Neoplastic diseases, including most: particularly the collection of diseases known as cancer, are major cause of mortality and morbidity of human and are the most difficult disease to treat. Although medical science and natural science has recently advanced so much, cancer still remains unresolved problem. In United States of America, cancer is surpassed only by cardiovascular diseases as the primary cause of adult death, one million and three hundred thousands of new cases of cancer develop yearly and five hundred and fifty thousands of men die of cancer every year. This means that one of every 2 or 3 American people falls victim to cancer. The four major cancers in United States of America include lung cancer, colorectal cancer, prostate cancer and breast cancer, and the risk of American people to get these 4 major cancer are shown in Table 1 (Bang Y J et al. Cancer: Current Diagnosis and Therapy. Hanuri Company:Seoul, 1999;69-107)
TABLE 1The risk of American men to get four major cancers (fromNational Cancer Institute of United States, SEER Data)Risk of gettingcancerRisk of dying ofType of cancerSex(%)cancer (%)Lung cancerMale8.67.1Female5.44.2ColorectalMale6.22.6cancerFemale5.92.6ProstateMale18.53.6cancerBreast cancerFemale12.63.6
The mechanism of development of human cancer is being clarified in more detail owing to advances of molecular biology and genetics; especially human genome project, functional genomics, nanotechnology and bioinformatics. Cancer is genetic disease, ie. Cancer develops secondary to genetic abnormality. Acquired genetic abnormality secondary to chemical carcinogen, UV light, irradiation or virus and hereditary genetic abnormality induces change (ie, mutation) into genetic information (DNA, RNA) of genome. When these mutation activate oncogenes and inactivate tumor suppressor genes, cancers may develop. Oncogene and tumor suppressor genes play key roles in regulation of signal transduction, cell cycle progression, cellular death and survival, accommodation with neighbor cells and angiogenesis is. Oncogenes induce proliferation, survival and escape from death, invasion of adjacent tissues and angiogenesis and thus stimulate development of cancer, whereas, tumor suppressor genes counteract oncogenes and thus inhibit development of cancer (Evan G et al. Matter of life and cell death, Science (1998) 281, 1317-1322; Harrington E A et al. Oncogene and cell death. Curr Opin Genet Dev (1994) 4, 120-129).
During the past twenty years, many medical scientists have focused on oncogenes and tumor suppressor genes and have tried to find genetic markers of cancer and tumor markers through investigation of oncogenes and tumor suppressor genes. Through these research, they have found important genes such as p53, Rb, p16 and other CDK inhibitors, which regulate cell cycle and cellular apoptosis (Macleod K et al. Tumor suppressor genes. Curr Opin Genet Dev (2000) 10, 81-93; Adams P D et al. Negative control elements of the cell cycle in human tumors. Curr. Opin. Cell. Biol. (1998); 10, 791-797), BRCA1 and BRCA2 which are closely related with hereditary breast cancer and hereditary ovarian cancer (Miki Y et al. A strong candidate for the breast and ovarian susceptibility gene BRCA1, Science (1994) 266, 66-71); Wooster R et al. Identification of the breast cancer susceptibility gene BRCA2. Nature (1995) 378, 789-792), and APC gene which is closely related with hereditary colorectal cancer (Kinzier K, et al, Lessons from hereditary colorectal cancer, Cell(1996) 87, 159-170). These findings, had greatly contributed to progress of cancer research. In addition, these research stimulated establishment of many research centers which tested specific gene mutation on a commercial basis, in particular, BRCA1 and BRCA2 in women with high risk of development of breast cancer and ovarian cancer owing to family history (Levine A J. p53, the cellular gatekeeper for growth and division, Cell(1997), 88, 323-331; Frank T S. Laboratory. identification of hereditary risk of breast and ovarian cancer, Curr. Opin. Biotech. (1999) 10, 289-294).
