Lung cancer and gene therapy Diana Zamora Ávila & Paul R Earl Immunology and Virology Department Biosecurity Laboratory, Level 3 Facultad de Ciencias Biológicas Universidad Autónoma de Nuevo León San Nicolás, 66451 NL, Mexico
Lung cancer is the most common cancer worldwide in both the indices of prevalence and mortality. Smoking is a risk factor highly associated with this type of cancer (85-90 %), and the exposure to tobacco smoke in the environment can cause cancer in nonsmokers. Certain agents like arsenic, asbestos, chromium, nickel and vinyl chloride encountered in the work environment increase the risk and also can cause cancer. In addition, the effects of smoking may be additive with some hazardous agents.
Classification The 2 main types of lung cancer are small cell and nonsmall cell neoplasms. 1/ Small cell pulmonary cancer constitutes 20 % of all these cancers, the cells multiplying rapidly and are able to metastisize into major organs like lymphatic ganglia, bones, brain, adrenal glands and liver. The primary tumor usually generates near the bronchi and expands to the center of the lungs. The main cause of this type of cancer is smoking.
2/ Nonsmall-cell pulmonary cancer represents almost 80% of all lung cancer. It diffuses more slowly than the small cell type. The 3 subtypes are 1/ carcinoma of squamous or epidermoid cells (30 %):, 2/ adenocarcinoma (40 %) y 3/ undifferentiated carcinoma with large cells (10 %). Some cancers start in the bronchioles and evolve from there through several years.
Lung cancer is a very aggressive disease in which less than 15 % (10-13 %) of the patients survive over 5 years. This is the lowest rate of all types of cancer. More, 80 % of cases do not respond favorably to chemotherapy or radiation. The main cause of poor or no recovery from lung cancer is the high rate of metastasis existing before diagnosis. In nonsmall cell lung cancer, about 2/3rds of the pacients are inoperable as a result of metastasis. In small cell lung cancer, metastases are present in most patients. The liver has 1/3 rd cases, and the brain is the first site.
Genetic therapy In genic therapy one procedure is introducing a gene(s) into somatic human cells. Two general strategies exist: 1/ genic therapy in vivo in which the target cells are introduced directly into the body, and 2/ therapy ex vivo in which the target cells are modified in vitro and later replaced. The genetic treatment of lung cancer is difficult as it is a multifactor disease. Thus various alternatives have been proposed for stimulating the immune system, including the transfer of suicidal cells, inactivation of oncogenes, gene replacement, tumor repressers and the transfer of pro-apoptotic genes.
A promising target for the development of novel anticancer strategies is telomerase , a ribonucleoprotein reverse transcriptase that extends human telomeres by a terminal transferase activity. Telomerase is highly active in the vast majority of human tumors, known since 1994. Its essential genes are hTR and hTER. However, telomerase has not yet advanced from the laboratory to the clinic. See www.nature.com/ clinicalpractice / onc . See also methioninase gene therapy and apoptosis as given by Yamamoto et al. (Cancer Gene Therapy 10, 445–450, 2003).
STIMULATION OF THE IMMUNE SYSTEM Tumor regression has been demonstrated in animal models that was mediated by the administration of cytokines such as interleukines (IL) –2, 4 , 6, 7, 1 2, stimulating factor of colonies of granulocytic macrophages (GM-CSF), tumor necrosis factor - (TNF- ), interferon- (IFN- ) & IFN- , nonetheless use in humans has been limited due to toxicity. The strategies based on cytokines can lead to the development of tumor cell vaccines that genetically incorporate modified fibroblasts or tumor cells that secrete cytokines.
TRANSFER OF KILLER GENES This type of genic therapy is based on the transduction of a gene able to convert a nontoxic compound into a toxic metabolite able to selectively kill tumor cells upon administration of the appropriate prodrug. The 2 genes used most for this type of therapy are thymidine kinase of herpes simplex virus HSV-tk and the gene of cytosine deaminase . Cytosine deaminase converts 5-fluorocytosine into an antimetabolite: cytotoxic 5-fluoroacyl. HSV-tk converts the gancyclovir into a toxic trifosfate metabolite. An adenoviral transduction in nonsmall cancer cells with HSV-tk followed by the administration of gancyclovir selectively kills the tumor cells.
INHIBITION OF ONCOGENES This type of therapy is based on the identicifation and inhibition of those genes critical for carcinogenesis. Oncogenes of the ras family are some of the more common oncogenes that are activated in lung cancer and therefore are targets for this type of therapy. In preclinical studies mediated by a plasmid with an antisense sequence with k-ras the mRNA is selectively blocked by mutation, and tumor growth is reduced both in vivo and in vitro in murine models.
