Early detection of primary lung cancer is difficult yet important since diagnosis at earlier stages is associated with significantly better survival. Circulating microRNA (miR) profiles have been proposed as promising diagnostic and prognostic biomarkers for cancer, including lung cancer. The goal in this study was to establish biomarkers for early detection of primary lung cancer (NSCLC) by measuring circulating miRs. We looked at paired serum and plasma samples of patients with early stage NSCLC and matched controls. We discovered differential expression of the following miRs: miR-146b, miR-221, let-7a, miR-155, miR-17-5p, miR-27a, and miR-106a, which were significantly reduced in the serum of NSCLC cases, while miR-29c was significantly increased. Even though we did not observe any significant differences in plasma, and expression levels in serum did not correlate well with levels of plasma, these tissue types are obtained differently, which could account for differences in the results. Moreover, reduced plasma expression of let-7b was modestly associated with worse cancer-specific mortality in all patients, whereas reduced serum expression of miR-223 was modestly associated with cancer-specific mortality in stage IA/B patients. Additionally, we looked at the expression of biologically relevant miRs (miR-21, miR-29b, miR-34a/b/c, miR-155, and let-7a) in formalin-fixed paraffin-embedded tumor specimens from the patients who participated in the International Adjuvant Lung Cancer Trial (IALT), the largest randomized study conducted to date of adjuvant chemotherapy in patients with radically resected NSCLC. The goal was to evaluate if the expression of these miRs represents prognostic and predictive value for survival. Importantly, the expression of certain miRs has been associated with chemoresistance. However, our results indicate that the miRs expression patters examined were neither predictive nor prognostic in this large patient cohort. Based on the previous miR microarray data of lung cancer specimens from NSCLC adenocarcinoma patients, we investigated the expression of 5 miRs whose expression were significantly altered in lung cancer and were associated with cancer-specific mortality. Three of those miRs (miR-17, miR-21, and miR-155) were potential oncogenic miRs, showing increased expression in tumors with high expression levels being associated with poor prognosis. We investigated their expression in early-stage lung adenocarcinoma snap-frozen tissues from NSCLC patients that originated from Maryland, Norway and Japan in association with tumor progression and survival. We found elevated expression of all three miRs to be associated with worse cancer-specific mortality in the Maryland cohort. However, only miR-21 showed the same association in the Norwegian cohort and worse relapse-free survival in the Japanese cohort. Additionally, more advanced stage tumors expressed significantly higher levels of miR-21 compared with TNM stage I patients, in which group miR-21 was also associated with worse cancer-specific mortality and relapse-free survival, independent of other clinical factors. We found that elevated miR-21 expression is independently associated with worse prognosis in 3 independent NSCLC adenocarcinoma cohorts from different regions of the world. This finding has a translational relevance;miR-21 expression has potential utility as an early-stage prognostic biomarker for NSCLC adenocarcinoma patients. Although we did not observe a significant increase of miR-21 expression in FFPE tissues, this could be due to lower quality of RNA extracted from those tissues, as has been reported previously. Moreover, we have undertaken a systems biology approach to study the effect of miRs in cancer. Each miR can control translation of hundreds of different coding messengers, and a single messenger can be controlled by more than one miR. We combined differential expression, genetic networks and DNA copy number alterations. We confirmed, or discovered, miRs with comprehensive roles in cancer: has-miR-103/106 were downregulated in cancer, whereas has-miR-30 became most prominent;has-miR-17/92 family was amplified and the has-miR-143/145 cluster deleted in cancer;has-miR-30 and has-miR-204 were found to be physically altered at the DNA copy number level. We reported the first miR network from normal tissues, but also built miR networks for coupled cancerous and noncancerous tissues, and identified cancer variations in miR networks. Finally, we superimposed DNA variations onto expression data to generate a comprehensive miR alteration map in cancer. In addition to our studies with miRs as important biomarkers in lung cancer, we investigated a tumor suppressor gene, LKB1/STK11, which has a serine-threonine kinase activity and is located on chromosome 19p13.3. Chromosome 19p is the second most common region of loss in lung tumors of smokers, and LKB1 mutations are found in 30% of lung cancer cell lines and in a smaller portion of primary lung adenocarcinomas. In this study, we used several complementary genetic approaches to assess the KLB1 locus in primary NSCLC. Our data identified total inactivation of the LKB1 gene by either homozygous deletion (HD) or loss of heterozygosity (LOH) with somatic mutation in 39% of tested samples, whereas loss of chromosome 19p region by HD or LOH at the LKB1 region occurred in 90% of NSCLC. Furthermore, we investigated the role of tumor estrogen receptors (ERs) and serum estrogen in lung cancer with a hypothesis that ERs and functional single nucleotide polymorphisms (SNPs) in the estrogen biosynthesis pathway are associated with poorer lung cancer survival. Serum estrogen, progesterone, tumor messenger RNA expression of hormone receptors and germ line DNA polymorphisms were analyzed for associations with lung cancer survival. Patients in the highest tertile of serum estrogen had worse survival in all three cohorts investigated in this study. Furthermore, the variant allele of estrogen receptor alpha (ER-alpha) polymorphism (rs2228480) was significantly associated with increased tumor ER-alpha levels and worse survival in all cohorts. Results were independent of gender and hormone replacement therapy. Thus, we reported a significant association of increased serum estrogen with poorer survival among lung cancer male and female patients. Additionally, we investigated a very important mechanism, cellular immortalization, which is one of the hallmarks of cancer and is dependent on the activity of a telomere length maintenance mechanism (TMM), either telomerase or alternative lengthening of telomeres (ALT). We examined 43 malignant pleural mesotheliomas (MPMs). The TMMs are widely regarded as potential targets for cancer therapies and telomerase inhibitors have entered clinical trials. We asked what proportion of MPMs use ALT and/or telomerase. We found that 43 of 43 MPMs were telomerase-positive[+] and Alt-negative[-]. To investigate whether MPM cells are unusually susceptible to activation of telomerase, we examined activation of the TMMs in an in vitro model of cellular immortalization, in which normal pleural mesothelial cells were transduced with SV40. We discovered that pleural mesothelioma cells are capable of activating either TMM in vitro, and the observation that 100% MPMs were telomerase[+] suggests that there are factors in vivo that select for telomerase activity during oncogenesis of this tumor type. We concluded that MPM is a tumor that could be considered for anti-telomerase therapy. Genes and mechanisms described in our studies, important to lung cancer initiation, progression and outcome, present potential molecular prognostic and diagnostic biomarkers in lung cancer.
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