nude mice to form breast tumors. Groups of eight tumor-bearing mice then received weekly intravenous injection of liposomal CT90-BikDD, CMV-BikDD, pGL3 vector (mock treatment) or no treatment (D5W). Tumor growth was suppressed significantly in groups that received CT90-BikDD or CMV-BikDD (P<0.05), compared to that in both mock treatment (pGL3) and no treatment (D5W) groups (Figure 6a). As shown in Figure 6b, the mean survival times of mice in CT90-BikDD, CMV-BikDD, pGL3 and D5W groups were 25.25±1.83, 23.25±1.74, 17.5±1.3 and 16.25±0.98 weeks, respectively. Both CT90-BikDD and CMV-BikDD treatments yielded significant survival benefit for MDA-MB-231 xenograft-bearing mice compared to the no treatment (D5W) or mock treatment (pGL3), indicating that CT90-BikDD provided therapeutic effect comparable to that of CMV-BikDD (Figure 6b, lower panel). The tumor-suppressing effects of liposome–CT90-BikDD complex on other human breast cancer cells were also examined using mice bearing MDA-MB-468 or MDA-MB-435 xenografts, and similar results were observed (data not shown).

Figure 6
figure6

CT90-BikDD gene therapy suppressed tumor growth and yielded survival benefit in an animal model. (a) Mice carrying MDA-MB-231 breast cancer xenografts received treatment with liposome–CT90-BikDD, liposome–CMV-BikDD, liposome–pGL3 (empty vector) or 5% dextrose in water by intravenous injection. Each treatment group had 10 mice. The mice were treated once a week (QW) for 8 weeks, and tumor size was measured twice a week. (b) Survival records of MDA-MB-231 breast tumor-bearing mice described in (a). Treatments were stopped in the eighth week, and the mice were kept alive until reaching morbid status defined by institutional regulations. The number of mice surviving each week was recorded and is shown in the upper panel. The mean survival times and statistical significance from the Kaplan–Meier analysis and log-rank test are shown in the lower panel. N.S., not significant.

We then asked whether the in vivo differential expression profiles of CT90-luc and CMV-luc (Figure 4d) were mirrored by CT90-BikDD and CMV-BikDD. To address this issue, expression of BikDD mRNA in tumor and heart tissues was examined by in situ hybridization (Figure 7). The deep brown staining all over heart tissue from a CMV-BikDD-treated mouse (Figure 7a, upper left panel) indicated a very high expression level of BikDD induced by the CMV promoter, whereas CT90-BikDD treatment induced relatively weak BikDD expression in the heart tissues (Figure 7a, upper right panel). No significant difference in density or level of BikDD expression could be detected between the tumor specimens from mice treated with CT90-BikDD and those treated with CMV-BikDD group (Figure 7b). The negative control using sense BikDD showed no brown staining (Figure 7a and b, lower panels), indicating that the positive signals in the antisense groups came from BikDD expression. These data demonstrate that systemically administered liposome–CT90-BikDD complex can direct selective BikDD expression in breast tumors. Importantly, expression of BikDD in normal organs such as heart was much lower in the CT90-BikDD-treated mice than in the CMV-BikDD-treated mice. Thus, CT90-BikDD possesses antitumor activity comparable to that of CMV-BikDD but may produce fewer side effects because of its lower level of expression in normal tissues.

Figure 7
figure7

CT90-BikDD directed selective expression of BikDD in breast cancer cells in vivo. CT90-BikDD or CMV-BikDD liposome complex was injected into mice carrying MDA-MB-468 breast cancer xenografts. The mice were killed 72 h after injection, and their tumors and hearts were removed and fixed. In situ hybridization was performed on the tissue sections to detect BikDD mRNA expression as described in Materials and methods. Representative slides of heart and tumor tissues are shown. (a) BikDD expression in heart tissues from CMV-BikDD- and CT90-BikDD-treated mice (left and right panels, respectively). The samples in the upper panels were stained with antisense BikDD probes, and the deep brown color indicates positive signal. The lower panels show the negative control experiments stained with sense BikDD probes. (b) BikDD expression in tumor tissues from CMV-BikDD- and CT90-BikDD-treated mice (left and right panels, respectively). The samples in the upper panels were stained with antisense BikDD probes, and the arrows indicate positive cells. The lower panels show the negative control experiments stained with sense BikDD probes.

Discussion

In gene therapy, the cytotoxicity and immunoginecity of the gene delivery vector, as well as its potential to induce host gene mutations, are critical safety factors. Most of the viral vectors induce a systemic immune response, limiting their use in repeated injections. The presence of viral proteins in normal cells increases the risk of cytotoxicity. Some viral vectors have been reported to induce malignant diseases in clinical trials and preclinical studies41, 42 Compared to viral vectors, liposomes have lower toxicity, lower immunogenicity and almost no risk of inducing host gene mutations. As the goal of this study is to develop a breast cancer-targeting gene therapy regimen for practical application, we adopted a liposome that has been tested in clinical trials, the DOTAP:Chol cationic liposome, as our gene delivery vehicle.

