Affiliations of authors: H. Yu, Section of Cancer Prevention and Control, Feist-Weiller Cancer Center, Louisiana State University Medical Center, Shreveport; M. R. Spitz, J. Gu, X. Wu (Department of Epidemiology), W. K. Hong (Department of Thoracic/Head and Neck Medical Oncology), The University of Texas M. D. Anderson Cancer Center, Houston; J. Mistry, Diagnostic Systems Laboratory, Inc. Webster, TX.
Correspondence to: Xifeng Wu, M.D. Ph.D., Department of Epidemiology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Houston, TX 77030.
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ABSTRACT |
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INTRODUCTION |
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The interaction between IGFs and IGF-IR is regulated by the IGF-binding proteins (IGFBPs). Six IGFBPs (IGFBP-1 to IGFBP-6) with high affinity for IGFs have been identified and characterized (2). The binding proteins normally inhibit the action of IGFs by blocking the binding of IGFs to their receptor; however, under certain circumstances, they can enhance IGF action by protecting IGFs from degradation (7-9). The dual regulatory effects of the IGFBPs are further modulated by many factors including the IGFBP proteases, which include prostate-specific antigen (PSA) and cathepsin D (2,10,11). Cell culture studies indicate that the antiproliferative effects of retinoic acid (a metabolite of vitamin A) and of wild-type p53 protein are mediated through increased expression of IGFBP-3, which in turn inhibits the mitogenic effect of IGFs on cell proliferation (12-15).
Cell culture experiments (16-19) have demonstrated that most lung cancer cell lines (small-cell and non-small-cell) are able to express IGFs and their binding proteins. Although IGFs are known to be potent mitogens for lung cancer cells and are present in lung tissue, evidence that IGFs can influence the development of lung cancer remains unknown. To examine the hypothesis that IGFs and their major binding protein in plasma play a causal role in lung cancer, we compared plasma levels of IGF-I, IGF-II, and IGFBP-3 in patients with newly diagnosed lung cancers and in age-, sex-, race-, and smoking status-matched control subjects.
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MATERIALS AND METHODS |
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The patients and control subjects were selected consecutively from an ongoing case-control study of lung cancer conducted in the Department of Epidemiology at The University of Texas M. D. Anderson Cancer Center. The study subjects were described in detail elsewhere (20). Briefly, the case subjects were consecutive patients with lung cancer registered in the Departments of Thoracic Surgery and Thoracic Medical Oncology at The University of Texas M. D. Anderson Cancer Center. These patients were newly diagnosed with histologically confirmed primary lung cancer. However, histologic rereview is not completed. They had been referred for diagnosis or definitive treatment and had received no previous radiotherapy or chemotherapy. After the patients were informed about the study and agreed to sign an informed consent form for participation, an in-person interview with the use of a structured questionnaire was scheduled.
The control subjects were identified from a control-pool database established from registrants of a large, private, multispecialty health care provider, Kelsey-Seybold Clinic, which involves a health maintenance organization, managed care, and fee-for-service patients in the Houston metropolitan area. There are more than 40 000 individuals enrolled in our potential control database. Control subjects were frequency matched to the case patients by sex, age (within 5 years), and ethnicity (white, black, or Hispanic), with a 1 : 1 ratio. Each randomly selected control subject was contacted by telephone to confirm his or her willingness to participate, and an appointment was scheduled at a Kelsey-Seybold Clinic site convenient to the participant. If the person refused to participate or was deemed ineligible, another potential control subject was selected. Since the study is ongoing and control subject selection is not conducted concurrently with case patient accrual, perfect 1 : 1 matching has not yet been achieved. Furthermore, some subjects did not have sufficient plasma specimens available for the study. Therefore, there are some discrepancies among the matching variables between case patients and control subjects. We adjusted these differences in our data analysis. There are no differences in consent rates between case patients and control subjects. The study was approved by the Institutional Review Boards at The University of Texas M. D. Anderson Cancer Center and the Kelsey-Seybold Foundation.
Specimen Collection
After the interview, 10-mL blood specimens were drawn from each participant through venipuncture. The blood was collected in a heparinized tube and transported immediately to the laboratory, where the specimens were separated and processed. The plasma was collected after centrifugation of the blood at 1500 rpm for 10 minutes at room temperature and was stored at -80 °C. To assess the degradation of IGF-I and IGFBP-3 in stored plasma, a previous study compared levels of IGF-I and IGFBP-3 in stored heparinized plasma and in fresh specimens. No difference was found between the two types of specimen (21).
