HER-2/neu is overexpressed in 25–30% of human breast cancers. We prepared an anti-HER-2/neu hammerhead ribozyme expressed by a recombinant adenovirus (rAdHER-Rz). Human breast cancer cell lines were transduced with high efficiency, resulting in decreased HER-2/neu expression. In vivo injections of rAdHER-Rz into BT-474 tumors established in nude mice inhibited tumor growth to 20% of mock-treated controls. Similar in vivo effects were shown in MCF-7 cells, which do not overexpress HER-2/neu. The growth inhibitory effects of rAdHER-Rz were greater than those of an antisense-expressing vector. These results suggest the utility of anti-HER-2/neu ribozymes as a rational strategy for gene therapy of breast cancer.
The HER-2/neu proto-oncogene (also called c-erbB-2) encodes a 185 kDA transmembrane receptor homologous with the epidermal growth factor receptor (EGFR).12 Although EGFR can transform mouse NIH3T3 cells only in the presence of ligand and receptor,3 HER-2/neu overexpression can transform NIH3T3 cells in a ligand-independent manner.45 The HER-2/neu protein is constitutively phosphorylated and demonstrates tyrosine kinase activity without the presence of ligand when overexpressed in NIH3T3 cells.6 A possible role of HER-2/neu in human epithelial malignancies such as breast and ovarian cancers has been postulated through its amplification and overexpression.7 HER-2/neu overexpression occurs in 25–30% of examined tumors.89 Moreover, there appears to be a correlation between high levels of HER-2/neu expression and poor clinical outcome, particularly in patients with positive axillary lymph nodes.101112
Because the HER-2/neu protein is not expressed in most normal human tissues,1314 down-regulating HER-2/neu expression may be an important strategy for cancer therapy. Previous studies have shown that monoclonal antibodies,15161718 a single chain antibody engineered for intracellular expression,19202122 and triplex2324 or antisense oligonucleotides targeting HER-2/neu252627 are capable of down-regulating its expression. However, other than those using monoclonal antibodies, few studies have explored the efficacy of such modalities to suppress breast cancer cell growth in vivo.
Catalytic RNAs such as hammerhead ribozymes have demonstrated utility in attenuating eukaryotic gene expression and have been examined in preclinical gene therapy models.28 Our group has explored the efficacy of anti-oncogene ribozymes in reversing the malignant phenotype of human bladder carcinoma cells,282930 malignant melanoma cells,3132 and ras-transformed NIH3T3 cells. These studies have targeted the mutated H-ras gene at codon 12 given its transforming potential.3334
In this study, we designed hammerhead ribozymes to cleave the normal HER-2/neu transcript. In order to achieve efficient gene delivery in vivo, we prepared a recombinant adenovirus encoding the ribozyme driven by the cytomegalovirus (CMV) promoter (rAdHER-Rz). We present the studies examining the efficacy of rAdHER-Rz in suppressing the growth of human breast cancer cells.
To select an effective anti-HER-2/neu ribozyme, we designed three ribozymes targeting GUC sequences at codon 71/72 (Rz-1), codon 212/213 (Rz-2) and codon 219 (Rz-3), all located close to the translation initiation site. The ribozyme sequences were subcloned into the pHβAPr-1-neo plasmid, and the plasmid introduced into MCF-7 and BT-474 cells by electroporation. The growth inhibitory effects of the ribozymes were analyzed using randomly selected stable transformants. The ribozyme targeting codon 71/72 (Rz-1, Figure 1) showed the most potent growth inhibitory effect in vitro on breast cancer cells (data not shown), and was thus selected for recombinant adenovirus construction.
