|Year : 2015 | Volume
| Issue : 6 | Page : 125-129
Prognostic impact of CUG-binding protein 1 expression and vascular invasion after radical surgery for stage IBnonsmall cell lung cancer
J Zhao, Y Zhao, Y Xuan, W Jiao, T Qiu, Z Wang, Y Luo
Department of Thoracic Surgery, The Affiliated Hospital of Qingdao University, Qingdao 266003, P. R, China
|Date of Web Publication||24-Dec-2015|
Department of Thoracic Surgery, The Affiliated Hospital of Qingdao University, Qingdao 266003
Source of Support: None, Conflict of Interest: None
Background: Nonsmall cell lung cancer is the leading cause of cancer mortality worldwide because of distant metastasis and frequent recurrence. Only few reliable and easily accessible tumor markers have been clinically implemented to the early nonsmall cell cancer prognosis. OBJECTIVE: The purpose of this study is to detect the expression of CUG-binding protein (CUGBP1) and assess the prognostic significance of CUGBP1 in early stage (IB) lung adenocarcinoma patients. Materials and Methods: Using quantitative reverse transcription-polymerase chain reaction (PCR) and immunohistochemistry (IHC) analysis, we detect the expression of CUGBP1 and assess their correlation with clinicopathological parameters by Chi-square test. Time to progression (TTP) was used as a recurrent index and was evaluated by univariate and multivariate analysis in the Cox hazard model. Results: Using PCR and IHC analyses, the expression of CUGBP1 and CUGBP1 messenger RNA (mRNA) had a close relationship with differentiation and vascular–invasion (VI). However, there were no significant differences between the CUGBP1 mRNA expression and CUGBP1 protein expression in IBlung adenocarcinoma. Using univariate and multivariate survival analyses, we found that CUGBP1 and VI were independent prognostic factors for IBstage adenocarcinoma individuals postsurgically. Conclusions: High expression of CUGBP1 could enhance the recurrence rate of adenocarcinoma and predicts an adverse postsurgical survival of TTP. Combination of CUGBP1 and VI detecting could be considered as indication to predict prognosis of IBstage adenocarcinoma in the clinical trial.
Keywords: CUG-binding protein 1, lung adenocarcinoma, prognosis, recurrence, vascular-invasion
|How to cite this article:|
Zhao J, Zhao Y, Xuan Y, Jiao W, Qiu T, Wang Z, Luo Y. Prognostic impact of CUG-binding protein 1 expression and vascular invasion after radical surgery for stage IBnonsmall cell lung cancer. Indian J Cancer 2015;52, Suppl S2:125-9
|How to cite this URL:|
Zhao J, Zhao Y, Xuan Y, Jiao W, Qiu T, Wang Z, Luo Y. Prognostic impact of CUG-binding protein 1 expression and vascular invasion after radical surgery for stage IBnonsmall cell lung cancer. Indian J Cancer [serial online] 2015 [cited 2019 Aug 23];52, Suppl S2:125-9. Available from: http://www.indianjcancer.com/text.asp?2015/52/6/125/172509
| » Introduction|| |
Nonsmall cell lung cancer (NSCLC), accounting for 1.2 million deaths each year, is the primary cause of cancer mortality in both sexes, with overall 5-year survival rate of only around 10–15%. Despite therapeutic advances, the high mortality of patients with NSCLC has not been substantially reduced over the past years, largely because of the potential of invasion, metastasis, and frequent recurrence. Another important reason for poor outcomes of the patients is that NSCLC is often detected after it has spread beyond the primary site at the time of diagnosis. Numerous studies of different biomarkers that predict which patients will have recurrences, have been performed, but only few reliable and easily accessible tumor markers have been clinically implemented currently. Therefore, the discovery of biomarkers and their application in conjunction with NSCLC cancer prognosis, which could to a large extent help improve patient care and quality of life in an earlier time, are extremely urgent.,
CUG-binding protein 1 (CUGBP1), a member of the CELF (CUGBP and embryonic lethal abnormal vision-like factor) family of RNA-binding proteins, was discovered as a protein which interacts with RNA CUG repeats that are expanded in patients with DM1 (myotonic dystrophy)., In subsequent studies, CUGBP1 was shown to be multifunctional, regulating many posttranscriptional processes including alternative splicing, deadenylation, messenger RNA (mRNA) decay, and translation. Involving in embryonic and cardiac development, skeletal muscle and adipose tissue differentiation, and germ cell formation, CUGBP1 may also play a great role in tumor genesis and the deterioration of certain tumors. Our scientific research institution had found that both CUGBP1 mRNA and CUGBP1 proteins were over-expressed in NSCLC, and as an independent prognosis factor, CUGBP1 mRNA predicts a shorter postsurgical survival in NSCLC.
