Yang SP et al / Acta Pharmacol Sin 2003 Apr; 24 (4): 289-295

Review

Advancements of antisense oligonucleotides in treatment of breast cancer1

YANG Shuan-Ping, SONG San-Tai, SONG Hai-Feng2,3

Affiliated Hospital, 2Department of Pharmacology, Institute of Radiation Medicine, Academy of Military Medical Sciences, Beijing 100850, China

1 Project supported by National Natural Science Foundation of China, No 30070895.

3 Correspondence to Assoc Prof SONG Hai-Feng. Phn 86-10-6693-0304. Fax 86-10-6821-4653. E-mail song_hf@hotmail.com

Received 2002-01-26 Accepted 2002-12-05

KEY WORDS breast neoplasms; antisense oligonucleotides; oncogenes

ABSTRACT

Breast cancer is one kind of multi-gene related malignancy. Overexpression of some oncogenes such as HER-2 (c-erbB-2, Neu), bcl-2/bcl-xL, protein kinase A (PKA), and transferrin receptor gene (TfR gene), etc significantly affect the prognosis of breast cancer. It was shown that specific suppression of the overexpressed genes above resulted in the improvement of the therapy of breast cancer. Antisense interference, one of useful tools for inhibiting the overexpression of specific oncogenes, was involved in the therapy of breast cancer in recent years. Data indicated that antisense oligonucleotides (ON) could inhibit specially the expression of the target genes on mRNA or protein levels in most of cases; some ON candidates showed encouraging therapeutic effects in vitro and in vivo on breast cancer cell lines or xenografts. Furthermore, the combination use of the antisense ON and normal chemotherapeutic agents indicated synergistic antitumor effects, which was probably the best utilization of antisense ON in the treatment of breast cancer.

INTRODUCTION

The notion that gene expression could be modified through use of exogenous nucleic acids derives from studies by Paterson et al[1], who first used single-stranded DNA to inhibit translation of a complementary RNA in 1977. In the following year, Stephenson and Zamecnik[2] showed that a short (13nt) DNA oligonucleotide reverse complementary in sequence (antisense) to the Rous sarcoma virus could inhibit viral replication in culture. In 1983, the existence of naturally occurring antisense RNA, and their role in the regulation of gene expression was proven[3]. These observations were particularly important because they lent credibility to the belief that antisense was more than a laboratory phenomenon and encouraged belief in the hypothesis that reverse mentary could be used in living cells to manipulate gene expression. The developing of this technique directly resulted in appearance of a new class of drugs, antisense oligonucleotides (ON). According to the central dogma, genetic information was transmitted from DNA to mRNA by transcription, from mRNA to protein by translation. The intent effectively silence the gene of interest by preventing synthesis of the protein that it encodes is nonetheless attractive because mRNA is much more accessible and is efficient to be manipulated than DNA[4]. At present, a larger body of studies have focused on destabilizing mRNA directing at various targets that play a role in cancer[5,6], cardiovascular disease, viral disease, and inflammatory, accompanying with gene mutation and/or overexpression.

Most tumors accompany with gene mutation and/or overexpression, which result in the activation of oncogene. This is a very important process during the pathogenesis of the tumors. Breast carcinoma is the most common malignancy in women in western countries. Data from Eastern countries also indicated that the morbidity of breast cancer is still rising year by year. Almost all breast cancers were found to accompany with gene mutation and/or overexpression. Plenty of studies showed that there were definite relations between gene overexpression and pathogenesis, development and prognosis of breast cancer[7]. Presently, genes that are found to be in relation to breast cancer include oncogenes, eg, HER-2 (c-erbB-2, Neu), bcl-2, c-myc, and ras, etc; tumor suppressor genes eg, p53; estrogen receptor gene, progestogen receptor gene, estrogen regulation protein gene, growth factor receptor gene, and cyclin, etc. Antisense drugs have the characteristics of high selectivity and affinity to its targets, quickly taking effects, relatively definite mechanism, to be easy to evaluate the destination and less side effects, etc. These characteristics make antisense treatment an attractive strategy to selectively modulate the expression of genes involved in the pathogenesis of diseases. Breast cancer provides this class of new drug an ideal platform for evaluating the effects of antisense interference and for making break through. Not only can antisense suppress the overexpressed genes, but it is still of great value in studying the mechanisms of gene overexpression.

