DICER1, DROSHA and mirnas in patients with non-small cell lung cancer: implications for outcomes and histologic classification For Peer Review

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1 Carcinogenesis Advance Access published January, DICER, DROSHA and mirnas in patients with non-small cell lung cancer: implications for outcomes and histologic classification Journal: Carcinogenesis Manuscript ID: CARCIN--0.R Manuscript Type: Original Manuscript Date Submitted by the Author: 0-Jan- Complete List of Authors: Díaz-García, C. Vanesa; Hospital Universitario de Octubre, Oncology; Instituto de Investigación Hospital de Octubre, Oncology Agudo-López, Alba; Hospital Universitario de Octubre, Oncology; Instituto de Investigación Hospital de Octubre, Oncology Pérez, Carlos; Hospital Universitario de Octubre, Oncology; Instituto de Investigación Hospital de Octubre, Oncology López-Martín, José; Hospital Universitario de Octubre, Oncology Rodríguez-Peralto, José; Hospital Universitario de Octubre, Pathology; Instituto de Investigación Hospital de Octubre, Oncology de Castro, Javier; Hospital Universitario La Paz, Oncology Cortijo, Ana; Hospital Universitario de Octubre, Oncology Martínez-Villanueva, Miriam; Hospital Universitario de Octubre, Oncology Iglesias, Lara; Hospital Universitario de Octubre, Oncology García-Carbonero, Rocío; Hospital Universitario Virgen del Rocío, Oncology Fresno-Vara, Juan; Instituto de Genética Médica y Molecular-INGEMM, Laboratory of Molecular Pathology and Oncology Gámez-Pozo, Angelo; Instituto de Genética Médica y Molecular-INGEMM, Laboratory of Molecular Pathology and Oncology Palacios, José; Hospital Universitario Virgen del Rocío, Pathology Cortés-Funes, Hernán; Hospital Universitario de Octubre, Oncology Paz-Ares, Luis; Hospital Universitario Virgen del Rocío, Oncology Agulló-Ortuño, Mª Teresa; Hospital Universitario de Octubre, Oncology; Instituto de Investigación Hospital de Octubre, Oncology Downloaded from at Pennsylvania State University on February, Keywords: lung cancer, interference machinery, mirnas, prognosis

2 Page of Carcinogenesis DICER, DROSHA, and mirnas in NSCLC DICER, DROSHA and mirnas in patients with non-small cell lung cancer: implications for outcomes and histologic classification Short Title: DICER, DROSHA, and mirnas in NSCLC. C. Vanesa Díaz-García,, Alba Agudo-López,, Carlos Pérez,, José A. López-Martín,, J. Luis Rodríguez-Peralto,, Javier de Castro, Ana Cortijo,, Miriam Martínez-Villanueva,, Lara Iglesias, Rocío García-Carbonero, Juan A. Fresno Vara, Angelo Gámez-Pozo, José Palacios, Hernán Cortés-Funes, Luis Paz-Ares,, M. Teresa Agulló-Ortuño,, *, Oncology Department. Hospital Universitario de Octubre, Madrid, 0, Spain. Instituto de Investigación Hospital de Octubre, Madrid, 0,Spain Pathology Department. Hospital Universitario de Octubre, Madrid, 0, Spain. Oncology Department. Hospital Universitario La Paz, Madrid, 0, Spain. Oncology Department. Hospital Universitario Virgen del Rocío, Sevilla, 0, Spain. Laboratory of Molecular Pathology and Oncology. Instituto de Genética Médica y Molecular- INGEMM. Instituto de Investigación Hospital Universitario La Paz-IdiPAZ, Madrid, 0, Spain. Pathology Department. Hospital Universitario Virgen del Rocío, Sevilla, 0, Spain. Present address: Clinical Research Support Office. MD Anderson Cancer Center, Madrid, 0, Spain. Present address: Clinical Pathology Department. Hospital Universitario Virgen de la Arrixaca, Murcia, 0, Spain. * To whom correspondence should be addressed. Tel: + 0; Fax: + 0; agullo@ho.es. Correspondence may also be addressed to Mª Teresa Agulló-Ortuño, Tel: + 0; Fax: + 0; agullo@ho.es These authors contributed equally to this work. Downloaded from at Pennsylvania State University on February,