Due to the frequent recurrence and mortality of lung adenocarcinoma, we furthermore detected the expression of CUGBP1 mRNA and CUGBP1 proteins in a series of 98 IB stage lung adenocarcinoma individuals in the present study, then evaluated CUGBP1 and clinicopathological parameters' prognostic significance.
| » Materials and Methods|| |
From 2008 to 2012, 147 early stage IB adenocarcinoma patients, received curative surgical resection (lobectomy) at the Department of Thoracic Surgery in Afflliated Hospital of Medical College, Qingdao University, were enrolled in our investigation. Moreover, 49 were excluded because of receiving preventive systematic chemotherapy (pemetrexed and cisplatin) postoperatively. All the leaving 98 patients had no associated co-morbidities and did not receive chemotherapy until the recurrence postsurgically. Computed tomography (CT) was performed every 2 months after the surgery and approximately half (46 patients) was known to have a recurrence (including local recurrence, distance metastasis, and lymph node metastasis) until the moment of carrying out the statistical analysis. Written and informed consent was obtained from all patients and the investigation was approved by Ethical Committee of our hospital. The clinical and pathological characteristics of subjects analyzed in the present investigation include: Sex, age, smoking status, smoking level, TNM-stage, and differentiation [Table 1]. AJCC 7th edition of pathologic staging was used to group patients to stage IB. Recently, a few reports have indicated that vascular invasion (VI) was an independent prognostic factor in lung adenocarcinoma, so we take VI as a new clinicopathological parameter to evaluate its prognostic significance.
|Table 1: Distribution of 98 individuals according to CUGBP1 and CUGBP1 mRNA expression status|
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Extraction of RNA and quantitative analysis by reverse transcription-polymerase chain reaction
Total RNA was isolated using Trizol reagent (Invitrogen, USA) according to the manufacturer's protocol. The concentration and purity of the RNA samples were determined by measuring the optical density in 260/280 nm ratios with a spectrophotometer. 10 ng of total RNA preparations from each sample was reverse-transcribed to complementary DNA, stored at −80°C until analysis. Quantitative polymerase chain reaction (PCR) was performed by monitoring the fluorescence of SYBR green dye on a Stratagene Mx3000P ® real-time PCR system in a total 20-μl reaction volume with the cycling conditions of: 95°C for 10 s, followed by 40 cycles of 95°C for 5 s, 60°C for 20 s, and 72°C for 15 s. The relative expression level of CUGBP1 mRNA was normalized by comparing with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene using appropriate primer. The forward primers for CUGBP1 mRNA and GAPDH were: 5'- GTCAGTGGTGGACCTGACCT-3' and 5'-TGACTTCAACAGCGACACCCA-3' and reverse primers were: 5'-AGGGGTCTACATGGCAACTG-3' and 5'-CACCCTGTTGCTGTAGCCAAA-3'. All primers were synthesized and purified by reverse-phase HPLC at the experimental center of our hospital. Samples were also subjected for separation on 2% agarose gel electrophoresis for ensuring the correct product amplification. Fluorescence data from each sample were analyzed with the 2−ΔΔCT method.