In this review, we focused on the recent advancements of antisense ON targeting at the genes that have definite relations with breast cancer. We will still concern some problems presently faced with.

SELECTION OF BREAST CANCER-RELATED GENES AS POTENTIAL TARGETS FOR ANTISENSE OLIGONUCLEOTIDES

HER-2/c-erbB-2/Neu The level of the Mr 185 000 HER-2 protein, encoded by the HER-2 oncogene, was elevated in approximately 30 % of cases involving human breast carcinoma[8]. Overexpression of HER-2 is widely considered as a poor prognosis following a tumor resection[9] and may be associated with increased resistance to cancer chemotherapy[10]. Herceptin, a humanized antibody generated against the HER-2 has been proven to be very effective in a phase II clinical trial involving patients with advanced stages of breast cancer, most of them expressing HER-2 at the highest levels[11]. This is the first therapeutic approach aimed at reducing the level of an oncogene product critically involved in breast cancer progression.

Bertram et al[12] selected two different regions of the HER-2 mRNA as potential antisense targets. These are the translation start region and the 3' translated region. The results showed that this design was very effective in reducing HER-2 expression in both SK-BR-3 and MCF-7 cell lines at a concentration as low as 2 mol/L. Subsequently, Vaughn et al[13] evaluated the effects of another 15-mer phosphorothioate antisense oligonucleotides that included the start codon of HER-2 mRNA on HER-2 expression and cell cycle. Such antisense treatment also downregulates HER-2 mRNA and protein levels in a sequence-specific manner, and the HER-2 downregulation is accompanied by an accumulation of SK-BR-3 cell in G1 phase of the cell cycle. Their research also indicated that the protein expression response to the PS antisense ON was biphasic, with a maximal downregulation achieved in the middle of the dosage range delivered by liposomes. Recently, the effect of the same sequence of antisense ON combined with several traditional chemotherapeutic agents, including doxorubicin hydrochloride, cis-platinum, and 5-fluorouracil on breast carcinoma cells growth were observed. The results showed a synergistic antitumor effect on BT-474 human breast carcinoma cells, one kind of high HER-2-expressed tumor cell line. However, in contrast to BT-474 breast carcinoma cells, there is no enhancement of these effects on low HER-2-expressed MCF-7 breast carcinoma cells with the same treatment[14]. This suggested that downregulation of HER-2 expression was able to increase the sensitivity of BT-474 cells to the cytotoxic effects of several traditional chemotherapeutic agents and this effect was directly in relation to the basic HER-2 expression level. The same reports also investigated the effect of this antisense ON in human tumor xenograft models in vivo. The results showed that systemic treatment with HER-2 antisense ON also significantly inhibited the growth of BT-474 xenografts in nude mice, the combination treatment using HER-2 antisense ON and doxorubicin resulted in an enhanced antitumor effect in vivo[14]. All these results indicate a potential clinical perspective of these antisense ON in the breast cancer therapy.