Effects of rAdHER-Rz on breast cancer cell growth in vitro
To test the growth inhibitory effects of the anti-HER-2/neu ribozyme on BT-474 and MCF-7 cells, we transduced rAdHER-Rz into BT-474 and MCF-7 cells at 5, 50 and 500 multiplicities of infection (MOI) in vitro. BT-474 cells overexpress HER-2/neu while MCF-7 cells express small quantities of the protein. The virus inhibited in vitro growth of BT-474 cells significantly at a dose of 500 MOI (Table 1). Similar results were obtained with MCF-7 cells (data not shown). BT-474 cells transduced by rAdCMVLacZ (encoding the β-galactosidase gene) were stained with X-gal to evaluate the transduction efficacy of the recombinant virus. We found that virtually 100% of the BT-474 cells were transduced by 500 MOI rAdCMVLacZ as evidenced by blue staining at 48 and 72 h (data not shown). To confirm that the growth inhibitory effect was specific for the ribozyme, we transduced several types of control recombinant adenovirus (rAdHER-As, rAdHER-mRz, rAd-HrasRz, rAd-vector and rAdCMVLacZ) into BT-474 cells. We found that only rAdHER-As significantly inhibited breast cancer cell growth although the effect was not so strong as that of rAdHER-Rz (Table 2).
Molecular effects of rAdHER-Rz infection
We next examined whether the anti-HER-2/neu ribozyme could effectively cleave target RNA and down-regulate its expression by Northern blot analysis. We studied the expression of c-erbB-2 mRNA, cleaved c-erbB-2 mRNA products and anti-c-erbB-2 ribozyme in BT-474 cells treated with 500 MOI of rAdHER-Rz (Figure 2a and b). We used 5′-endlabeled A1 and A2 primers, which are antisense and sense strands of the cleavage site, respectively. Theoretically, 5′ and 3′ cleaved mRNAs are labeled by the antisense and sense probe, respectively. Anti-c-erbB-2 ribozyme was expressed at 1.0 kb and the predominant mRNA species detected included the 2.0 kb and 0.3 kb fragments (Figure 2a). The expressed ribozyme cleaved the c-erbB-2 mRNA into two fragments. Northern blot analysis revealed that expression of anti-c-erbB-2 ribozyme or anti-c-erbB-2 antisense by the recombinant adenovirus down-regulated c-erbB-2 expression in vitro (Figure 2b).
To test the effects of the ribozyme in vivo, we treated subcutaneously established 1 × 107 BT-474 cells with three weekly injections of 10 MOI rAdHER-Rz. Tumor tissue was obtained 3 days after the last injection and total RNA was isolated. Expression of adenovirus-mediated RNA was determined by RT-PCR analysis of tumor tissue obtained from vector-only treated tumors (Figure 3a, lane 3), rAdHER-Rz treated tumors (lane 4), and other control adenoviruses. No adenoviral sequences were detected in PBS-injected tumors (lane 2). Finally, the effects of rAdHER-Rz infection of HER-2/neu expression was examined in vivo. Northern analysis of mRNA from ribozyme-treated tumors revealed that expression of the HER-2/neu gene was reduced to 59% of that observed in control PBS-treated tumors (Figure 3b).
Effects of rAdHER-Rz on breast cancer cell growth in vivo
To examine the effects of rAdHER-Rz treatment on breast cancer growth, BT-474 cells were treated with 500 MOI rAdHER-Rz or rAdCMVLacZ. After transduction, 1 × 107 cells were inoculated subcutaneously into the flanks of nude mice. BT-474 cell growth in vivo was significantly inhibited by rAdHER-Rz in comparison with control rAdCMVLacZ (Figure 4a).
We next wished to examine the therapeutic efficacy of rAdHER-Rz on established tumors. Implanted BT-474 tumor nodules reach a stable size around day 14 when the cells are inoculated with an estradiol pellet and matrigel, with a tumor take approaching 100%. One to 100 MOI rAdHER-Rz were tested and 10 MOI found to be optimal for subsequent studies (data not shown). The recombinant adenovirus was administered intratumorally weekly for 5 weeks starting on day 14. Treatment of BT-474 tumors with rAdHER-Rz resulted in significant inhibition of tumor growth (Figure 4b), such that by day 50 tumor size was 20% of control PBS-treated tumors (Figure 5). rAdHER-As administration also inhibited tumor growth, but the effect was not as strong as that of rAdHER-Rz. Other control adenoviral vectors, including the unrelated rAd-Hras-Rz targeting the activated H-ras gene, did not show any significant tumor inhibitory effect.