3 Page of DICER, DROSHA, and mirnas in NSCLC Abstract The clinical and functional significance of RNA interference machinery in lung cancer is poorly understood. Besides, micrornas have the potential to serve both as biomarkers and therapeutic agents, by personalizing diagnosis and therapy. In this study, we investigated whether the expression levels of DICER and DROSHA, components of the RNA-interference machinery, can predict survival, and whether microrna expression profiles can differentiate histologic subtypes in non-small cell lung cancer (NSCLC). Levels of DICER, DROSHA and five different micrornas was measured in NSCLC specimens (N=) by qrt-pcr assay, and correlated with clinical outcomes. Low expression of DROSHA was associated with an increased median survival (. vs. months; P=0.0). Also, high DROSHA expression was associated with decreased median survival in the following subgroups: adenocarcinoma (P=0.0), grade III tumors (P=0.0), and low stage patients (P=0.0). In multivariate analyses we found two independent predictors of reduced disease-specific survival: high DROSHA expression (HR=.; P=0.0), and advanced tumor stage (HR=.; P=0.0). In general, the overall tumor mirna expression was downregulated inour cohort compared to normal tissues. Expression level of hsa-let-a (P=0.00) and mir- (P=0.00) mirna were significantly higher in squamous cell carcinoma than in adenocarcinoma samples. The present study supports the value of the expression profiling of the components of the mirna-processing machinery in the prognosis of NSCLC patients, especially DROSHA expression levels. In addition, differential expression of mirnas, such as hsa-let-a and mir- may be helpful tools in the histologic subclassification of NSCLC. Downloaded from at Pennsylvania State University on February,

4 Page of Carcinogenesis DICER, DROSHA, and mirnas in NSCLC Introduction Lung cancer is the leading cause of cancer-related death worldwide (). Pulmonary neoplasias are classified as small (SCLC) or non-small cell lung cancer (NSCLC), the latter comprising a heterogeneous group which is also subclassified in different morphological varieties mainly, squamous cell carcinoma (SCC), adenocarcinoma (ADC) and large cell carcinoma (LCC). The emerging treatment revolution determined by new targeted therapies stresses the importance of accurate tumor subtyping as a mandatory step in the clinical work-up of these patients. Moreover, the discovery that gene expression can be altered through RNA interference () has stimulated research on the role of RNA interference in the development of cancer. Regulation of gene expression through RNA interference occurs by means of microrna (mirna) or small interfering RNA (sirna). Expression profiling of many micrornas in various normal and diseased tissues has demonstrated unique spatial and temporal expression patterns. Some mirnas have been functionally characterized as oncogenes or tumor suppressor genes (, ). Also, mirnas are involved in tissue differentiation during both normal development and carcinogenesis, and mirna expression profiles have been seen not only as a new class of diagnostic and prognostic tools but also as candidates for pharmacological targeting (, -0). Most importantly, mirna expression signatures in both tumor cells and peripheral blood cells from cancer patients, can predict outcome (-). The biogenesis of mirna has been reviewed extensively (Supplementary Figure ) and its pathway components could be either misexpressed or mutated in tumors. A clear consequence is that mirna overexpression could result in downregulation of tumor suppressor genes, whereas their underexpression could lead to oncogene upregulation. Their functional association with cancer, small gene size and potential to simultaneously affect a multitude of genes makes them unique candidate loci for conferring cancer susceptibility, as a small genetic change in a mirna sequence can theoretically lead to widespread phenotypic effects. The RNases DICER and DROSHA serve as key regulatory proteins in mirna biogenesis pathway and their alterations may contribute to widespread mirna deregulation in cancer (). Thus, we examined whether altered levels of DICER and DROSHA mrna, components of the RNAinterference machinery, are associated with clinical outcome in lung cancer. Furthermore, we checked out the role of several mirnas in differentiating between the two major NSCLC subtypes in human tumor samples, and their potential as biomarkers for lung cancer risk stratification in order to validate the reliability of these novel markers in routine diagnostic samples. Downloaded from at Pennsylvania State University on February,

5 Page of DICER, DROSHA, and mirnas in NSCLC Materials and methods Tumor Samples Patients were eligible if they had been diagnosed NSCLC, surgically resected, never received radiotherapy or chemotherapy and if there was evaluable tumor tissue available. From 00 to 0, we selected patients at centers for participation in the study: de Octubre University Hospital (N=), La Paz University Hospital (N=) and Virgen del Rocio University Hospital (N=). In addition, we obtained eight benign ephitelial-lung tissues from de Octubre University Hospital Tissue Bank. Specimens were snap-frozen at -0ºC until used. Histologic preparations of tumor sections were examined by pathologists without any information about the outcome, and only tissue samples with tumor content >0% were selected. The study was approved by the local Ethics Committees and Commissions of Research (Comité Ético de Investigación Clínica del Hospital Universitario de Octubre), and permission was obtained for the use of all samples. Real-time quantitative reverse transcription-pcr Samples were blinded and coded before any laboratory procedure was started. Quantitative RT-PCR was performed with the use of the TaqMan gene-expression assay and TaqMan MicroRNA assay kits (Applied Biosystems) according to the manufacturer s instructions. Amplifications were carried out on the Applied Biosystem 00 RT-PCR system, and relative gene expression values were calculated by the Ct method (Sequence Detection System.0.). Results from analysis, done in triplicate, are expressed relative to expression levels of TATA box binding protein (TBP) for DICER and DROSHA, and small nuclear RNA U (RNUB) for mirnas. The final mrna levels were converted to ratios of decreased expression ( ) or increased expression (>) relative to levels of respective mrna or mirna in normal lung epithelium. Immunohistochemical Analyses The immunohistochemical experiments (IHC) were performed on µm-thick formalin-fixed and paraffin embedded (FFPE) tissue sections. All steps were performed on the Leica Bond III automated system (Leica Microsystems) according to the manufacturer s instructions. In brief, specimens were deparaffinized and antigen was retrieved on the instrument. All slides were incubated with primary antibodies against DICER (diluted :0, Abcam) or DROSHA (:00, Abcam) for minutes, Downloaded from at Pennsylvania State University on February,