However, ideal genetic marker remains not to be found for acquired solid tumors which constitute most of human cancers. In addition, no molecular marker common to all human cancer has been found so far. It is p53 gene that shows highest frequency of mutation in all forms of human cancer, but even for p53 gene, the frequency of mutation or deletion is only 30 to 50%, which suggests that p53 is inappropriate for use as a molecular diagnostic marker of human cancer in clinical practice (Levine A J et al. p53, the cellular gatekeeper for growth and division, Cell (1997) 88, 323-331). Oligonucleotide DNA chip which detects mutation of p53 gene has recently been tested in patients with lung cancer, but only 40% of the lung cancer tissues showed mutation of p53 gene, which indicates limitation of analysis of mutation of p53 gene as a diagnostic tool of lung cancer (Ahrendt S A et al. Rapid p53 sequence analysis in primary lung cancer using an oligonucleotide probe array, Proc Natl Acad Sci U.S.A (1999) 96, 7382-7387). So far, no marker has been found to be of practical value for the clinical management of lung cancer, stomach cancer, colorectal cancer and breast cancer which form more than 50% of all human cancers.
Affymetrix company (www.affymetrix.com) has recently manufactured Human Cancer G110 Array, a new type complementary DNA(cDNA) chip which detects expression of about 100 oncogenes and tumor suppressor genes which have been found so far. However, it is questionable whether this DNA chip pan detect all human cancer, due to the fact that the genes found so far are at most 5-10% of the genes related to all human cancer.
Despite progress of surgery, chemotherapy, radiation therapy and immunotherapy, the success rate of treatment of human cancer except some hematologic cancer and childhood malignancies has not been remarkably improved during last several decades. The main reason for the poor treatment outcome of human cancer lies in the delayed diagnosis of cancer in advanced status when it has already metastasized and cure is hard to attain rather than limited efficacy of current therapy for cancer. Nowadays the prevention of cancer takes a key place in clinical science as does treatment of cancer.
Primary method of cancer prevention is so called chemoprevention which aims to delay or inhibit multistep development of cancer by change of life style, diet or drugs. The chemoprevention is appropriate in particular for asymptommatic people with high risk of cancer because of family history or past medical history of cancer. For example, a clinical study of chemoprevention is under way to administer retinoic acid to patients in status of long term remission from lung cancer after therapy. However, we still do not exactly know either the etiology of cancer (except smoking) or effective chemopreventive drugs, and we have no reliable marker to identify the efficacy of chemopreventive agents, all of which limit the practical value of chemoprevention of cancer.
The secondary method of cancer prevention is early detection or screening of cancer. The fate of individual cancer patient, ie. cure rate and long term survival, is primarily determined by volume and stage of tumor at the time of diagnosis; The cure rate and survival rate is highest among cancers in stage 1 or stage 2. In fact, we can expect cure of cancer only when it is diagnosed in early stage, ie. stage 1 and/or stage 2. Therefore, medical society makes every effort to detect cancer from the general public in early stage. Screening methods of cancer include inspection (skin, oral cavity, external genitalia, uterine, cervix), palpation (breast, oral cavity, thyroid, anus and rectum, prostate, testicle, uterus, lymph nodes), clinical chemistry tests such as, Papanicolaou smear and tumor markers including serum prostate specific antigen (PSA) or α-feto protein, radiologic study such as barium enema study of colon, chest X ray, and endoscopic examination. Table 2 shows the cancer screening methods recommended by American Cancer Society.
