There was a very intriguing paper published online by Clinical Cancer Research on May 9 last week that should be of interest for those involved in lung cancer research. The authors are Mohamed Kabbout, Melinda Garcia, Junya Fujimoto, et al. from M.D. Ancerson Cancer Center in Houston and the title of the article is "ETS2 mediated tumor suppressive function and MET oncogene inhibition in
human non-small cell lung cancer."
In brief, the authors find that there is significant downregulation of
the ETS2 transcription factor in lung adenocarcinomas compared to normal
lung tissues. ETS2 expression is linked to various canonical
cancer-associated pathways in lung cancer cells, in particular the HGF
pathway. Moreover, knockdown of ETS2 is associated with up-regulation
of the ZEB1, SNAI2, RAB3B, SERPINB5 and SPDEF genes
with concomitant with elevated cell migration and invasion. Last, low ETS2 protein is predictive of shorter time-to-recurrence in NSCLC and lung adenocarcinoma. These data suggest that ETS2 functions as a tumor-suppressor in lung cancer cells by, at least in part, regulation of the migration- and invasion-promoting transcriptome.
More details. . .
Gene expression profiling showed significant decreased expression of ETS2 transcription factor transcripts in lung adenocarcinoma (ADC) compared to normal controls and quantitative RT-PCR confirmed decreased ETS2 expression. Subset analysis should significantly decreased ETS2 expression in those ADCs from smokers compared to those from never-smokers.
They analyzed ETS2 expression by IHC in a microarray set of 201 NSCLC (135 ADC, 66 squamous cell carcinoma-SQC) tumors. Low ETS2 expression was associated with shorter time-to-recurrence in stage I and all stages of patients. Multivariate Cox proportional hazards analysis showed that, after adjusting for stage, low ETS2 expression was an independent predictor of shorter time-to-recurrence in NSCLC and ADC.
H441 human lung cancer cell lines transfected with small interfering RNA (siRNA) specific to ETS2 showed significantly reduced ETS2 transcript compared to controls. ETS2 knockdown significantly increased cell growth, migration, and invasion. Conversely, ETS2 overexpression in H1299 lung cancer cells showed significantly decreased cell migration and invasion. Of interest, the magnitude of these changes following knockdown or overexpression of ETS2 was greater in measures of migration and invasion compared to cell growth--suggesting a tumor suppressor function for ETS2 at least in vitro.
Gene expression profiling of H441 lung cancer cell lines transfected with scrambled siRNA (control) versus ETS2-specific siRNA showed significant differential expression in 1,816 transcripts. Functional analysis by Ingenuity Pathways Analysis (IPA) showed ETS2 knockdown modulated key oncogenic pathways, including HGF, integrin, tissue factor, semaphorin, and MAPK signaling. Notably, HGF was the top modulated canonical pathway identified following knowdown, and further, an HGF-mediated gene-interaction network was among the among the top significant gene networks following topological arrangement of the genes found to be differentially expressed by IPA.
Western blot analysis and ELISA both showed increased phosphorylation MET following ETS2 knockdown. Importantly, co-transfection of H441 lung cancer cells with MET-specific siRNA abrogated cell migration, invasion, and, to a lesser effect, cell growth mediated by knockdown of ETS2 expression alone. Furthermore, co-transfection of cells with MET-specific siRNA significantly attenuated the induction of SNAI2, RAB3B, SPDEF and SERPINB5 mediated by knockdown of ETS2 alone as well as reduced expression of ZEB1. These data demonstrate that ETS2 suppresses key features of the lung cell malignant phenotype, at least in part, through inhibition of the MET oncogene.
The authors also find that ETS2 suppresses EGF-mediated signaling in lung cancer cells. HGF treatment increased ETS2 protein expression and phosphorylation of MET and ERK1/2 MAPK in H441 cells. Conversely, expression of ETS2, phospho-MET, and phospho-ERK1/2 was attenuated by co-treatment of H441 cells with the MET TKI PHA-665752. HGF treatment also significantly increased the expression of SNAI1, RAB3B, SPDEF, ZEB1, and SERPINB5 genes. Moreover, the expression of these genes was significantly higher in HGF-treated cells transfected with ETS2-specific siRNA compared to similarly treated control cells. Conversely, overexpression of ETS2 in H1299 cells decreased HGF-induced phosphorylated MET levels and decreased HGF-induced cell migration in the wound healing assay.
Finally, NSCLC (p=0.01) or ADC (p=0.02) patients with relatively low ETS2 protein expression and higher phospho-MET membrane immunoreactivity showed shortest tiime-to-recurrence compared to other patient groups, specifically those with higher ETS2 and higher phospho-MET