Measurements of IGFs and IGFBP-3
Three commercially available immunoassay kits (DSL, Webster, TX) were used in the study to determine the plasma levels of IGF-I, IGF-II, and IGFBP-3 through enzyme-linked immunosorbent assay. Cross-reaction of the antibodies with other members of the IGF family is not detected at physiologic concentrations, according to the manufacturer. The intra-assay and inter-assay precision is between 4.5%-8.6% and 3.3%-6.8% of the coefficient of variation, respectively, for the IGF-I assay; between 3.4%-6.7% and 5.9%-7.9% for the IGF-II assay; and between 7.3%-9.6% and 8.2%-11.4% for the IGFBP-3 assay.
The assays were performed following the instructions of the manufacturer (DSL) and without knowledge of case-control status. To separate IGFs from their binding proteins, we mixed plasma specimens with acid-ethanol extraction buffer before measurement. The extraction procedure has been evaluated, and the efficiency of the extraction was identical to that for acid-column chromatography. For IGFBP-3, the specimens were diluted 100-fold in an assay buffer before the test. To assess the impact of freeze-thaw cycles on the values of IGF-I, IGF-II, and IGFBP-3 in heparinized plasma, we measured each of 10 plasma specimens once per freeze-thaw cycle for five cycles. Levels of IGF-I, IGF-II, and IGFBP-3 in plasma remained constant over these freeze-thaw cycles.
Statistical Analysis
The correlations among the three growth factors were examined by use
of the Spearman correlation coefficient. The distributions of the
studied variables between the case patients and control subjects were
compared by use of the 2 test for categorical data and
the two-sample Student's t test for numerical data. All
P values were two-sided. Associations were considered
statistically significant at P<.05. Since the distributions
of IGF-I and IGFBP-3 in the population were positively skewed, the
levels of IGFs and IGFBP-3 were analyzed categorically on the basis of
their quartile distribution in the control group (Table
1).
To assess the strength of the association
between lung cancer risk and the growth factors, we calculated the odds
ratio (OR) and its 95% confidence interval (CI) with the use of
unconditional logistic regression analysis (22). The logistic
regression model was developed as both univariate and multivariate
models. In the multivariate analysis, the following variables were
included in the model: sex, age, ethnicity (white, black, or Hispanic),
cigarette smoking status (never, former, or current), body mass
index (BMI = kg of body weight/m2 of height), and family
history of any cancer (yes or no in their first-degree relatives). The
interactions between IGF-I and IGF-II and between IGFs and IGFBP-3 were
also examined in the logistic regression model by use of the product of
the two given variables.
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RESULTS |
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Table 2 summarizes the categorical distributions of
the three IGF variables together with other variables measured in the
two study populations. Because the control subjects were selected to
match the patients on sex, age, race, and cigarette smoking status, no
statistically significant differences were observed between the two groups for
these variables. The BMI was slightly higher in the control subjects than in
the patients, and the difference was statistically significant (P =
.03). Patients in the highest fourth quartile of IGF-I level made up 36.3%
of the total, compared with 24.8% of control subjects (P = .04).
For IGF-II and IGFBP-3, there were no differences between patients and control
subjects in the quartile distributions.
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The distribution of the case patients and control subjects within the
four categories of IGF-II did not differ (P = .75, Table 2).
In the logistic regression analysis, the risk of lung cancer was
modestly elevated in the highest quartile compared with the lowest
quartile of IGF-II, but the difference was not statistically
significant (OR = 1.33; 95% CI = 0.77-2.31; P = .31,
Table 3
). When we adjusted for IGF-I and IGFBP-3 and their interactions
in the model, we found no significant association between IGF-II and
disease risk (data not shown).
There was no association between cigarette smoking status (never, former, or current smoker) and levels of IGFs and IGFBP-3 among the control subjects (data not shown). We also examined pack-years of smoking, duration of smoking, and the total number of cigarettes smoked in relation to plasma levels of IGF-I, IGF-II, and IGFBP-3. None of the correlations analyzed were shown to be significant (data not shown), suggesting that levels of IGFs and IGFBP-3 in plasma were not influenced by cigarette smoking.
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DISCUSSION |
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Recently, two prospective studies reported higher plasma levels of IGF-I in association with increased risks of prostate cancer in men (21) and of breast cancer in premenopausal women (23). We were impressed with the striking similarities between these studies and our own, although three different types of cancers were investigated and our study was a retrospective analysis. There was a substantial association between IGF-I levels in plasma and risks of all three cancers. All three studies consistently showed a strong, dose-response relationship between increased risks of these cancers and elevated levels of IGF-I. The effect of IGF-I tended to be more significant when adjustment was made for levels of IGFBP-3 in the analyses. For prostate and lung cancers, IGFBP-3 also showed some protective effects; however, by itself, IGFBP-3 did not demonstrate such an effect. Also, for both prostate and lung cancers, no association was found for IGF-II.