GENES SUPPRESSERS OF TUMORS Another genic therapy strategy based on work with gene tumor suppressers, in contrast to oncogenes, is with the 2 alleles of a tumor suppresser gen that should be eliminated or inactivated by inducing tumor growth. Theoretically, replacement of only one copy of the tumor supresser gene in cells with a loss of homozygotic function can restore the ways of normal growth and cellular proliferation. One of the genes most commonly mutated (50-70 % of cases) is p5 3 that can be inactivated by overexpression of MdM-2.
TRANSFER OF PRO-APOPTOTIC GENES Cells with multiple genetic alterations are usually eliminated by apoptosis. For survival, they depend upon the overexpression of antiapoptotic molecules like bcl-2, bcl-xL or survivin. The downregulation of such proteins may reduce the apoptotic threshold of cells. The 2 major apoptotic signaling pathways are 1/ the mitochrondrial pathway and 2/ the death receptor pathway. Cells having condensed and fragmented chromatin demonstrate apoptotosis.
TRANSFER OF GENES Many systems have been used for the administration of genes in the treatment of cancer such as use of adenovirus and associated viruses, poxvirus, herpes simplex, but all of these can provoke an immune responce against the vector, in the manner required to use different strains or different routes of administration. An ideal technique for genic transfer should be nontoxic and have great efficiency for various types of cells with 1 or more genes for which the transfer process is selective.
NONVIRAL SYSTEMS Due to the low efficiency of the transfection of naked DNA, various methods have been used for increasing the efficiency like microinjection, electroporation and precipitation with calcium, but unfortunately these methods are usually inapplicible in vivo and generally result in a transient expression of the gene. The efficiency can be raised by using a gen gun to incorporate the DNA by bombarding the skin.
VIRAL SYSTEMS In comparison with the previous systems, this type of system shows an efficiency of better tranduction both in vitro and in vivo. The viral vectors used the most are defective retroviruses, adenoviruses and ones associated with adenoviruses. Others are herpes simplex, vaccinia, avipox & baculoviruses. Among the disadvantages encountered in the use of viruses as vectors are toxicity, the production of foreign transient proteins and limitations in the size of the gene to be transferred. The virus vectors most widely used with adenoviruses are easy to produce in vitro efficiently and with surety.
TRANSFER OF GENES IN LUNG CANCER AEROSOL TECHNIQUE Most drugs and DNA complexes have been administered by conventional routes: oral or intravenous. The biodistribution of drugs by means of these strategies are diseminated and the quantity that is deposited in the lung is small. Another important aspect to consider in these types of systems is the toxicity that is observed after injection. The ability to express trangenes in the lung in a selective manner can facilitate the development of genic therapy for a variety of human diseases.
Polyethylene amine (PEI) is a polycation able to condense DNA and to protect against the degradation of DNAases así como de liberarlo in vitro and in vivo, this polymer forms polyionic complexes with DNA to establish electrostatic cooperative interactions with its ammonium groups and the phosphate groups of the DNA. More, it is possible to reach high levels of gene expression if human albumin, albumin from mouse serum or ovialbumin and human igG are added to this compound.
Furthermore, liposomes formulated with 9-nitrocamptotecine (9NC-DLPC), which is an inhibitor of topoisomerase 1 and inhibits the growth of subcutaneously induced tumors like the metastasis of lung cancer in the murine model, observing a synergistic effect through the combination of the administration of gene p53 and PEI. Recently, the expression of Wilms' tumor gene (WT1) has been encountered in lines lung cancer cells in 5/11 (45 %) by the test for reverse transcriptase polymerase chain reaction (RT-PCR), 2/5 (40 %) in SLCL and in 3/6 of NSCLC. In another study, WT1 overexpression was found in 12/15 cell lines of lung cancer. The protein of WT1 is an attractive target for immunotherapy.
STIMULATION OF THE IMMUNE SYSTEM BY WT1 Recent advances in immunology and molecular biology have permitted the identification of many antigens associated with tumors (TAAs), and the episomes that are recognized by cytotoxic HLA class I T lymphocytes in different kinds of neoplasms. One of the TAAs identified is the product of gene WT1 that allows the possibility of slowing down the cancer based on administration of this peptide.
In a phase I clinical study, Tsuboi et al. (Microbiol Immunol 48: 175-84, 2004) applied immunotherapy based upon WT1 by intradermal injection of 3 mg of the peptide of WT1 of 9 bases that stimulated HLA-A2402, the vaccination in an interval of 2 weeks, resulting in the reduction of the CEA and xSLX tumor markers and a transient decrease in the tumor size being the primary clinical evidence that shows that vaccines against WT1 are an alternative for pulmonary cancer patients.
The future Lung cancer is one of the most aggressive and fatal neoplasms. Today it is possible to diagnose earlie, and we have an improved panel of anticancer drugs. Nevertheless, less than 15 % of patients survive more than 5 years. The most recent trend is to explore genic therapy and therapeutic vaccines as alternatives for treatment. With these new tools perhaps the patient's life can be prolonged reducing the cost of treatment.