6 Page of Carcinogenesis DICER, DROSHA, and mirnas in NSCLC followed by incubation with a mouse-rabbit-horseradish peroxidase polymer and, - diaminobenzidine substrate. The stained slides were scored by two investigators who were unaware of the RT-PCR results. Staining intensity was given as no or very weak staining (score 0), weak to moderate (score ) and deep staining (score ) at x00 magnification. The percentage of positive cells was scored: 0 (0%-0%), (%-%), (%-0%), and (%-00%). We defined the IHC staining score as the sum of the intensity and percentage scores. Tumors were categorized into lowexpression group (score= 0~), and high-expression group (score= ~). The scores from two independent investigators were compared and disagreements were resolved by consensus. Validation Analysis The relation between levels of DICER and DROSHA mrna and survival among patients with lung cancer was examined in existing microarray data sets of samples (GEO accession number: GSE, and GSE0), that have been profiled with an Affymetrix GeneChip assay (HG-U Plus.0) (, ). Probe sets _at and _at were used to measure DICER and DROSHA expression, respectively. Statistical Analysis Statistical analysis was performed using SPSS.0 (Chicago, IL, USA). Kaplan-Meier plots were constructed and log-rank test was used to determine differences in survival and disease-free survival curves. Multivariate analyses with a Cox proportional hazard model were used to examine the effects of DICER, DROSHA and mirna expression on death while adjusting for other covariates. Student s t-test was used for comparison between two groups and U Mann-Whitney test was used to compare groups which did not conform to the assumption of normality (Kolmogorov-Smirnov test). Contingency tables and Fisher s exact test were used to statistically evaluate the relationship between death and categorical variables. All the statistical tests were conducted at the two-sided 0.0 level of significance. Results DICER and DROSHA are deregulated in NSCLC Downloaded from at Pennsylvania State University on February,

7 Page of DICER, DROSHA, and mirnas in NSCLC Levels of DICER mrna in cancer specimens followed a normal distribution (P= 0.). However, the distribution of DROSHA mrna levels was bimodal (P= 0.000) with two peaks in the ratio (0. and.). DICER and DROSA expression varied among cancer specimen; % had decreased DICER mrna and % had increased DROSHA mrna (Supplementary Table ). We did not find a significant correlation between DICER and DROSHA expression levels in NSCLC tumors (P= 0.0). The protein levels of DICER and DROSHA were also examined by immunohistochemical staining in a subgroup of samples (Supplementary Figure ). The results were consistent with the mrna expression levels for both DICER (Spearman s rho= 0., P= 0.000) and DROSHA (Spearman s rho= 0.0, P= 0.00). Analysis of Clinical Associations Table lists the baseline characteristics of all patients with NSCLC. Clinical outcome data were obtained from patient records. At a median follow-up of months (range -), overall -year survival was.% and patients (.%) had relapsed. At the end of the study, most patients were alive without disease (.%). In univariate analyses, neither DICER nor DROSHA mrna levels were significantly associated with age, tobacco consumption, tumor histology, tumor grade or tumor stage (Table ). Death from any cause was associated with high levels of DROSHA mrna (. ± 0. vs. ± 0., P= 0.0;.% of dead patients), but we found no relationship to the levels of DICER mrna (P= 0.). We examined whether the expression levels of DICER and DROSHA were associated with patient survival after surgery: Kaplan-Meier survival curves show that, even though the median overall survival (OS) was substantially reduced among patients whose tumor had low level of DICER mrna, did not reach statistical significance (P= 0., Figure.A). On the other hand, Kaplan-Meier curves demonstrated that the probability of survival was significantly lower for the group of patients with high levels of DROSHA mrna (. vs.. months; P= 0.0, Figure.B). Comparison among other subgroups of patients is shown in Suplementary Figure. We did not find any significant difference in the expression levels of DICER or DROSHA between SCC and ADC histology (P= 0.0 and P= 0. respectively). Nevertheless, the median OS was significant reduced among ADC patients with high levels of DROSHA expression (not reached, vs. months, P= 0.0) (Figure.C), and thus, risk of death was associated with high levels of Downloaded from at Pennsylvania State University on February,