Finally, the effects of rAdHER-Rz on the growth of MCF-7 cells were tested. Five weekly intratumoral injections of 10 MOI rAdHER-Rz into MCF-7 tumor nodules inhibited the tumor growth and appeared to be superior to the antisense-expressing construct (Figure 6). Other control adenoviral vectors did not show any significant tumor inhibitory effect.
In this study, we explored the efficacy of a recombinant adenovirus encoding the anti-HER-2/neu ribozyme as a potential gene therapy agent of human breast cancer. We have demonstrated that expression of the ribozyme results in suppression of target gene expression. We have also shown that this reduced expression results in diminished in vivo growth of human breast cancer cells. The contribution of the hammerhead ribozyme to inhibition of breast cancer cell growth was superior to that of the adenoviral vector alone or recombinant adenoviruses encoding antisense HER-2/neu (lacking the catalytic motif), a disabled ribozyme targeting HER-2/neu, or a ribozyme targeting an unrelated gene. Finally, we have presented data suggesting the antitumor efficacy of rAdHER-Rz in breast cancer cells regardless of the endogenous level of HER-2/neu expression.
The mechanism underlying inhibition of tumor cell growth by ribozyme-mediated cleavage of HER-2/neu mRNA remains unknown. Three distinct mechanisms of breast cancer growth inhibition are plausible. First, decreases in overexpression of HER-2/neu may induce apoptosis of breast cancer cells as observed in anti-HER-2/neu single chain antibody-treated cells.22 Second, decreases in HER-2/neu may induce differentiation of the breast cancer cells. Third, injection of recombinant adenovirus may cause an immunological reaction resulting in cell death. Pathological examination of the adenovirus-treated tumors revealed that there was neither differentiation induction of the tumor tissues nor an inflammatory cell infiltration (data not shown). It is therefore possible that a reduction of HER-2/neu expression by the ribozyme led to altered signal transduction pathways, which either mitigated its cell growth or induced apoptosis.
Interestingly, the growth inhibitory effects of ribozyme (as well as antisense) expression appeared to be more profound in vivo than in vitro. It is difficult to draw direct comparisons between the experiments, as a higher dose of the recombinant adenoviruses was utilized in the in vitro studies, while five treatments were used in the mouse studies. Nevertheless, it is tempting to speculate that residual immune effector cells (such as macro- phages) could play a role in the improved antitumor effects noted in vivo.
In both in vivo models of tumor cell growth, the antitumor effects of the ribozyme-expressing adenovirus were greater than its antisense-expressing counterparts, possibly owing to the catalytic nature of the ribozyme. Interestingly, in the intratumoral injection model, both the antisense and mutant ribozyme constructs showed significant antitumor activity over the adenoviral vector alone. This suggested an intermediate level of activity for the antisense constructs, likely resulting from sequence-specific inhibition of HER-2/neu translation.
Surprisingly, the inhibition of cell growth achieved by rAdHER-Rz was similar in both BT-474 and MCF-7 cells. These cell lines were selected because they represent high versus low expressing HER-2/neu lines, respectively. While this finding may have implications regarding the relevance of the level of HER-2/neu overexpression in breast cancer cell lines (despite its apparent clinical significance912), it potentially underscores the utility of targeting HER-2/neu expression as a broad-based strategy for therapy of breast cancer.