The consistency of the findings for IGF-I prompts us to speculate that IGF-I either may have a carcinogenic effect or may be a powerful growth promoter and that circulating IGF-I levels may serve as a biomarker for assessing lung cancer risk. It may also be possible that an increased plasma IGF-I level is part of the phenotype of certain types of cancer that require IGF-I to maintain their high rate of proliferation and growth. Results from cell culture studies and animal experiments have suggested that IGF-I is a potent mitogen for a variety of cancer cells, including breast, prostate, lung, colon, and liver cells (1,24-26). IGF-I increases DNA synthesis and up-regulates the expression of cyclin D1, thereby accelerating the cell cycle from G1 to S phase (27). While stimulating cell proliferation, IGF-I also shuts down the apoptotic pathway (3,4). Because the actions of IGF-I are mediated through the IGF-IR, removing the receptor from the cell membrane could abolish its mitogenic and apoptotic effects (2,28,29). In addition, IGF-IR is involved in cell transformation, and interruption of IGF-IR expression on the cell membrane blocks cell transformation induced by a tumor virus or an oncogene product (28).
The interaction between IGF-I and IGF-IR is regulated by the IGFBPs. In the univariate analysis, of two of the studies cited above, this protein failed to show any association with the risk of prostate or lung cancer. However, when analyzed together with IGF-I, IGFBP-3 appeared to be associated with a reduced risk of both prostate and lung cancers, but the binding protein also appeared to enhance the associations between risk of these cancers and plasma IGF-I level. These observations in epidemiologic studies are compatible with the results from in vitro and in vivo studies, demonstrating that IGFBP-3 suppresses the mitogenic and apoptotic effects of IGF-I on cancer cells. This suppression is explained by the fact that IGFBP-3 prevents the interaction between IGF-I and IGF-IR because of IGF-I's higher binding affinity for the binding protein than for the receptor. Recent experiments (30) also suggest that IGFBP-3 may inhibit cell growth independently of IGF-I.
The relationship between IGF-I and IGFBP-3 in lung cancer may shed light on the action of two antiproliferative molecules whose effects have been studied in lung cancer, retinoic acid and p53. Mutation of the p53 tumor suppressor gene (also known as TP53) has been linked to the development of many cancers, including lung cancer (31). One of the main functions of the p53 protein is to slow down cell divisionwhich allows cells to repair DNA damage or to initiate apoptosis if the damage is irreversible. The suppression of cell division by p53 is speculated to be mediated through IGFBP-3, because wild-type p53 protein is shown to increase IGFBP-3 expression. IGFBP-3 subsequently suppresses the mitogenic effect of IGF-I, which results in the inhibition of cell proliferation (13). The possible link between IGF-I and p53 is further supported by an observation that the function of p53 protein is suppressed by IGF-I. As a transcription factor, p53 protein must be intranuclear to exert its action. When cells undergo division induced by IGF-I, p53 protein is expelled from the nucleus (32). In addition, p53 protein down-regulates the expression of IGF-IR (15). The growth of bladder tumors induced by p-cresidine in p53-deficient transgenic mice was suppressed by decreasing serum levels of IGF-I through diet restriction, and restoring IGF-I levels in serum resulted in resumption of tumor growth and progression (33). This study also indicated that tumor growth control by IGF-I was related to IGF-I's mitogenic and anti-apoptotic effects.
Cell culture studies (12,14,34) have found that retinoic acid stimulated the production of IGFBP-3, which in turn inhibited the action of IGF-I. Findings from our study support such a relationship between IGF-I and IGFBP-3 and, furthermore, indicate that monitoring changes in IGFBP-3 and IGF-I levels in the blood may help to evaluate the effectiveness of vitamin supplements as chemopreventive agents.
In our study, the BMI was lower in the case subjects than in the control subjects, and the difference was statistically significant (P = .03). However, this difference should not have any impact on the association between IGF-I and lung cancer risk, because the ORs did not show substantial changes when we adjusted for BMI in the analysis. Furthermore, no correlation between IGF-I and BMI has been observed in previous studies (19,35,36). Because this is a case-control study, findings from our study need to be further confirmed by prospective cohort studies. Nevertheless, similarities between our study and two cohort studies on different cancer sites lend support to our speculation that IGF-I may be involved in the disease's development. If our observations can be confirmed in prospective studies, the measurement of plasma levels of IGF-I and IGFBP-3 will have potential utility in assessing lung cancer risk and/or in monitoring the effectiveness of chemoprevention interventions.
Supported by Public Health Service grants U19CA68437 (to W. K. Hong), R01CA55769 (to M. R. Spitz), and 1R03CA70191 (to X. Wu) from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. Dr. Hong is an American Cancer Society Clinical Research Professor.
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Manuscript received May 29, 1998; revised November 4, 1998; accepted November 17, 1998.
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