8 Page of Carcinogenesis DICER, DROSHA, and mirnas in NSCLC DROSHA mrna in ADC patients (. ± 0. vs. ± 0., P= 0.0; 0% of dead ADC patients). However, these relationships were not found in SCC patients (data not shown). Similar to the histology, there were no differences in DICER or DROSHA expression between high- and low-grade tumors (P= 0. and P= 0.). Nevertheless, the median OS was significant reduced among grade III patients with high levels of DROSHA expression (not reached vs. months, P= 0.0) (Figure.D). Neither were differences between high- and low-stage tumors (P= 0., DICER and P= 0., DROSHA), although the OS was significantly reduced among low stage (I or II) patients with high levels of DROSHA expression (P= 0.0, Figure.E). To validate our findings, we used previously reported microarray data to compare expression of DICER and DROSHA with OS, in two different cohorts of lung cancer patients. In the first validation cohort () and similar to our initial findings, increased survival was associated with high expression of DICER mrna (not reached vs. months, P= 0.0) and low DROSHA mrna (. vs. months, P= 0.0) (Figure.F). Also, in the second validation cohort (), increased OS was associated with high expression of DICER mrna (P= 0.000) and low expression of DROSHA mrna (P=0.00), (Figure.G). The latter validation cohort consisted of patients with very early ADC stage (I or II) and the median survival time was not reached. In multivariate analyses (including age, sex, tumor stage, grade, histology, and DICER and DROSHA mrna levels), only tumor stage (III-IV vs others, P=0.0) and DROSHA mrna expression (P=0.0) were selected as independent prognostic variables (Table ). An increased DROSHA mrna level was a significant predictive factor for poor prognosis (HR=.; %CI,.0 to.; P=0.0), whereas DICER expression level did not show a significant association with survival (HR= 0.; %CI, 0. to.; P=0.). mirna expression profiling Several mirnas have recently been linked to tumorigenesis in many types of tumors (). In view of this, we decided to study in samples from our cohort in which sufficient RNA were available (N= ), the expression of some of the most relevant lung cancer-related mirna described in the literature. In particular, we studied hsa-let-a, mir-0c, mir-, mir- and mir- expression levels by qrt- PCR. The overall tumor mirna expression was downregulated compared to normal tissue (Supplementary Table ). The qrt-pcr analysis revealed that hsa-let-a (P=0.00) and mir- Downloaded from at Pennsylvania State University on February,

9 Page of DICER, DROSHA, and mirnas in NSCLC (P=0.00) expression levels were significantly higher in SCC than in ADC, (Figure.A). The only mirna with higher expression levels in ADC was mir- (P=0.), and at least -fold upregulation of mir expression was found in.% of cases. There were no significant differences in mirna expression levels depending on the tumor stage or tumor grade. Given the different mirna profiles by histology, we conducted survival analyses in the two histology groups separately. We did not find significant differences in neither OS nor disease free survival according to the mirna expression level in both histological subtypes. However, OS in our total population was higher in those patients with higher hsa-let-a (. ±. vs. ±. months, P=0.0) and higher mir-0c (. ±. vs 0. ±. months, P= 0.0) expression levels. In our study, increased DROSHA expression was associated with poor prognosis in lung cancer patients. Curiously, despite a.-fold increase in DROSHA expression in lung cancer samples, all studied mirna expression levels were decreased in this subpopulation. Interestingly, when we divided our population according to DICER and DROSHA expression levels (low and high), the mirna expression differences between the two histological types occurred only when DICER or DROSHA levels were below benign ephitelial-lung tissues levels (Figure.B). We also calculated pairwise correlation values for the mirnas and DICER and DROSHA: hsa-let- a showed a high correlation value with mir- (ρ= 0.0), with mir-0c (ρ= 0.), and with mir- (ρ= 0.). mir-0c and mir- showed a Spearman s correlation value of 0. (Supplementary Table ). There was no correlation between DICER and DROSHA expression (ρ= 0.), however there was an inversely correlation between DICER and hsa-let-a (ρ= -0.), and mir-0 (ρ= -0,0), and mir- (ρ= -0.). Also, DROSHA showed an inverse correlation value with hsa-let-a (ρ= -0.), and with mir- (ρ= -0,) (Supplementary Table ). Discussion The production of mature microrna involves a cascade of events that are closely linked to the functions of DICER and DROSHA. Likewise, it is possible that deregulated mirna expression, observed in several types of tumor (), is secondary to defective RNA silencing machinery. In our study, we found that DICER and DROSHA expression levels are variable among NSCLC specimens, and play a significant role in OS, especially DROSHA mrna expression. Levels of gene and proteomic expression were examined by qrt-pcr and IHC, with high values of correlation. Downloaded from at Pennsylvania State University on February,