Ribozyme targeting of HER-2/neu gene expression has recently been examined by other groups.353637 In one study, an adenovirus encoding a ribozyme targeting codons 663 and 664 was shown to deplete HER-2/neu mRNA in ovarian carcinoma cells by 75% 24 h after infection in vitro.35 Time-course studies demonstrated peak ribozyme expression 3 days after adenoviral infection. Transient inhibition of HER-2/neu protein was observed on days 3–5 after adenoviral infection. The effects of the ribozyme-containing adenovirus on tumor cell growth were not reported. Two other studies have examined the effects of anti-HER-2/neu ribozymes using transfectants in ovarian carcinoma cells.3637
The studies reported here are the first to demonstrate the therapeutic efficacy of anti-HER-2/neu ribozymes against breast cancer cells in vivo. In our models, breast cancer cell growth was suppressed whether by previous in vitro infection with or subsequent in vivo administration of rAdHER-Rz. Moreover, decreased tumor growth was observed in two cell lines with divergent levels of endogenous HER-2/neu. The adenovirus-based approach may have clinical utility in treating local/ regional disease, such as locally recurrent breast tumors involving the chest wall, for which limited treatment options currently exist. For successful application to gene therapy of metastatic breast cancer, improved systemic delivery approaches will be required which mediate high-level ribozyme expression in metastatic tumor cells. In conclusion, these studies support the use of ribozyme targeting of HER-2/neu expression in future gene therapy of breast cancer.
Materials and methods
The human E1A transcomplementary cell line 293 was obtained from Dr F Graham (McMaster University, Hamilton, Ontario, Canada). Human breast cancer cell lines BT-474 and MCF-7 were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA). 293 Cells were grown as described previously.30 Breast cancer cells were grown in RPMI 1640 supplemented with 10% FCS and 1% penicillin/streptomycin. 2 mM L-glutamine and 10 mg/l bovine insulin were added to complete the medium for BT-474. Medium and supplements were obtained from Gibco BRL (Gaithersburg, MD, USA).
Three independent anti-HER-2/neu ribozymes against the GUC sequences at codon 71/72 (Rz-1; R1/R2), codon 212/213 (Rz-2; R3/R4) and codon 219 (Rz-3; R5/R6) were designed. The oligonucleotide sequences include SalI and HindIII linker sites and are as follows:
R1: 5′-TCG ACA GGA AGC TGA TGA GTC CGT GAG GAC GAA ACA GGC TA-3′
R2: 5′-AGC TTA GCC TGT TTC GTC CTC ACG GAC TCA TCA GCT TCC TG-3′
R3: 5′-TCG ACG GCT CTC TGA TGA GTC CGT GAG GAC GAA ACA ATC CA-3′
R4: 5′-AGC TTG GAT TGT TTC GTC CTC ACG GAC TCA TCA GAG AGC CG-3′
R5: 5′-TCG ACG GCA CAC TGA TGA GTC CGT GAG GAC GAA ACA GTG CA-3′
R6: 5′-AGC TTG CAC TGT TTC GTC CTC ACG GAC TCA TCA GTG TGC CG-3′
Rz-1 was selected for incorporation into the adenoviral vector. In addition, recombinant adenoviruses encoding a corresponding anti-HER-2/neu mutant ribozyme (mR1/mR2) and anti-HER-2/neu antisense (A1/A2) targeting the same sequences were synthesized. The mutant ribozyme has a one-base mutation, resulting in loss of cleavage activity. The structures of these inserts are shown in Figure 1. Oligonucleotide sequences are as follows:
mR1: 5′-TCG ACA GGA AGC TCA TGA GTC CGT GAG GAC GAA ACA GGC TA-3′
mR2: 5′-AGC TTA GCC TGT TTC GTC CTC ACG GAC TCA TGA GCT TCC TG-3′
A1: 5′-TCG ACA GGA AGG ACA GGC TA-3′
A2: 5′-AGC TTA GCC TGT CCT TCC TG-3′
Virus construction by cotransfection was carried out as described previously.30 pACCMVpLpARS(+) and rAdCMVLacZ were kind gifts from Dr R Gerald (University of Texas, Houston, TX, USA).