The surgically resected specimens were fixed routinely with 15% formalin and cut serially into slices of 5–7 um thick. The monoclonal antibody (MAb) of CUGBP1 was bought from ABcom Company and the test was performed according to manufacturer's instruction. Immunohistochemistry (IHC) was performed as described in literature. Briefly, after dewaxing and hydration, the slides were rinsed in phosphate-buffered saline (PBS) and blocked endogenous peroxidase activity with 3% hydrogen peroxide for 10 min. Antigen retrieval was achieved with preheated 10 mmol/L (pH 6) citrate buffer for 20 min to 95°C. After heating 3 times in a microwave oven for 5 min at 750 W in citrate buffer (0.01 M, pH 6.0), the sections were incubated with 1% (v/v) hydrogen peroxidase in methanol for 30 min to block the endogenous peroxidase activity. Then, the specimens were incubated with the anti-CUGBP MAb (diluted 1:200 in PBS) at 36°C for 80 min using PBS instead of antibody served as negative control and the secondary antibodies 50 min at 36°C in a humid chamber. Finally, reaction products were visualized using 3, 3'-diaminobenzidine, and the sections were then counterstained with hematoxylin.
To assess VI, Elastica van Gieson staining was also performed, and the levels of VI was classified simply as VI+, when tumor cells were identified in the vessel lumens and VI− when they were not. The evaluation of VI was performed prospectively by more than two pathologists, and if there was a discrepancy, the consensus was made by discussion. In this study, VI involves arterial invasion and venous invasion according to the Staging Manual for Thoracic Oncology (2009).
Assessment of the CUG-binding protein expression
The expression of CUGBP1 mRNA was divided into two groups of “low” (negative) and “high” (positive) according to the median level. For obtaining immunohistochemical results, each slide of CUGBP1 was scored in a blinded fashion by two pathologists according to the manufacturer's recommended criteria. Immunohistochemical staining was assessed by measuring both the intensity of the staining (0, nonstaining; 1, yellow staining; 2, brown yellow staining; and 3, brown staining) and extent of staining (0, 0%; 1, 0–10%; 2, 10–50%; and 3, 50–100%). The scores for the intensity and extent of staining were multiplied to give a weighted score, grouped in two categories where scores of 0–3 were considered negative and 4–9 considered positive, for each case.
We selected 60 years and 50 pack-years as cutoffs value to discuss continuous variable such as age and smoking level (pack-years), respectively. The Chi-square test was applied to analyze the association between CUGBP1 and clinicopathological characteristics. Furthermore, we used time to progression (TTP) as the prognostic index to evaluate the prognostic significance of all the clinicopathological variables in accordance with our experimental requirements. About the parameter of Smoking, we further used the cut-off of 50 pack-years as a new parameter to evaluate the significance of recurrence and prognosis. The value of the independent prognostic factors was assessed by multivariate analysis using the Cox hazard model. All statistical analyses were performed with the SAS 9.2 software and statistical tests were two-sided with P < 0.05 as the significant level unless otherwise specified.
| » Results|| |
Distribution of 98 individuals according to CUG-binding protein 1 mRNA and CUG-binding protein 1 expression status
Seventy-three female and 25 male patients were selected to our investigation, with the mean age of 62.3 years (range: 43–76 years). As shown in [Table 1], the expression of CUGBP1 and CUGBP1 mRNA had a close relationship with differentiation and VI in our current investigation. Using PCR and IHC analyses, there were no significant differences between the CUGBP1 mRNA expression and CUGBP1 protein expression in IB lung adenocarcinoma (Z = 1.4706, P = 0.2253) [Table 2]. Using IHC staining, CUGBP1 expression of the lung adenocarcinoma was located in cyteblast [Figure 1].