10 Page of Carcinogenesis DICER, DROSHA, and mirnas in NSCLC We found that low DICER expression levels were associated with poor outcome, although this was not statistically significant. Previously, downregulation or lower expression of DICER was found to be significantly associated with NSCLC survival (), advanced tumor stages of ovarian carcinomas (), or epithelial skin cancer (), while upregulation of DICER has been described for prostate adenocarcinomas and lung carcinomas (, ). Chiosea et al. () showed that in lung ADC, DICER was downregulated in areas of invasion and in cases of advanced disease. Similar to Sand et al. (), we found a highly significant upregulation of DROSHA expression in NSCLC samples. This result supports the hypothesis that patients with lung cancer may show an alteration in DROSHA expression levels as DROSHA was overexpressed in both the SCC and ADC histologic groups. Moreover, we show an association between high DROSHA expression and poor clinical outcome in both high grade tumors and early stage tumors, suggesting that DROSHA exerts its influence most profoundly within these subgroups of tumors. High DICER and high DROSHA expression were correlated with poor prognostic factors in prostate cancer and esophageal carcinoma (, ). There are several plausible explanations for the divergent expression patterns of DICER and DROSHA among different solid tumors and how they relate to clinical and pathologic variables. Direct interactions with other components of the RNAinterference cascade could result in compensatory alterations of DICER or DROSHA expression in the presence of mutated cofactors such as genes for DGCR, XPO, and AGO (, -). The findings described in this study indicated that the expression levels of DROSHA appeared to have a significant impact (P= 0.0) on the postoperative survival of NSCLC patients, independent of disease stage (P= 0.0). Although there are some differences in the statistical significance levels, we believe that our study has been validated in two independent cohorts of patients, since Kaplan-Meier survival analysis of these validation cohorts showed a significant survival disadvantage for patients whose tumors expressed low DICER and high DROSHA mrna levels. Thus, increased expression of DROSHA appears to be clinically useful for the prognosis of lung cancer patients. In addition, other factors may underlie the potential biological effects of increased DROSHA expression in lung cancer cells. Several large-scale mirna expression studies have indicated that global mirna levels are reduced in tumor samples in comparison with normal tissues (). Our results are consistent with this, and the five mirna studied here show lower expression levels in our set of tumor samples than in normal Downloaded from at Pennsylvania State University on February,

11 Page 0 of DICER, DROSHA, and mirnas in NSCLC 0 tissues. Interestingly, the rate of reduction was much more pronounced in ADC than in SCC tumors, with statistically significant differences in the expression levels of hsa-let-a and mir- between both histological types. This pattern suggests that these mirnas may be more relevant for ADC tumorigenesis because they exhibit poor expression in this tumor type. Even more interesting is that these differences of expression between the two histological types only hold when DICER or DROSHA expression levels are below normal tissues values. That is, different mirna expression profiles between ADC and SCC subtypes are evident mainly when the mirna machinery is defective. On the other hand, we must take into account the inverse correlation between DICER and hsa-let-a, mir-0 and mir- expression levels, and also between DROSHA and hsa-let-a and mir- expression levels. A possible explanation could be that these mirnas suppress, somewhat, the expression of DICER and DROSHA, but this assertion must be tested further, and new experiments should be designed. We must consider that an examination of any individual or small series of mirnas may not reflect the complexity of changes in mirna expression participating in clinical tumor biology (). In addition, mirna could have varying regulatory effects independent of alterations in the RNA-processing machinery (0-). Posttranscriptional regulation of mirna processing also plays an important role in the regulation of mirna expression (, ), but a connection of these modifications to tumorigenesis has not yet been definitive. Moreover, one-half of the mirnas have been aligned to genomic fragile sites or regions associated with cancer. Finally, several studies suggest that mirna function is determined by the specific cellular context (). More important, mirna overexpression could result in downregulation of tumor suppressor genes, whereas their underexpression could lead to oncogene up-regulation. For this reason, some mirnas have been shown to function as oncogenes while others such as tumor suppressors: mir- and mir- are examples of mirna described as oncogenes, and hsa-let-a and mir-0c as tumor suppressor mirna (). In our set of patients, OS was higher in patients with hsa-let-a or mir-0c overexpressed, and these results are in accordance with the function as tumor suppressors of these mirnas. We found higher mir- expression levels in SCC compared to ADC samples, and this difference was highly significant statistically. Similarly Bandi et al. () detected overexpression of mir- in % of NSCLC tumors. Tan et al. () described a -microrna classifier that can distinguish cancerous SCC lesions from adjacent normal tissues; however none of the mirna described match those Downloaded from at Pennsylvania State University on February,

12 Page of Carcinogenesis DICER, DROSHA, and mirnas in NSCLC studied here. They also showed that hsa-mir- directly targets the DICER -UTR and repressed their expression. Moreover, high expression of hsa-mir- was associated with poor survival, which is consistent with our data, given that low levels of DICER correlates with shortened postoperative survival. Voortman et al. () studied a panel of mirna by qrt-pcr in FFPE tumor specimens from International Adjuvant Lung Cancer Trial (IALT) patients. No significant association was found between any of the tested mirnas and survival, and expression patterns examined were neither predictive nor prognostic in this large patient cohort. In conclusion, the present study supports the value of the expression profiling of the mirnaprocessing machinery components in the prognosis of NSCLC patients, especially DROSHA expression levels. Whether alterations in the mirna machinery are associated with the risk of lung cancer or whether DICER and DROSHA expression levels are concomitantly altered due to lung cancer, remains unclear and needs to be addressed further in future studies. In recent years, no biomarker has generated as much interest as mirnas, which have been considered for a variety of purposes, and we have proved that differential expression of some mirna such as hsa-let-a, and mir- are reliable tools in the subclassification of NSCLC. Thus, mirnas could become powerful therapeutic tools in the near future. However, the validation of these findings in multiple cohorts and the testing of their applicability to different ethnic populations are also required. Supplementary material Supplementary Figures, and, and Supplementary Tables, and, can be found at Funding This work was supported by Instituto de Salud Carlos III (grant numbers PI0, PI to L.P.- A., PI0 to R.G.-C., PI00 to J.P.), Spain. A.A.-L. and C.P. are fellows supported by Ministerio de Sanidad y Política Social (TRA-), Spain. V.D.-G. is supported by Fundación Mutua Madrileña (0/0), Spain. Downloaded from at Pennsylvania State University on February,