Bcl-2 and bcl-xL The bcl-2 family of proteins play major roles in regulating apoptosis and include both anti- and pro-apoptotic members. Bcl-2 and bcl-xL are members of anti-apoptotic proteins. Expression of these proteins is significantly higher in primary breast cancers, where they might play a pivotal role in tumor initiation, progression, and resistance to chemotherapy and radiotherapy[15,16]. There is a more than 10-year history of antisense researches targeting at bcl-2 mRNA. G3139, which is being tested in clinical trials, is one of the few antisense ON targeting at the translational start region of the bcl-2 mRNA[17]. Chi et al observed the effects of G3139 on cell viability, bcl-2 protein expression, apoptosis of high bcl-2 protein expressing, estrogen receptor (ER) positive MCF-7 and low bcl-2 expressing, and ER negative MDA435/LCC6 human breast cancer cell lines. They found that treatment with G3139 in vitro caused a specific reduction of bcl-2 protein levels and increased apoptosis in both cell lines. These results suggested that the relative degree of down-regulation of the bcl-2 protein was more important than the absolute reduction[18]. In the same work, combined treatment with G3139 and cytotoxic agents resulted in additive cytotoxicity in both cell lines. Furthermore, the initial clinical results of G3139 were still encouraging[19], 21 patients with bcl-2-positive relapsed NHL received a 14-d sc infusion of G3139, bcl-2 protein was reduced in 7 of 16 assessable patients, indicating its potential antitumor activity. oligonucleotide 4259 directed at breast cancer is the 2-O-methoxy-ethoxy antisense oligonucleotide targeting coding region of the bcl-xL mRNA. Treatment of MCF7 cells with oligonucleotide 4259 at a concentration of 600 nmol/L for 20 h decreased bcl-xL mRNA and protein levels by more than 80 % and 50 %, respectively and induced cell apoptosis. Moreover, the results that oligonucleotide 4259 resulted in similar effects in the breast carcinoma cell lines T-47D, ZR-75-1, and MDA-MB-231 were also observed in Simoes-Wust and his colleagues' experiments[20]. Oligonucleotide 4625 is another antisense ON targeting at a region of high homology shared by bcl-2 and bcl-xL mRNA. This oligonucleotide is also 2-MOE modified, 100 % complemented to bcl-2 mRNA and with 3 mismatches to bcl-xL mRNA. The results showed that 4625 treatment reduced bcl-2 and bcl-xL mRNA levels in a dose-dependent manner. In addition, the initial data of 4625 on breast carcinoma xenografts finished by Gautschi et al was also encouraging [21], oligonucleotide 4625 statistically significantly inhibited the growth of breast carcinoma xenografts by 51 %, it also reduced bcl-2 and bcl-xL protein levels and induced tumor cell apoptosis.

Protein kinase A (PKA) There are two types of PKA, PKAI, and PKAII, which share a C subunit but contain different regulatory R subunits, RI and RII, respectively. Through biochemical studies and gene cloning, four isoforms of the R subunits, RI, RI, RII, and RII have been identified. PKA plays a pivotal role in the control of cell growth and differentia-tion [22]. PKAI is involved in cell proliferation and neoplastic transformation, and is required for the G1>S transition of the cell cycle. PKAI is overexpressed in the majority of human cancers, correlating with worse clini-pathological features and prognosis in breast cancer patients [23]. Conversely, PKAII is preferentially expressed in normal tissues. Several studies investigated the RI antisense targeting against NH2-terminus 8-13 codons. Srivastava et al[24,25] sequentially evaluated the effects of this RI antisense ON on tumor cell growth inhibition, mRNA and protein expression levels and apoptosis induction in different breast cancer cell lines. Their results indicated that this kind of antisense ON inhibited the growth of MDA-MB-231 breast cancer cells in a dose-dependent manner. The growth inhibitory effects correlated with a decrease in the RI mRNA and protein levels. Similar growth inhibitory effects of this antisense ON were observed in MCF-7 breast cancer cell line, the growth inhibition was accompanied by an apoptosis induction, downregulation of RI and upregulation of RII protein expression. In the same study, they also found an interesting phenomenon that daily treatment of low doses of RI antisense ON for 3 d which were tested to be ineffective doses for single use still significantly inhibited breast cancer cell growth. To evaluate the effect of blocking PKAI on MCF-10A cell sensitivity to taxanes, Ciardiello et al [26] treated these cells with taxol or taxotere in combination with the PKAI antisense ON. Their results indicated that treatment with this agent was able to overcome the effect of HER-2 overexpression on MCF-10A cell sensitivity to taxol and taxotere.