TABLE 2Cancer screening methods: Guideline recommended byAmerican Cancer Society (1993).TargetScreeningAge of screeningScreeningCancermethodSexpopulationfrequencyProstateDigital rectalMale50 years or afteryearlycancerexaminationSerumMale50 years or afterYearlyPSA assayBreastSelfFemale20 years or aftermonthlycancerexaminationClinicalFemale20-40 years/Every 3breast40 years oryears/examinationafterYearlyMammographyFemale50 years or afterYearlyColorectalStool occultMale and50 years or afterYearlycancerblood testfemaleColonoscopyMale and50 years or afterEvery 3 tofemale5 yearsUterinePap smearFemale18 years or afterYearlycervixPelvixFemale18-40 years/Every 1-3cancerexamination40 years or afteryears/YearlyEndometrialFemalePostmenopausal,DependingbiopsyHigh risk womenon doctor'srecom-mendationLungChest X rayNot recommended as a routine studycancerSputumcytology
The cancer screening tests listed in Table 2 have been shown actually to improve the treatment outcome of target cancers. In particular, serum PSA assay is widely used for the screening, diagnosis, follow up after therapy of prostate cancers (Rimer B K et al. Cancer Screening. In DeVita V T Jr, Hellman S, Rosenberg S A. eds. Cancer. Principles and Practice of Oncology. fifth ed., Lippincott-Rave:Philadelphia, 1997; 619-631).
Detection of expression of specific gene in blood has recently been used to identify specific cells and diagnosis of specific diseases, especially cancer. For example, detection of benign or malignant prostatic epithelial cells which express PSA or prostate specific membrane antigen (PSMA) from blood by using reverse transcription, polymerase chain reaction (RT-PCR) assay has been shown to be of value for the staging of cancer, ie. detection of metastatic cancer (which has been called molecular staging) as well as diagnosis of prostatic cancer. Presence of cancer cells within blood does not indicate metastasis by itself, but highly suggests metastasis (Katz A E et al. Molecular staging of prostate cancer with the use of an enhanced reverse transcriptase-PCR assay; Israeli R S et als. Sensitive nested reverse transcription polymerase chain reaction detection of circulating prostatic tumor cells: comparison of prostate specific membrane antigen and prostate specific antigen-based assays. Cancer Research (1994) 54: 6306). However, no pan-tumor molecular marker has been found so far which show abnormality in most human cancers and thus is of practical value for the diagnosis and staging of cancer in clinical practice.
Lung cancer ranks the first of all human cancers both in the incidence and death rates in United States of America: About 180,000 new cases of lung cancer develop yearly, about 160,000 patients die of lung cancer and overall 5-year survival rate of patients with lung cancer is only around 10%. Most of human lung cancers are bronchogenic carcinomas, which is primarily classified into small cell carcinoma and non-small cell carcinoma. The small cell carcinomas are a single type, while the non-small cell carcinomas consist of adenocarcinoma, squamous cell carcinoma, large cell carcinoma, bronchioalveolar carcinoma. Primary cause of lung cancer is smoking and the amount and duration of smoking is directly correlated with incidence and death rates of lung cancer.
The risk of getting lung cancer increases twenty-folds and risk of death of lung cancer becomes 13% on smoking 25 cigarettes daily for 10 years. Of remark is that the risk of lung cancer increases not only in primary or direct smokers but also in secondary or indirect smokers. The screening for lung cancer is indicated both in primary smokers and secondary smokers and also in men who had been exposed to lung carcinogen such as asbestos.
Classical methods for the screening of lung cancer include chest radiography (simple X ray) and sputum cytology examination, however the former and the latter has a diagnostic sensitivity for lung cancer of only 30% and 40-60%, respectively. It is hard for these two studies to detect lung cancer in early stage and to significantly improve treatment outcomes of lung cancer, and for this reason these two studies were excepted from a list of recommended screening tests for cancers by American Cancer Society.
Owing to lack of effective screening methods, ninety percent of cases of lung cancer are nowadays diagnosed in, advanced status(stage III or IV), in which cases most of the patients die within 2 years after diagnosis and 5 year survival rate is less than five percent despite aggressive chemotherapy and irradiation therapy (Choi S J et al. eds. Lung: neoplasia and cancer. In Current Diagnosis and Therapy. Han-Uri Publishing Co.:Seoul. 1999;323-332). In contrast, if lung cancer can be detected by screening study in occult carcinoma status when patient has no symptoms and radiologic study of the lung shows no cancerous lesion, the cure rate of cancer is more than eighty percent. In fact some reports showed evidence that treatment outcomes of lung cancer are remarkably improved even by limited classical screening study of chest radiography and sputum cytology examination and that there were significant differences in 5 year survival rate between lung cancers detected by screening study (35%) and those diagnosed by lung cancer-related symptoms (13%) (Berlin N I et al. Early lung cancer detection: Summary and conclusions, American Review of respiratory diseases (1984) 30, 565).