|Figure 1: Immunohistochemistry staining for CUBGP1 from IBstage lung adenocarcinoma patients: CUGBP1 expression was located in cyteblast (a: CUGBP1 [+]; b: CUGBP1 [−]). CUGBP1: CUG-binding protein 1|
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The survival significance of clinicopathological variables in 98 IB stage lung adenocarcinoma patients
The mean TTP of all the IB stage adenocarcinoma patients was 27.9 months (range: 1.4–48 months). Moreover, the mean follow-up was 26 months until carrying out the statistical analysis. Using univariate analysis, our data indicated that the survival rates had close relationship with differentiation (χ2 = 6.1928, P = 0.0452), VI (χ2 = 6.8113, P = 0.0091), and CUGBP1 (χ2 = 8.6495, P = 0.0033) [Table 3]. Finally, the multivariate analysis of all these factors influencing TTP using a Cox regression model revealed that the CUGBP1 expression (P = 0.0145, hazard ratio [HR] =2.177, 95% confidence interval [CI]: 1.166–4.062), VI (P = 0.0321, HR = 1.991, 95% CI: 1.061–3.736) were independent prognostic factors [Table 4]. The survival curves from a Cox model according to CUGBP1 and VI were shown in [Figure 2].
|Table 4: Cox multivariate analysis of prognostic factors of IB adenocarcinoma|
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|Figure 2: Postsurgical survival curves from a Cox model according to CUGBP1 and vascular invasion (a: The mean time to progression of CUGBP1 [−] was 27.5 months, while CUGBP1 (+) was 23.1 months; b: The mean time to progression of vascular invasion [−] was 28 months, while vascular-invasion [+] was 25.2 months). CUGBP1: CUG-binding protein 1|
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| » Discussion|| |
As leading cause of cancer death, it is of great challenge to clinicians and basic scientists to find out the molecular markers associated with the progression and prognosis of NSCLC, a tumor type with high metastatic potential. Prognostication of early stage lung cancer to identify cases likely to recur can help guide the administration of potentially harmful adjuvant therapies postsurgically., Due to the introduction of CT and magnetic resonance imaging into the clinic, the detection rate of early stage nonsmall cell cancer increases significantly, whereas no biomarkers can be used in the clinical practice of recurrence and prognosis currently., It is a doubt that whether IB stage lung adenocarcinoma needs preventive systematic chemotherapy postsurgically to the thoracic surgeons. Largely because of the frequent recurrence and mortality of lung adenocarcinoma, the aim of this study is to confirm CUGBP1 as an effective molecular marker that related to the recurrence and a preliminary guide of systematic chemotherapy postsurgically for IB stage adenocarcinoma patients. In addition, as VI is the initial step of distant metastasis and a few reports have indicated that VI is prognostically significant in adenocarcinoma, we detect the relevancy of VI with recurrence in our investigation.
CUGBP1 (CUGBP1, also named CELF1, CUGBP and embryonic lethal abnormal vision-like factor 1) functions as potent posttranscriptional regulators of gene expression, particularly by altering mRNA stability and translation. Meanwhile, some researches come up with a hypothesis that CUGBP1 may also play a great role in tumor genesis and deterioration of certain tumors. Zheng et al. found that the disruption of the network consists of CUGBP1 and its binding target transcripts could result in tumorigenesis and tumor progression., Our scientific research institution had found both CUGBP1 mRNA and CUGBP1 proteins were over-expressed in NSCLC, and as an independent prognosis factor, CUGBP1 mRNA predicts an adverse postsurgical survival in NSCLC. In this study, we further detected the expression of CUGBP1 mRNA in a series of IB adenocarcinoma patients. From the present statistical results, we can see that the expression of CUGBP1 is significantly associated with differentiation and VI. However, no significant differences were found between the CUGBP1 mRNA expression and CUGBP1 protein expression.
