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. 2016 May 31;7(22):33316-30.
doi: 10.18632/oncotarget.8955.

Oncogenic ALK Regulates EMT in Non-Small Cell Lung Carcinoma Through Repression of the Epithelial Splicing Regulatory Protein 1

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Free PMC article

Oncogenic ALK Regulates EMT in Non-Small Cell Lung Carcinoma Through Repression of the Epithelial Splicing Regulatory Protein 1

Claudia Voena et al. Oncotarget. .
Free PMC article

Abstract

A subset of Non-Small Cell Lung Carcinoma (NSCLC) carries chromosomal rearrangements involving the Anaplastic Lymphoma Kinase (ALK) gene. ALK-rearranged NSCLC are typically adenocarcinoma characterized by a solid signet-ring cell pattern that is frequently associated with a metastatic phenotype. Recent reports linked the presence of ALK rearrangement to an epithelial-mesenchymal transition (EMT) phenotype in NSCLC, but the extent and the mechanisms of an ALK-mediated EMT in ALK-rearranged NSCLC are largely unknown. We found that the ALK-rearranged H2228 and DFCI032, but not the H3122, cell lines displayed a mesenchymal phenotype. In these cell lines, oncogenic ALK activity dictated an EMT phenotype by directly suppressing E-cadherin and up-regulating vimentin expression, as well as expression of other genes involved in EMT. We found that the epithelial splicing regulatory protein 1 (ESRP1), a key regulator of the splicing switch during EMT, was repressed by EML4-ALK activity. The treatment of NSCLC cells with ALK tyrosine kinase inhibitors (TKIs) led to up-regulation of ESRP1 and E-cadherin, thus reverting the phenotype from mesenchymal to epithelial (MET). Consistently, ESRP1 knock-down impaired E-cadherin up-regulation upon ALK inhibition, whereas enforced expression of ESRP1 was sufficient to increase E-cadherin expression. These findings demonstrate an ALK oncogenic activity in the regulation of an EMT phenotype in a subset of NSCLC with potential implications for the biology of ALK-rearranged NSCLC in terms of metastatic propensity and resistance to therapy.

Keywords: ALK; EMT; ESRP1/2; lung cancer.

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. ALK-rearranged NSCLC are enriched in EMT markers
A. The enrichment of EMT signatures of MSigDB on the differentially expressed genes between ALK-rearranged tumors and normal samples based on the KS test. B. A heatmap of clustered data (both on the gene and sample levels) is shown, selected data of the EMT activated signatures genes were included. Blue color indicates GEO batch and yellow color indicates TCGA batch. Red color indicates ALK positive tumors and black color indicates normal samples. C. Human NSCLC cell lines harboring different genetic lesions were blotted with the indicated antibodies. D. Immunofluorescence staining for E-cadherin and vimentin (top and central panels) and bright-field images showing morphology (bottom panels) of ALK-rearranged NSCLC cell lines, H3122, H2228 and DFCI032. Nuclei were stained with DAPI.
Figure 2
Figure 2. ALK oncogenic activity regulates EMT in ALK-rearranged NSCLC
A. Top gene EMT related signatures of MSigDB CGP showing enrichment with the up-regulated and down-regulated genes of ALK based on hyper-geometric test. B. RT2 Profiler Array analysis of the H2228 cell line where EML4-ALK was inhibited for 24 hours with 300nM TAE-684 or crizotinib or knocked-down by shRNA for 72 hours. Histograms represent means of genes up- or down-regulated in all the three different treatments. Fold change levels are shown compared to controls (untreated cells). Dotted lines indicate upper or lower limits of significant changes.
Figure 3
Figure 3. EML4-ALK regulates ESRP1 and ESRP2
A-B. H2228 (A) and DFCI032 (B) were treated with crizotinib (300nM) for 24 hours and collected for qRT-PCR analysis to check mRNA expression of PRL-1 and SerpineE1. C. H2228 and the DFCI032 cell lines were treated with TAE-684 (300nM) for 24 hours. Total cell lysates were blotted with the indicated antibodies. D. H2228 and DFCI032 cell lines were treated with TAE-684 or crizotinib (300nM) for 48 hours and the collected for Western blot analysis. Total cell lysates were blotted with the indicated antibodies. E-F. H2228 (E) and DFCI032 (F) were treated with TAE-684 (150nM) and collected at 96h for qRT-PCR analysis to check mRNA expression of ESRP1 and ESRP2. One representative experiment out of two is shown. G. H2228 and DFCI032 cell lines were treated with 300nM TAE-684 for the indicated time. Cells were collected and blotted with the indicated antibodies. Two-tailed Student's t tests were used to calculate the p values shown. Data are represented as mean (±SEM). *, P<0.05; **, P<0.005.
Figure 4
Figure 4. ALK oncogenic activity sustains the mesenchymal phenotype in ALK-rearranged NSCLC
A-B. Real-time PCR analysis of mRNA levels of E-cadherin (A) and vimentin (B) in H2228 and DCI032 cells treated with TAE-684 or crizotinib (300nM) for the indicated time. mRNA expression values are calculated relative to controls. One representative experiment out of three is shown. C. H2228 and DFCI032 cells were treated with 300nM TAE-684 or crizotinib for the indicated time. Total cell lysates were blotted with the indicated antibodies. D. Representative hematoxilin-eosin (H&E) (left panels) and immunostaining with anti-E-cadherin (central panels) and anti-vimentin (right panels) antibodies on tumor xenograft sections from control (Ctrl) and mice treated with TAE-684. Data are from two independent experiments. Two-tailed Student's t tests were used to calculate the p values shown. Data are represented as mean (±SEM). For E-cadherin, *, P<0.05; **, P<0.005. For vimentin the p value was not significant.
Figure 5
Figure 5. EML4-ALK ectopic expression in immortalized bronchial epithelial cells induces a mesenchymal phenotype
BEAS-2B cell line were infected with GFP-retrovirus expressing either wild-type or kinase dead (KD) EML4-ALK. A. Cells are shown in phase contrast and under fluorescent detection to identify GFP-positive cells. B. Histogram displays the percentage of spindle-like cells over the total of GFP-reporter positive cells. C. Immunofluorescence staining for E-cadherin and vimentin (red fluorescence). Nuclei were stained with DAPI. D. Histogram shows the percentage of vimentin and E-cadherin positive cells over the total of GFP-reporter positive cells. One representative experiment out of two is shown. E. Cells were collected after infection and immunoblotted with the indicated antibodies. F-G. qRT-PCR analysis to detect mRNA levels of E-cadherin and vimentin (F) and ESRP1 (G). Two-tailed Student's t tests were used to calculate the p values shown. Data are represented as mean (±SEM). *, P<0.05; **, P<0.005; ****, P<0.0001.
Figure 6
Figure 6. EML4-ALK regulates E-cadherin expression through ESRP1 repression
A. H2228 and DFCI032 cell lines were transduced with a lentivirus expressing the human ESRP1, collected and blotted with the indicated antibodies. B. H2228 and DFCI032 were infected with pLKO expressing an shRNA targeting ESRP1 or a control shRNA (shCtrl). Cells were treated with ALK inhibitors for 96 hours and harvested for Western Blot analysis with the indicated antibodies.

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