13 Page of DICER, DROSHA, and mirnas in NSCLC Acknowledgements We would to thank Juan Carlos Rubio of Genomic Department (Instituto de Investigación Hospital de Octubre, Madrid) for his invaluable technical assistance. Conflict of Interest Statement: None declared. References. Jemal, A. et al. () Global cancer statistics. CA Cancer J Clin.,, -0. /. Fire, A. et al. () Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, (), 0-.. Farazi, T.A. et al. () mirnas in human cancer. J Pathol.,,0-.. Markou, A. et al. () Prognostic, therapeutic and diagnostic potential of micrornas in non-small cell lung cancer. Clin Chem Lab Med.,, -.. Lu, J. et al. (0) MicroRNA expression profiles classify human cancers. Nature,, -.. Iorio, M.V. et al. (0) MicroRNAs in cancer: small molecules with a huge impact. J Clin Oncol.,, -.. Mascaux, C. et al. (0) Evolution of microrna expression during human bronchial squamous carcinogenesis. Eur Respir J.,,-.. Landi, M.T. et al. (0) MicroRNA expression differentiates histology and predicts survival of lung cancer. Clin Cancer Res.,, 0-.. Liu, X. et al. () Involvement of micrornas in lung cancer biology and therapy. Transl Res.,, Fassina, A. et al. () Classification of non-small cell lung carcinoma in transthoracic needle specimens using microrna expression profiling. Chest,, 0-.. Yu, S.L. et al. (0) MicroRNA signature predicts survival and relapse in lung cancer. Cancer Cell,, -.. Showe, M.K. et al. (0) Gene expression profiles in peripheral blood mononuclear cells can distinguish patients with non-small cell lung cancer from patients with nonmalignant lung disease. Cancer Res.,, -0.. Roa, W. et al. (0) Identification of a new microrna expression profile as a potential cancer screening tool. Clin Invest Med.,, E.. Leidinger, P. et al. () Specific peripheral mirna profiles for distinguishing lung cancer from COPD. Lung Cancer,, -. Downloaded from at Pennsylvania State University on February,

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15 Page of DICER, DROSHA, and mirnas in NSCLC. Garofalo, M. et al. () micrornas: Master regulators as potential therapeutics in cancer. Annu Rev Pharmacol Toxicol.,, -.. Bandi, N. et al. (0) mir-a and mir- are implicated in cell cycle regulation in a Rbdependent manner and are frequently deleted or down-regulated in non-small cell lung cancer. Cancer Res.,, -.. Tan, X. et al. () A -microrna signature for lung squamous cell carcinoma diagnosis and hsa-mir- for prognosis. Clin Cancer Res.,, 0-.. Voortman, J. et al. (0) MicroRNA expression and clinical outcomes in patients treated with adjuvant chemotherapy after complete resection of non-small cell lung carcinoma. Cancer Res., 0, -. Downloaded from at Pennsylvania State University on February,

16 Page of Carcinogenesis DICER, DROSHA, and mirnas in NSCLC TABLES Table. Clinicopathologic characteristics of patients. a NA: not available Characteristic (N = ) N (%) Age at diagnosis (years) Mean. Range - Gender Male 0 (.) Female (0.) Tobacco (N = 0) Non smokers (0.0) Former smokers (.) Current smokers (.) Histology Squamous (.) Adenocarcinoma (.) Large cell (.0) Other (.) Histologic differentiation Well differentiated (grade I) (.) Moderately differentiated (grade II) (.) Poorly/un-differentiated (grade III) (.) NOS (Not otherwise specified) (.) Tumor Stage I (A and B) (.0) II (A and B) (.) III (A and B) (.) IV (.) NA a (0.) Disease status Alive with disease (0.) Alive without disease (.) Dead from disease (.) Downloaded from at Pennsylvania State University on February,