Transferrin receptor gene (TfR gene) Iron is required for the activity of ribonucleotide reductase, a key enzyme involved in DNA synthesis. TfR is the principal transport protein that mediates iron uptake into cells. TfR expression correlates with cellular proliferation and is found higher in rapidly dividing cells. The density of transferring receptors has also been corre lated with the rate of DNA synthesis and metastatic potential of tumor cells[27,28]. Aggressive breast carcinomas that usually carry a poor prognosis had significantly higher TfR expression compared to grade I lesions with better prognosis[29]. Yang et al synthesized a 24-mer nucleotide complementary to the sequence corresponding to the translational initiation AUG codon of the TfR mRNA. They found that after exposure to antisense TfR-ON 1 mol/L for 72 h, the number of live MCF-7, T47-D, and MDA-MB-231 tumor cells was significantly reduced. The IC50 (50 % inhibition of DNA synthesis) of TfR ON for the MCF-7, T47-D, and MDA-MB-231 cells were 0.5, 0.5, and 1.0 mol/L, respectively, whereas the IC50 to normal breast cells was 30 mol/L. Additionally, inhibitions of mRNA and protein synthesis induced by the same TfR antisense ON to these breast cancer cell lines were also observed by Yang and his coworkers[30].

V integrin gene Integrins are cell surface glycoprotein and consist of I heterodimers. At least 16 and 8 subunits have been described in mammals and these subunits can combine to form 22 different heterodimers, each with a specific recognition and binding affinity toward the various components of the extracellular matrix (ECM) milieu and cell adhesion molecules[31,32]. Integrins are not only implicated in cell-cell and cell-ECM interaction but have been shown to play a critical role in cell signaling, migration, differentiation, and tissue modeling[33]. There is considerable evidence for altered integrin levels in breast cancer cell lines. Levels of V integrin protein were increased and could play a major role in breast carcinoma metastasis[34]. Based on the above data, Townsend et al[35] designed a 18-mer V integrins antisense ON and tested the anti-adhesive potential and expression-inhibiting effects of the V integrin antisense ON on breast cancer cell line. They found that this antisense also significantly reduced V mRNA transcription and protein expression in a dose- and time-dependent manner and promoted apoptosis of MDA-MB-231 cells .

Other targets It was recently reported that the mouse double minute 2 (MDM2) gene was amplified in breast cancer. Recently, Wang et al[36] selected MCF-7 cell line containing wild-type p53 and MDA-MB-468 cell line containing mutant p53 to evaluate the effects of MDM2 antisense treatment on p53 and p21 protein levels in both cell lines. They found that in MCF-7 cells, p53, and p21 protein levels were elevated as a result of reduced MDM2 expression. On the other hand, level of the p53 protein remained unchanged in MDA-MB-468 cells after treatment with MDM2 antisense ON. The same study also examined the effectiveness of the MDM2 antisense ON treatment in vivo. In nude mice bearing MCF-7 or MDA-MB-468 xenografts, MDM2 antisense dose-dependently inhibited the tumor growth. In both these models, synergistically or additive therapeutic effects of MDM2 inhibition were also observed when MDM2 antisense ON were combined with some other chemotherapeutic agents commonly used in the clinics such as irinotecan, 5-fluorouracil, and paclitaxel (Taxol). In addition, thymidylate synthase (TS) which was a key enzyme involved in the synthesis of DNA had been targeted for cancer chemotherapeutic agents [37]. Vascular endothelial growth factor (VEGF) and protein kinase C (PKC) as candidate targets for breast cancer were also reported and need further investigations.

CLINICAL RESEARCH

The promising results of the use of antisense ON in vitro strongly stimulate the interests of the researchers to further investigate their effects in vivo, and consequently lead to development of several clinical trials. Antisense is rapidly moving from being a laboratory tool to becoming a full-fledged therapeutic strategy to treat various human diseases. At present, there are more than 20 clinical trials under way using antisense compounds directed at various targets that play roles in cancer[5,6], viral disease[38], and inflammatory disorders[39]. In July of 1998, the Food and Drug Administration (FDA) approved for marketing the first antisense-based therapeutic called fomivirsen which targets cytome-galoviral retinitis[40]. The investigations from Jansen and his colleagues[41] brought new hope for the idea of antisense, which showed that, besides the clinical benefit for patients with advanced melanoma, systemic treatment with antisense ON also results in the downregulation of the target protein within the target tissue. This finding will certainly stimulate the progress of antisense ON in clinical research in the treatment of breast cancer, since the results suggest that the principle of antisense works, not only with local treatment, as shown with fomivirsen[40], but also with systemic treatment with antisense ON. At present, the proof of clinical efficacy of antisense target at breast cancer is still missing. Though G3139 was undergoing clinical trails, this antisense compound was presently administered in non-Hodgkin's lymphoma[15]. Nevertheless, the initial encouraging antitumor effects of antisense ON target at HER-2 finished by Roh et al[14] suggested its potential clinical value in the near future. The beauty and future potential of antisense ON in the treatment of breast cancer also depends on the design of multiple drugs based on our increasing knowledge of genes and their functions. We believe that as the deepening of the basic researches and accumulating of more basic data, the application of antisense ON in the treatment of breast cancer in clinics will be finally realized.