Finally, we use the TTP to evaluate the prognostic significance of each parameter. Cox multivariate survival analysis revealed that CUGBP1 is an independent predictor that the elevated expression of CUGBP1 predicted an adverse survival after surgery. The underlying biological mechanisms that might explain the relationship between CUGBP1 expression and adenocarcinoma are unclear, but we believe CUGBP1 plays a great role in tumor genesis and tumors deterioration on the basis of our data. In addition to CUGBP1, we also found the clinical parameters of VI has significant prognosis independently in our series, which has a similar conclusion with the previous results. Bodendorf et al. have reported that adenocarcinoma frequently shows VI as an indicator of poor prognosis, and should be considered for inclusion in the staging criteria and indications for adjuvant chemotherapy. However, smoking status and smoking level were irrelevant to prognosis in our investigation, inconsistent with others scientific achievement. Janjigian et al. found the number of pack-years of smoking were the independent predictors of inferior survival while smoking status was not. However, Chen et al. found that both smoking status (former or current smokers) and intensity (pack-years smoked) at the time of SCLC diagnosis were significant survival predictors. Besides, differentiation also predicts adverse postsurgery prognosis independent of CUGBP1 and VI.
Although the extent to which the adverse survival for the adenocarcinoma patients with CUGBP1 expression depends on the administered therapy is not absolutely clear, our data indicate that low expression of CUGBP1 predicts an advantageous and consistent determinant of superior survival, regardless of other independent predictors. There is no direct evidence from the previous study, and formal clinical trials exist to guide treatment decisions for the individual patient on the basis of CUGBP1 expression, but this study provides a span-new insight into recurrence and prognosis among patients of lung adenocarcinoma. On the basis of our statistical data, we speculated that CUGBP1 should be routinely detected on the adenocarcinoma patients in the future clinical practice. Moreover, a combination of CUGBP1 and VI may be used to classify adenocarcinoma patients as having a low, intermediate, or high risk of recurrence, and a new direction for the treatment of IB stage adenocarcinoma patients that CUGBP1 (+) and VI (+) should receive preventive systematic chemotherapy postsurgically, can be applied in the future clinical trials. Meanwhile, as ours is a prospective, small series, single-center study, we next step to concentrate in the underlying biological mechanisms of CUGBP1 with VI and increase the sample quantity to validate our investigation results.
| » Conclusion|| |
In summary, our investigation revealed that high expression of CUGBP1 predicts an adverse postsurgical survival of IB stage adenocarcinoma. Moreover, the combination of CUGBP1 and VI detecting could be considered as indication to predict prognosis of IB stage adenocarcinoma in the clinical trial.
| » Acknowledgments|| |
This study is supported by the grant of Shandong Tackle Key Problems in Science and Technology (2010GSF10245); Shandong Excellent Young Scientist Research Award Fund Project (BS2010YY013); Shandong Natural Science Fund (Y2008C48).
| » References|| |
Kang S, Koh ES, Vinod SK, Jalaludin B. Cost analysis of lung cancer management in South Western Sydney. J Med Imaging Radiat Oncol 2012;56:235-41.
Bodendorf MO, Haas V, Laberke HG, Blumenstock G, Wex P, Graeter T. Prognostic value and therapeutic consequences of vascular invasion in non-small cell lung carcinoma. Lung Cancer 2009;64:71-8.
Ohta Y, Shimizu Y, Minato H, Matsumoto I, Oda M, Watanabe G. Results of initial operations in non-small cell lung cancer patients with single-level N2 disease. Ann Thorac Surg 2006;81:427-33.
von Plessen C. Improving chemotherapy for patients with advanced non-small cell lung cancer. Clin Respir J 2011;5:60-1.
Yildiz PB, Shyr Y, Rahman JS, Wardwell NR, Zimmerman LJ, Shakhtour B, et al.
Diagnostic accuracy of MALDI mass spectrometric analysis of unfractionated serum in lung cancer. J Thorac Oncol 2007;2:893-901.
Mori D, Sasagawa N, Kino Y, Ishiura S. Quantitative analysis of CUG-BP1 binding to RNA repeats. J Biochem 2008;143:377-83.
Ward AJ, Rimer M, Killian JM, Dowling JJ, Cooper TA. CUGBP1 overexpression in mouse skeletal muscle reproduces features of myotonic dystrophy type 1. Hum Mol Genet 2010;19:3614-22.