PCR and sequencing for verification of the recombinant adenovirus
The insert sequences were confirmed as described previously.30 P1 and P2 served as PCR primers, S1 as the sequencing primer. Wild-type adenovirus contamination was ruled out by PCR using primers P3 and P4. Primer sequences are as follows:
P1: 5′-GCG TGT ACG GTG GGA GGT CT-3′
P2: 5′-GTT TCG TCC TCA CGG ACT CAT-3′
P3: 5′-ATT ACC GAA GAA ATG GCC GC-3′
P4: 5′-CCC ATT TAA CAC GCC ATG CA-3′
P5: 5′-GCG TGT ACG GTG GGA GGT CT-3′
In vitro effects of the recombinant adenovirus
BT-474 or MCF-7 cells (2 × 104) were cultured in a 35-mm dish. The cells were incubated with 5 p.f.u. per cell (MOI), 50 MOI, or 500 MOI of rAdHER-Rz, or 500 MOI of rAdHER-mRz, rAdHER-As, rAdCMVLacZ, rAd-vector or rAd-HrasRz for 1.5 h. Cells were counted in a Coulter counter for 72 h. Cells treated with rAdCMVLacZ were fixed with glutaraldehyde (Sigma, St Louis, MO, USA) and stained by X-gal (Stratagene, La Jolla, CA, USA).
Tumor implantation into nude mice
Six-week-old female nu/nu mice (Charles River Breeding Laboratories, Portage, MI, USA) were maintained under standard pathogen-free conditions. One day before tumor cell injection, mice were implanted with a 17β-estradiol pellet (0.72 mg, 60-day release type; Innovative Research of America, Toledo, OH, USA). BT-474 or MCF-7 cells growing in log-phase were resuspended in PBS. BT-474 cells (1 × 107) in a volume of 0.1 ml were injected subcutaneously into the flanks with 0.1 ml of matrigel (Collaborative Biomedical, Bedford, MA, USA). MCF-7 injection was similar, but without matrigel. Tumor size was measured every other day; tumor volume was calculated as (length × width2)/2.
Intratumoral injection of the recombinant adenovirus
Ten MOI of the recombinant adenovirus in 0.1 ml PBS were injected intratumorally on day 14 after implantation, assuming that 1 cm3 of tumor contains 1 × 109 cells. Contralateral tumors were injected with PBS only. Five weekly treatments were performed. In a minority of cases, the bilateral tumors regressed spontaneously after the second injection, suggesting an immunological trigger. These cases were not included in the response assessments.
One to 10 μg poly (A)+ RNA were transferred to a Hybond N+ nylon membrane (Amersham, Buckinghamshire, UK) and cross-linked by Stratalinker (Stratagene). Hybridization took place at 68°C with QuickHyb (Stratagene) for 1 h. The HER-2/neu and PGK probes (both Oncogene Science, Cambridge, MA, USA) were α-32P dCTP-labeled using random primed labeling kit (Gibco). Ribozyme (primers R1/R2), antisense (primers A1/A2) and GAPDH (Oncogene Science) probes were 5′-endlabeled (Gibco) with γ-32P ATP (Dupont NEN, Wilmington, DE, USA). The highest washing stringency was reached with 0.1 × SSC/0.1% SDS at 60°C for 30 min. Semi-quantification was performed using a FluorS-MultiImager (BioRad, Hercules, CA, USA).
One μg of total RNA was denatured at 90°C for 5 min and then incubated with reverse transcriptase (Boehringer Mannheim, Indianapolis, IN, USA) at 42°C for 45 min primed by random hexamers (Gibco). The volume of the resultant cDNA sample was split 1:7 and used for separate PCR reactions (larger volume, primers RT-P1A/RT-P1B; smaller volume, β-actin control primers). The concentration of the reaction components used has been described previously.38 Thirty-five cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, followed by a final extension step of 72°C for 5 min, were performed. Visualization took place on a 1.5% TAE agarose gel. Primer sequences were as follows:
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T Suzuki is supported by the grant from Uehara Memorial Foundation for Research of Life Science, Japan. O Engebraaten is supported by the Norwegian Cancer Society, the US–Norway Fulbright Foundation for Educational Exchange, and the Thomas Fearnley, Heddy and Nils Astrup Foundation. M Kashani-Sabet is supported by the Leaders Society Clinical Career Development Award of the Dermatology Foundation. We thank A Harty for preparing the manuscript. D Curiel is supported by NIH grants ROI-CA60245 and ROI-CA74242.
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