PROBLEMS AND PERSPECTIVES

Despite the short history of antisense research, numerous companies have already invested billions of dollars in exploring the potential of its use in treatments of various human diseases. Nevertheless, there is still long way to go before antisense drugs becoming one kind of mature and extensively used drugs. The use of antisense ON to fight against breast cancer also faced its own problems. First of all is the design of the antisense, not all antisense drugs complemented to the targeted mRNA are effective. In spite of the significant progress using advanced technology to predict mRNA secondary structure and phylogenesis analysis[42,43], the selection of the target sequences in designing effective antisense ON remains problematic. Second, the oligonucleotide must be taken in by the target cell, tissue or organ in a quantity sufficient to invoke a biological response, antisense needs efficient system for drug delivery. Third is the stability of the antisense oligonucleotide, presently, many researchers try to enhance the stability of the antisense in vivo through modification of base, ribose, and oligonucleotide skeleton. Fourth, diverse clinical outcomes of antisense were gotten in different laboratories. It is important to note that there is currently a major dichotomy between in vitro and in vivo studies of antisense effects. Some nonsequence-specific effects of antisense still could not be ruled out. In addition, expensive cost, dose-depend-ent side effects such as hypotension, complement activation, lymphoid hyperplasia, splenomegaly, and prolongation of thromboplastin time are problems of antisense presently faced with[44].

Like all other new developing technologies, antisense technology is still not a mature technology. More expectations for antisense ON are quite reasonable. Present initial experiments indicated that inhibition of most genes in relation to breast cancer by specific antisense ON could inhibit breast cancer cell growth and reduce the expression of mRNA and protein in vitro and in vivo. These results are encouraging and thus suggest that to select antisense ON as a common measure to manipulate the target gene is a quite effective method in those cells, tissues or organs accompany with gene overexpression. The understanding of antisense conception will be sublimated as the accumulation of more data and may finally result in some new findings. Immune stimulation is generally recognized as an undesirable side effect of certain antisense ON. Nevertheless, CpG oligonucleotides, which are designed to provide optimum immune stimulation, are promising anticancer drugs. G3139 that contains two CG dinucleotides and a TC at the 5' end described above was successfully used as an immunostimulatory CpG oligonucleotide in animal tumor models[45]. This new function of antisense should certainly benefit its antitumor effects and be sure to increase the complexity of antisense mechanism. It seems that the potential applications of antisense ON, this kind of nontoxic drug in the treatment of breast cancer in the near future maybe include following situations: single administration as a new kind of antitumor drugs; provocation of immune stimulation to enhance the anti-tumor effects, especially for those patients with immune system deficiency; combination with chemotherapeutic agents to produce synergistic anti-tumor effects, which may be the most probably utilization of antisense ON.

In conclusion, at present critical time, there are opportunities as well as dangers for antisense researches. As the deepening of the basic research and accumulation of the experimental data, antisense, this new class of drug will become mature. It now appears that antisense technology, which represents a new class of drugs, has nearly reached maturity and will have an important clinical role in the treatment of breast cancer[46].

ACKNOWLEDGMENT We thank for assistance and advices on English grammar revising by GUO Zhi-Hong and Sic L CHAN (Laboratory of Neurosciences, Gerontology Research Center, National Institute on Aging, 5600 Nathan Shock Drive, Baltimore, Maryland 21224, USA).

REFERENCES