17 Page of DICER, DROSHA, and mirnas in NSCLC Table. Correlation of Clinical and Pathological Features with DICER and DROSHA Messenger RNA (mrna) Levels in Patients with lung cancer. Plus-minus values are means ± SD. P values were calculated after missing values were excluded. DICER mrna Level Low High P value Low High P value Variable (N=) (N=) (N=) (N=) Age yr.±..±. 0..±..±0. 0. Tobacco N (%) Non smokers (.) (.) 0. (.) (.) 0. Former smokers (.) (.) (.) (.) Current smokers (.) (.) (.) 0 (.) Histology N (%) Squamous (.) (.) 0. 0 (.) (.) 0. Adenocarcinoma (.) (.) (.) (.) Large cell (.) (0.) (.) (.) Others (.) (0.) (.) (.) Histol. Differentiation N (%) Well diff. (grade I) (.) 0 (.) 0. (.) 0 (.) 0. Moderately diff. (grade II) (.) (.) (.) (.) Poorly/undiff. (grade III) (.) (.0) (.) (.) Tumor stage N (%) I (.) (.) 0. (.) (.) 0. II (.0) (.) (.) (.0) III (.) (.) (.) (0.0) IV (.) 0 (.) (.) DROSHA mrna Level Downloaded from at Pennsylvania State University on February,

18 Page of Carcinogenesis DICER, DROSHA, and mirnas in NSCLC Table. Multivariate Analysis Results of Independent Prognostic Factors in Patients with NSCLC. Hazard Ratio for Death P value Factor (% CI) High DROSHA expression. (.0-.) 0.0 High DICER expression 0. (0.-.) 0. Increased age.0 (0.-.0) 0. Male. (0.-.) 0. Adenocarcinoma histology.0 (0.-.) 0. Tumor stage III or IV. (.-.) 0.0 High-grade tumor. (0.-.) 0. Downloaded from at Pennsylvania State University on February,

19 Page of DICER, DROSHA, and mirnas in NSCLC TABLE AND FIGURES LEGENDS Table. Clinicopathologic characteristics of patients. Table. Correlation of Clinical and Pathological Features with DICER and DROSHA Messenger RNA (mrna) Levels in Patients with lung cancer. Plus-minus values are means ± SD. P values were calculated after missing values were excluded. Table. Multivariate Analysis Results of Independent Prognostic Factors in Patients with Lung Cancer. Fig.. Kaplan-Meier survival curves and log-rank analysis for patients according to tumor expression of DICER and DROSHA. (A, B) Survival curves in the study cohort. (C) In adenocarcinoma patients. (D) In patients with tumor grade III. (E) In patients with lung cancer stage I or II. (F, G) Curves from validation analysis are also shown for the expression of DICER and DROSHA in independent cohorts of patients with lung cancer. Fig. : (A) Relative fold of hsa-let-a, mir-0c, mir-, mir- and mir- levels in lung ADC (bright gray) and SCC (dark gray) samples. MicroRNA expression profiles significantly differed between SCC and ADC. (B) Differences in hsa-let-a and mir expression levels in SCC (N= ) and ADC (N=) samples with low expression of DICER and DROSHA. Columns, representative images of reactions run in triplicate; bars, SD. P< 0.0. Downloaded from at Pennsylvania State University on February,

20 Page of Carcinogenesis Fig.. A % of Survival,0,,,, 0,0 0 Months after surgery Nº at Risk Events Months 0 0 Low DICER High DICER 0 C % of Survival,0,,,, 0,0 0 P= 0. P= 0.0 Months after surgery (AD) High DICER Low DICER 0 Low DROSHA High DROSHA Nº at Risk Events Months 0 0 Low DROSHA High DROSHA B % of Survival D % of Survival,0,,,, 0,0,0,,,, 0,0 0 0 P= 0.0 Months after surgery Nº at Risk Events Months 0 0 Low DROSHA High DROSHA P= 0.0 Low DROSHA 0 0 Months after surgery (Grade III) High DROSHA Low DROSHA High DROSHA Nº at Risk Events Months 0 0 Low DROSHA 0 High DROSHA Downloaded from at Pennsylvania State University on February,

21 Page of E % of Survival,0,,,, 0,0 0 0 Month after surgery (Stage I or II) Nº at Risk Events Months 0 0 Low DROSHA High DROSHA 0 0 F % of Survival,0,,,, 0,0 0 P= 0.0 First Validation Cohort P= 0.0 Months after surgery Low DROSHA High DROSHA High DICER 0 00 Low DICER 00 Nº at Risk Events Months 0 0 Low DICER 0 High DICER 0 % of Survival,0,,,,,0 0 P= 0.0 Months after surgery Low DROSHA High DROSHA 0 00 Nº at Risk Events Months 0 0 Low DROSHA High DROSHA Downloaded from at Pennsylvania State University on February,

22 Page of Carcinogenesis G % of Survival,0,,,,,0 0 Second Validation Cohort: Stage I-II lung AD P= Months after surgery High DICER Low DICER Nº at Risk Events Months 0 0 Low DICER 0 High DICER % of Survival,0,,,,,0 0 P= 0.00 Months after surgery Nº at Risk Events Months 0 0 Low DROSHA 0 High DROSHA Low DROSHA High DROSHA 00 Downloaded from at Pennsylvania State University on February,

