Exosomal MicroRNA: A Diagnostic Marker for Lung Cancer

Guilherme Rabinowits; Cicek Gerçel-Taylor; Jamie M. Day; Douglas D. Taylor; Goetz H. Kloecker

Disclosures

Clin Lung Cancer. 2009;10(1):42-46. 

In This Article

Abstract and Introduction

Abstract

Purpose: To date, there is no screening test for lung cancer shown to affect overall mortality. MicroRNAs (miRNAs) are a class of small noncoding RNA genes found to be abnormally expressed in several types of cancer, suggesting a role in the pathogenesis of human cancer.
Patients and Methods: We evaluated the circulating levels of tumor exosomes, exosomal small RNA, and specific exosomal miRNAs in patients with and without lung adenocarcinoma, correlating the levels with the American Joint Committee on Cancer (AJCC) disease stage to validate it as an acceptable marker for diagnosis and prognosis in patients with adenocarcinoma of the lung.
Results: To date, 27 patients with lung adenocarcinoma AJCC stages I-IV and 9 controls, all aged 21–80 years, were enrolled in the study. Small RNA was detected in the circulating exosomes. The mean exosome concentration was 2.85 mg/mL (95% CI, 1.94–3.76) for the lung adenocarcinoma group versus 0.77 mg/mL (95% CI, 0.68–0.86) for the control group (P <.001). The mean miRNA concentration was 158.6 ng/mL (95% CI, 145.7–171.5) for the lung adenocarcinoma group versus 68.1 ng/mL (95% CI, 57.2–78.9) for the control group (P <.001). Comparisons between peripheral circulation miRNA-derived exosomes and miRNA-derived tumors indicated that the miRNA signatures were not significantly different.
Conclusion: The significant difference in total exosome and miRNA levels between lung cancer patients and controls, and the similarity between the circulating exosomal miRNA and the tumor-derived miRNA patterns, suggest that circulating exosomal miRNA might be useful as a screening test for lung adenocarcinoma. No correlation between the exosomal miRNA levels and the stage of disease can be made at this point.

Introduction

Lung cancer is the leading cause of cancer-related death among men and women and these second leading cause of cancer in the United States. Approximately 70% of patients with lung cancer present with symptoms caused by locally advanced or metastatic disease that is not amenable to cure.[1] Despite advances in therapy, 5-year survival rates average 15% for all individuals with lung cancer.[2] To improve the outcome of these patients, multiple screening studies with imaging techniques and cytologic analysis of sputum have been performed, but none have shown to significantly affect overall mortality.[3,4]

MicroRNAs (miRNAs) are small noncoding RNA gene products discovered in diverse organisms thought to regulate other genes' expression.[5] It has been suggested that the miRNAs are involved in various biologic processes, including cell proliferation, differentiation, death, stress resistance, and fat metabolism.[6]

The genome-wide expression profiling of miRNAs has been shown to be significantly different among primary lung cancers and corresponding noncancerous lung tissues. A study demonstrating diagnostic miRNA signatures of non–small-cell lung cancer (NSCLC) revealed overexpression of 12 specific miRNAs (hsa-miR-17-3p, hsa-miR-21, hsa-miR-106a, hsa-miR-146, hsa-miR-155, hsa-miR-191, hsa-miR-192, hsa-miR-203, hsa-miR-205, hsa-miR-210, hsa-miR-212, and hsa-miR-214) when compared with normal lung tissue.[7]

Tumor-derived exosomes, small (50–100–nm) membrane vesicles of endocytic origin, were initially demonstrated in the peripheral circulation of patients with cancer in 1979.[8,9] Although the primary source of circulating exosomes in patients with cancer is the tumor, we have developed a novel approach for the separation of tumor-derived exosomes from those derived from normal lymphoid cells, using adherence to specific magnetic beads.[10,11] While tumor-derived exosomes exhibit some common, shared proteins, they also express an array of tumor antigens that reflect the originating tumor cells. Although exosome release can be demonstrated in many proliferating cell types, their release is exacerbated in tumor cells, as evidenced by their increased presence in plasma, ascites, and pleural effusions of patients with cancer.[12,13] This increased presence in serum and ascites fluids of patients with cancer and the overexpression of certain biomarkers have led investigators to propose a role for exosomes in diagnosis and biomarker analysis.[14]

MicroRNA (miR)-17-3p, miR-21, miR-106a, miR-146, miR-155, miR-191, miR-192, miR-203, miR-205, miR-210, miR-212, and miR-214 have previously been demonstrated to be upregulated markers for lung cancer. The graphs present the average intensities of duplicate samples.

Exosomes appear to play a central role in cell-to-cell communication and to affect target cells by stimulating them directly by surface-expressed ligands or by transferring molecules between cells. Ratajczak et al demonstrated the presence of exosomal RNA and showed the horizontal transfer of genetic information between cells.[15] Because the biologic effects of these exosomes were inhibited after pretreatment with ribonuclease, the involvement of RNA components was indicated. RNA molecules, after translocation from the nucleus to the cytoplasm, can bind to and be transported by membranous organelles or vesicles to specific intracellular sites, which might provide an explanation for the association of RNA populations with exosomes. Valadi et al demonstrated that released exosomes contain a subset of both cellular mRNA and miRNA, which could be transferred to target cells.[16] Our studies indicate that miRNA contained in tumor exosomes can suppress the mRNA for signal transduction components within T cells.[17] Because released exosomes contain RNA populations, including miRNA, it is possible that this exosomal miRNA reflects the miRNA signature of the parental tumor.

In this study, we evaluated the circulating levels of tumor exosomes, exosomal small RNA, and the aforementioned 12 specific exosomal miRNAs in patients with and without lung adenocarcinoma, correlating the levels with the American Joint Committee on Cancer (AJCC) disease stage to validate it as an acceptable marker for diagnosis and prognosis in patients with adenocarcinoma of the lung.

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