Ladd AN, Charlet N, Cooper TA. The CELF family of RNA binding proteins is implicated in cell-specific and developmentally regulated alternative splicing. Mol Cell Biol 2001;21:1285-96.
Jiao W, Zhao J, Wang M, Wang Y, Luo Y, Zhao Y, et al.
CUG-binding protein 1 (CUGBP1) expression and prognosis of non-small cell lung cancer. Clin Transl Oncol 2013;15:789-95.
Wang R, Geng J, Wang JH, Chu XY, Geng HC, Chen LB. Overexpression of eukaryotic initiation factor 4E (eIF4E) and its clinical significance in lung adenocarcinoma. Lung Cancer 2009;66:237-44.
Usui S, Minami Y, Shiozawa T, Iyama S, Satomi K, Sakashita S, et al.
Differences in the prognostic implications of vascular invasion between lung adenocarcinoma and squamous cell carcinoma. Lung Cancer 2013;82:407-12.
Tamura A, Hebisawa A, Hayashi K, Sagara Y, Fukushima K, Kurashima A, et al.
Prognostic significance of thrombomodulin expression and vascular invasion in stage I squamous cell carcinoma of the lung. Lung Cancer 2001;34:375-82.
Goldstraw P. Staging Manual in Thoracic Oncology. An International Association for the Study of Lung Cancer Publication. Florida: Editorial Press; 2009.
Garcia-Lujan R, Conde-Gallego E, Lopez-Ríos F, Martin de Nicolas JL, Sanchez-Céspedes M, García-Quero C, et al.
Analysis of the molecular expression profile of non small cell lung carcinoma associated to chronic obstructive pulmonary disease. Histol Histopathol 2009;24:417-23.
Reungwetwattana T, Eadens MJ, Molina JR. Chemotherapy for non-small-cell lung carcinoma: From a blanket approach to individual therapy. Semin Respir Crit Care Med 2011;32:78-93.
Bunn PA Jr. Early-stage NSCLC: The role of radiotherapy and systemic therapy. J Natl Compr Canc Netw 2004;2 Suppl 2:S31-40.
Vincent MD, Kuruvilla MS, Leighl NB, Kamel-Reid S. Biomarkers that currently affect clinical practice: EGFR, ALK, MET, KRAS. Curr Oncol 2012;19 Suppl 1:S33-44.
Marco S, Tomasini P, Greillier L, Barlesi F. Anti-angiogenic factors in thoracic oncology: Successes, failures and prospects. Rev Mal Respir 2011;28:1216-29.
Mizuno T, Ishii G, Nagai K, Yoshida J, Nishimura M, Mochizuki T, et al.
Identification of a low risk subgroup of stage IB lung adenocarcinoma patients. Lung Cancer 2008;62:302-8.
Zheng Y, Miskimins WK. CUG-binding protein represses translation of p27Kip1 mRNA through its internal ribosomal entry site. RNA Biol 2011;8:365-71.
Beisang D, Rattenbacher B, Vlasova-St Louis IA, Bohjanen PR. Regulation of CUG-binding protein 1 (CUGBP1) binding to target transcripts upon T cell activation. J Biol Chem 2012;287:950-60.
Janjigian YY, McDonnell K, Kris MG, Shen R, Sima CS, Bach PB, et al.
Pack-years of cigarette smoking as a prognostic factor in patients with stage IIIB/IV nonsmall cell lung cancer. Cancer 2010;116:670-5.
Chen J, Jiang R, Garces YI, Jatoi A, Stoddard SM, Sun Z, et al.
Prognostic factors for limited-stage small cell lung cancer: A study of 284 patients. Lung Cancer 2010;67:221-6.
Risch A, Plass C. Lung cancer epigenetics and genetics. Int J Cancer 2008;123:1-7.
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[Table 1], [Table 2], [Table 3], [Table 4]