23 Page of Fig.. A B Relative mirna expression 0 SCC Relative mirna expression 0 SCC ADC ADC P=0.00 P=0.00 P=0.0 P=0. P=0. let-a mir-0c mir- mir- mir- P=0.0 Low DICER P=0.00 P=0.00 Low DROSHA P=0.00 let-a mir- let-a mir- hsa-let-a mir- SCC ADC SCC ADC Low DICER.±0. 0.±0..±0..0±0. Low DROSHA.±0. 0.±0..±..±0. Downloaded from at Pennsylvania State University on February,

24 Page of Carcinogenesis Supplementary Figure. mirna biogenesis pathway illustrative overview. micrornas (mirnas) are transcribed to produce a nucleotide transcripts, called pri-mirna. It is cropped to form a pre-mirna hairpin (-00 nucleotides) which is transported to the cytoplasm through an export complex. Finally, the pre-mirna is processed to produce two mirna strands, a mature mirna sequence, approximately -0 nucleotides in length, and its short-lived complementary sequence, which is denoted mir* and sometime called the passenger strand. The thermodynamic stability of the mirna duplex termini and the identity of the nucleotides in the overhang determine which of the strands is incorporated into the RNA-inducing silencing complex (RISC). RISC is thought to direct mirna-mediated destabilization or mirna-mediated translational repression of mirna without perfectly complementary mrna targets. Partial complementarity mirna encoding gene DROSHA pri-mirna NUCLEUS GEMIN GEMIN AGO - mature mirna CYTOPLASM RISC RNA Pol II DGCR pre-mirna XPO TRBP DICER mir/mir* duplex RAN Perfect complementarity ORF AAAAA ORF AAAAA Translational repression Splicing DROSHA-independent Cleavage of target mrna DICER-independent AGO Downloaded from at Pennsylvania State University on February,

25 Page of Supplementary Figure : Immunohistochemical staining of NSCLC samples. These assays were performed using formalin-fixed, paraffin-embedded lung cancer tissues (FFPE). Immunohistochemistry was performed on a subset of lung cancer biopsies selected due to their varying levels of DICER (upper panels) and DROSHA (lower panels) mrna expression. Results were consistent with those obtained in DICER and DROSHA mrna expression levels by q-rt-pcr. We did not find any significant difference in the expression of DICER or DROSHA between SCC and ADC histology. DICER DROSHA High expression Low expression Downloaded from at Pennsylvania State University on February,

26 Page of Carcinogenesis Supplementary Figure : As compared with other subgroups (low DICER and high DROSHA expression), patients with tumors with high levels of DICER mrna and low levels of DROSHA mrna were associated with an increased median survival (not reached, vs.. months; P=0.0). Also, tumors with low DICER and low DROSHA mrna levels were associated with an increased median survival as compared with tumors with low DICER and high DROSHA mrna levels (not reached vs.. months; P= 0.0). % of Survival,0,,,, 0,0 0 P= 0.0 High DICER Low DROSHA Low DICER Low DROSHA Months after surgery 0 High DICER High DROSHA Low DICER High DROSHA 00 Nº at Risk Events Months 0 0 Low DICER, Low DROSHA High DICER, High DROSHA 0 Low DICER, High DROSHA High DICER, Low DROSHA 0 Log Rank (significance) Low DICER Low DROSHA High DICER High DROSHA Low DICER High DROSHA High DICER, High DROSHA 0. (0.0) Low DICER, High DROSHA. (0.0) 0. (0.) High DICER, Low DROSHA 0. (0.). (0.0). (0.0) Downloaded from at Pennsylvania State University on February,

27 Page of Supplementary Table. Distribution of DICER and DROSHA mrna levels in lung cancer tumors. N (%) N= Median Ratio (range) Individual Low DICER (.) 0. ± 0.0 (0.-0.) High DICER (.). ± 0.0 (.0-.) Low DROSHA () 0. ± 0.0 (0.-.00) High DROSHA (). ± 0. (.0-.) Joint Low DICER and DROSHA () Low DICER and High DROSHA () High DICER and DROSHA () High DICER and low DROSHA () Downloaded from at Pennsylvania State University on February,

28 Page of Carcinogenesis Supplementary Table. Downregulation of mirnas in NSCLC samples compared to normal tissue. % of total cohort % of ADC % of SCC hsa-let-a... mir-0c..0. mir-..0. mir-... mir-... Downloaded from at Pennsylvania State University on February,

29 Page of Supplementary Table. Correlation coefficients between micrornas, DICER and DROSHA expression levels. DICER ρ a DROSHA ρ a hsa-let-a ρ a mir-0c ρ a mir- ρ a mir- ρ a mir- ρ a DICER * -0.0** -0.* DROSHA -0.** ** hsa-let-a 0.** 0.0** 0. 0.** mir-0c 0.** 0. 0.** mir * mir- 0.** mir- a ρ= Spearman s coefficient. N= samples. *p< 0.0, **p< 0.0. Downloaded from at Pennsylvania State University on February,