In this study, we identified the breast cancer-specific activity of the topoIIα promoter. We also found that ICBs of the topoIIα promoter respond to cyclin A/Cdk2 in a breast cancer-specific manner. Cyclin A/Cdk2 complex is required during S phase,38 which coincides with increasing topoIIα expression. Cyclin A has been shown to phosphorylate and activate NF-YA,43 the transcription factor that binds to ICBs and activates topoIIα promoter.44 Furthermore, interrupting the association between cyclin A and cdk2 can specifically suppress breast cancer cell growth.45, 46 Together with our results, these data indicate that cyclin A/Cdk2 is a specific upstream activator of topoIIα promoter in breast cancer cells. Interestingly, among the deletion mutants of topoIIα promoter, only the minimal −90 promoter was potentiated successfully by the CMV enhancer, resulting in activity comparable to that of the CMV promoter while retaining specificity for breast cancer. This demonstrated that other regulatory sites in the upstream region of the topoIIα promoters may have adverse interaction with the enhancer sequence.

Tumor vasculature is usually irregular and has dilated lumens and fragile basement membranes, forming a leaky structure.47, 48, 49 Increased permeability would facilitate movement of liposome complex from vessels into tumor interstitium. Actually, it has been reported that cationic liposome complex is preferentially accumulated in tissues with high density of capillaries (e.g. thyroid or ovary), chronic inflammation and tumors.50 When the luciferase gene is delivered by liposome through tail vein injection in a mouse model, however, the principal sites of luciferase activity are the lung, heart and liver, and the luciferase activity in lung per microgram of tissue protein is one to two order higher than that in any other organs.14, 15, 16, 17 This fact could reflect that gene delivery efficiency depends not only on the interstitial retention, but also on the uptake of tissues, nuclear transport of DNA and tissue-specific promoter activity.51 In this study, a similar biodistribution pattern was observed when the DOTAP: Chol–CMV-luc complex was injected through the tail vein of tumor-bearing mice (Figure 4d, upper panels). However, CT90-luc induced a much lower luciferase activity in normal organs, especially lung and heart, and higher levels of activity in tumors than CMV-luc. Therefore, CT90 fits our two criteria for a promoter in systemic cancer gene therapy described in Introduction.

Proapoptotic genes can kill cells efficiently regardless of their proliferation rate; thus, they are good candidate therapeutic genes for cancer gene therapy. bik is a BH3-only proapoptotic gene that has been recognized as an essential initiator of apoptosis.52, 53 Recently, we found that mutation of threonine 33 and serine 35 of Bik to aspartic acid increases its apoptotic activity in vitro and its antitumor activity in animal models. The Bik mutant BikDD is more potent in inducing apoptosis than wild-type Bik.26 In our mouse models of orthotopic breast tumor xenografts, treatment with CT90-BikDD or CMV-BikDD yielded comparable efficacies in tumor growth suppression (Figure 6a) and survival benefits (Figure 6b). More importantly, the CT90 promoter drives much lower levels of gene expression than the CMV promoter in normal organs such as heart (Figure 7a), indicating that it may reduce the risk of side effects of therapy. As CMV promoter-driven constructs have been used widely in clinical trials of cancer gene therapy (according to The NIH/FDA Genetic Modification Clinical Research Information System (GeMCRIS; www4.od.nih.gov/oba/rac/GeMCRIS/GeMCRIS.htm) and The Journal of Gene Medicine Clinical Trial site (www.wiley.co.uk/genetherapy/clinical/)), the potential of the CT90 promoter to reduce side effects of cancer gene therapy has very important clinical implications. In gene therapy protocols using a potent proapoptotic gene, CT90 may be a better choice of promoter than CMV for both therapeutic efficacy and safety.

In summary, we identified the breast cancer-specific activity of the topoIIα promoter and its activating signal. On the basis of this finding, we have generated a CT90 promoter by linking topoIIα with an enhancer element of CMV promoter, and used it to develop a systemic breast cancer gene therapy delivered by liposome–DOTAP: Chol–CT90-BikDD. Systemically administered liposome–CT90-BikDD complex can direct BikDD expression in breast cancer cells in vivo, effectively suppress tumor growth and prolong the survival of tumor-bearing mice. This preclinical study shows that liposome–CT90-BikDD complex has a potential to be developed further for breast cancer treatment.

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Acknowledgements

We thank Ms Chu-Li Weng for the technical assistance. C-PD is an awardee of Department of Defense Breast Cancer Research Project Training Grant. K-M Rau is a visiting scientist from Chang Gung Memorial Hospital, Kaohsiung Hsien, Taiwan. This work was supported by Susan G Komen Breast Cancer Foundation Research Grant, US Army Breast Cancer Research Program Center of Excellence, Breast Cancer Research Foundation and MDACC Cancer Center Supporting Grant (CA16672).

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Day, C., Rau, K., Qiu, L. et al. Mutant Bik expression mediated by the enhanced minimal topoisomerase IIα promoter selectively suppressed breast tumors in an animal model. Cancer Gene Ther 13, 706–719 (2006). https://doi.org/10.1038/sj.cgt.7700945

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Keywords

  • breast cancer
  • liposome
  • topoisomerase IIα promoter
  • BikDD

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