Diagnostic study and follow up study after therapy of lung cancer leaves much room for improvement. Accurate diagnosis of lung cancer is in reality not easy. It is hard to detect early stage lung cancer by chest X ray and sputum cytology examination and, even after detection of lung mass, it is not easy to differentiate between lung cancer and benign lung mass and between primary lung cancer and metastatic lung cancer. Definitive diagnosis of lung cancer is usually made by bronchoscopic biopsy, brush biopsy, bronchoalveolar lavage cytology examination, percutaneous needle aspiration cytology examination, mediastinoscopic biopsy, lymph node biopsy or pleural biopsy, but sometimes requires even open lung biopsy. The diagnosis is often ambiguous even after radiologic study and biopsy, in particular for solitary pulmonary nodules with diameter of less than 5 mm as being important in clinic (Ginsberg R J et al. Cancer of the lung. Section 2. Non-small cell lung cancer. In DeVita V T Jr. Hellman S, Rosenberg S A. eds. Cancer. Principles and Practice of Oncology, 5th ed., Lippincott-Raven: Philadelphia, 1997; 858-910).
The next step after diagnosis of lung cancer is staging work up, ie., study of extent of cancer. The conventional staging methods for lung cancer include: computerized tomography (CT) scan, bronchoscopy, thoracoscopy, mediastinoscopy, and biopsy and cell examination using them, but all of these methods have limited accuracy and endoscopic studies are invasive. Lung cancer commonly invades pleura band and thus induce pleural effusion, in which cases, pleural fluid cytology examination and/or pleural biopsy are performed to identify the cause of pleural effusion, but reveals definitive diagnosis in only about half of the cases. Therefore, staging method for lung cancer definitely leaves much room for improvement.
The appropriate follow up study is essential after therapy for lung cancer which can accurately define the results of therapy, detect residual or recurrent cancer in a sensitive and rapid way. The current follow Up study of lung cancer include radiologic study such as CT scan and endoscopic examination, but it is almost impossible to detect microscopic residual or recurrent cancer by these study. Therefore, novel method for follow up of lung cancer is urgently necessary.
Appropriate maintenance of membrane water permeability is a fundamental requirement of all living organisms. Aquaporin (AQP) is a family of water channel proteins of membranes through which water are transported into and out of cells. AQP exists in all type of living organisms which include microorganisms, plants, mammalians. Ten types of mammalian AQP, ie., from type 1 to type 10 AQP, have been identified so far, whereas, more than 100 types of AQP exist in plants in which transport of water are more critical for the survival than in mammalians. AQP1 was the first type to be isolated in erythrocytes. Human type AQP1 was cloned for the first time by one of the present inventors (Moon C et als. Cloning of human aquaporin 1 gene, J Biol Chem (1993) 268, 15772-15778). Two functional groups of AQP are now being recognized. The first, including AQP1, AQP2, AQP4, AQP5, AQP6, AQP8 and AQP10 are permeable only to water, as classically defined. A second group, including AQP3, AQP7 and AQP9 are highly permeable to water, but also are permeable by glycerol (King L S et al. Aquaporin in health and disease, Molecular Medicine Today (2000) 6, 60-65). The present inventors have recently found the evidence that AQP also plays important roles in cell cycle regulation, signal transduction, delayed early response to growth factors, and gas exchange in hypoxic condition.
