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A.C. Borczuk
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P1.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 233)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.04-047 - The Inhibitory Effects of CDK4 and MDM2 on Migration and Invasion in Human Non-Small Cell Lung Cancer Cells (ID 2833)
09:30 - 09:30 | Author(s): A.C. Borczuk
- Abstract
Background:
Cyclin-dependent kinase 4 (CDK4)/RB and mouse double minute 2 (MDM2)/p53 are the two main regulators of the tumor-suppressor pathways that control cellular responses to potentially oncogenic stimuli. CDK4 inhibits RB by triggering its phosphorylation, leading to releasing the G1-S restriction point. MDM2 inhibits the transcription activity of p53 by blocking the transfer of p53 from cytoplasm to nucleus and by accelerating ubiquitination of p53. Our preliminary SNP microarray analysis using lung specimens from non-invasive tumor (adenocarcinoma in situ) and invasive tumor (lepidic predominant adenocarcinoma) showed the amplification of chromosome 12q13–15, including CDK4 and MDM2 gene regions. The aim of the present study was to determine the mechanistic implications of CDK4 and MDM2 in lung adenocarcinoma migration and invasion.
Methods:
Using siRNAs specific for CDK4 and MDM2, the expressions of CDK4 and MDM2 were knocked down in the human non-small cell lung cancer cell lines A549, H460, H1299, SK-Lu-1 and H23, which harbor wild-type RB yet contain other aberrations in p53 (wild-type in A549, H460, absent in H1299, and mutated in SK-Lu-1, H23). Cell proliferation (AlamarBlue staining), mobility (scratch assay), and invasion (transwell-matrigel chamber system) were investigated.
Results:
The knockdown of CDK4 (5.5, 18.5, 2.2, 22.8 and 8.3% compared to scrambled siRNA in A549, H460, H1299, SK-Lu-1 and H23, respectively) significantly inhibited cell proliferation in H23 and SK-Lu-1, and decreased cell migration in SK-Lu-1 and H460. It also repressed cell invasion in H460, SK-Lu-1 and A549. The decreased expression of MDM2 (43.4, 69.6, 6.4, 27.3, 8.7% compared to scrambled siRNA in A549, H460, H1299, SK-Lu-1 and H23, respectively) dramatically inhibited cell proliferation in H1299, SK-Lu-1 and H23, and diminished cell migration in H23, A549 and SK-Lu-1. It also hindered cell invasion in H460 and H23.
Conclusion:
These findings suggest CDK4 and/or MDM2 pathways may play critical roles in cell proliferation, mobility and invasion, and furthermore, the targeting CDK4 and/or MDM2 may provide therapeutic benefit to lung cancer patients.
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P3.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 235)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/09/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P3.04-069 - Exportin-5 (XPO5) in Lung Adenocarcinoma: A New Biomarker of Invasion in Pathology Specimens (ID 1283)
09:30 - 09:30 | Author(s): A.C. Borczuk
- Abstract
Background:
The WHO/IASLC classification of lung adenocarcinoma (LADC) emphasizes the distinction of adenocarcinoma in situ (AIS) and minimally invasive adenocarcinoma (MIA) from their invasive counterparts. The distinction between lepidic-pattern lesions, in particular AIS/MIA and lepidic-predominant adenocarcinoma (LPA), is difficult in small biopsies and cytology specimens. Currently, there are no biomarkers of lung invasion in this setting.
Methods:
The WHO/IASLC classification of LADC was used for all components of this study. Gene expression (GE) data from 58 LADC samples including 33 samples of AIS/MIA and LPA identified two predominant clusters of 553 differentially expressed genes (p<0.01, FDR<0.06). The 317 genes upregulated in LPA localized to 6 regions on chromosomes 1, 2, 6 and 17 (Gene Set Enrichment Analysis). Expression data was compared to copy number (CN) data of AIS/MIA and LPA pooled from a re-annotated Cancer Genome Atlas data set along with prior annotated Affy 6.0 SNP array data (total 1086 LADC samples including 43 AIS/MIA and 26 LPA). Two regions (6p and 17q) contained genes with increased expression and CN increase in LPA. The XPO5 gene at 6p21 was selected for further study. Immunohistochemistry (IHC) for the XPO5 protein product Exportin-5 (XPO5, Sigma-Aldrich, St. Louis, USA) was performed on 686 lung cancers (NSCLC), on tissue microarrays and read independently by two pathologists. Nuclear (N) and cytoplasmic (C) positivity was scored for intensity (0-3) and percentage; an H-score was calculated for each (0-300, N-score and C-score). A total score (T-score) was calculated from the sum of the N-and C-scores (0 to 600). Statistical analysis was performed using the independent-samples Kruskal-Wallis test and pairwise analysis. Cox regression was used for survival analysis (continuous variable and quartile regressions), as well as Kaplan-Meier curves, logrank statistic.
Results:
XPO5 at 6p21 showed upregulation in LPAs by CN, GE and IHC. High XPO5 IHC T-scores correlated with CN, with a median T-score of 300 in tumors with CN gain vs. 50 in tumors without gain. High T-scores were seen in the following invasive patterns of NSCLCs as compared to AIS/MIA: acinar-ADC, solid-ADC, papillary-ADC, large cell carcinoma and squamous cell carcinoma; mean T-scores ranged from 144.7-251.4 in these groups vs. 48.3 and 72.1 in AIS and MIA, respectively. Importantly, T-scores correlated with overall survival for all-stage (n=686) and stage I (n=307) analyses, with higher scores predicting inferior survival. While IHC scores did not show statistically significant staining in LPA as compared to AIS/MIA, a qualitative difference was noted in some cases with acquisition of cytoplasmic positivity in the invasive component of LPAs.
Conclusion:
XPO5 is a candidate biomarker of invasion in LADC. GE and CN data along with IHC staining patterns in 686 NSCLC samples show upregulation of XPO5 in invasive tumors and in tumors with poor survival. In addition to its application in small biopsies, this marker may be of particular use in cytology specimens, where there is significant morphologic overlap between lepidic-pattern tumors and well-differentiated invasive patterns of LADC.