AQP proteins exist in cell membranes, and to adapt to water channel function, its structure has six transmembrane domains and five connecting loops (loop A-E). The. Amino terminal (NH2 terminal) and carboxy terminal (COOH terminal) portion of AQP are located inside cytoplasm. Loop B and E of AQP contains signature motif Asn-Pro-Ala, which is called NPA, and adjacent cysteine. Two NPA motifs and cysteine combine to become center of the water channel (Walz T et als. Three-dimensional electron density map of human aquaporin 1 at 6 A resolution. Nature (1997) 387, 624-627; Lee M D et al. The human aquaporine-5 gene. J. Biol. Chem (1996) 271, 8599-8604).
Each type of 10 mammalian aquaporins has a distinct tissue and cellular distribution and plays a diverse and specific role depending on the type of tissues and cells where it is located. AQP1 is located in erythrocytes, kidney, lung, eye, choroid plexus, biliary tract, nonfenestrated endothelia. AQP1 is abundant in proximal tubules and descending thin limb of Henle's loop segments, actively reabsorbs most of glomerular, filtrate and thus greatly contributes to concentration of urine. AQP2 is located in collecting duct epithelia of kidney, secreted in response to stimulation of antidiuretic hormone and thus contribute to concentration of urine. Deficiency of AQP2 produces nephrogenic diabetes insipidus which is characterized by failure to concentrate urine. AQP3 is located in renal collecting duct, gastrointestinal tract, airway epithelia, corneal epithelium and brain. AQP4 is abundant in glial cells and ependymal cell of brain tissue, but is also located in retina and airway epithelia. AQP5 is located in salivary gland, lacrimal gland and lung, in which plays an important role in production of saliva, tear and airway secretions. AQP6 is located in proximal tubular epithelia and collecting duct epithelia of kidney and characteristically acts as intracellular water channel and also is involved in regulation of acid base balance. AQP7 and AQP8 are expressed in germ cells and sperms. AQP9 is abundant in adipocytes (Deen P R T et al. Epithelial aquaporins., Current Opinion in Cell Biology (1999) 10, 435-442; King L S et al. Aquaporin in health and disease, Molecular Medicine Today (2000) 6, 60-65; Agre P. Aquaporin water channels in kidney. J. American Society of Nephrology (2000) 11, 764-777).
AQP plays important roles particularly in kidney, lung, brain, eye and eythrocytes. The lung has exceptionally high epithelial and endothelial permeability. Appropriate removal and supply of water in the airway, vascular and interstitial compartments of the lung are essential for normal gas exchange and lung defence. AQP is actively involved in the maintenance of liquid layer of surface of airway epithelia, which is essential for normal mucosal ciliary action, and also involved in appropriate supply of water to airway which prevents dehydration of airway and ensures adequate dehydration of expired air. Four water channels, including AQP1, AQP3, AQP4 and AQP5 have been indentified in the lung of rats and mice. AQP1 (Genebank No. NM-000385) is abundant in apical and basolateral membrane, of microvasculature and pleural membrane. AQP5 (Genebank No. NM-001651) is abundant in apical membrane of type 1 alveolar pneumocytes and secretory cells of airway submucosal gland. AQP3 (Genebank No. NM-004925) and AQP4 (Genebank No. U63623) are expressed in epithelial cells of airway and nasopharynx. AQP is also reported to be involved in CO2 exchange of alveolar cells, which suggest that AQP may act as a gas channel (Nielsen S et al. Aquaporin in complex tissue II., Cellular and subcellular distribution in respiratory tract and glands of rat., American J. Physiology (1997) 273, 1549-1561; King L S et al. Aquaporin-1 water channel protein in lung: ontogeny, steroid-induced expression, and distribution in rat., J. Clin Invest (1996) 97, 2183-2191). However, distribution and function of each type of AQP in the human lung remain to be indefinite. In addition, role of AQP in human cancer, in particular lung cancer, remains to be indefinite.
Considering the prior art up to now, there is a need for the development of new tumor markers, which is useful for screening, diagnosis, and follow-up study after treatment for human cancer including lung cancer.