Systems analysis reveals a transcriptional reversal of the mesenchymal phenotype induced by SNAIL-inhibitor GN-25
- Asfar S Azmi†1Email author,
- Aliccia Bollig-Fischer†2,
- Bin Bao1,
- Bum-Joon Park3,
- Sun-Hye Lee3,
- Gyu Yong-Song4,
- Gregory Dyson2,
- Chandan K Reddy5,
- Fazlul H Sarkar1, 2Email author and
- Ramzi M Mohammad2, 6Email author
© Azmi et al.; licensee BioMed Central Ltd. 2013
Received: 12 April 2013
Accepted: 22 August 2013
Published: 3 September 2013
HMLEs (HMLE-SNAIL and Kras-HMLE, Kras-HMLE-SNAIL pairs) serve as excellent model system to interrogate the effect of SNAIL targeted agents that reverse epithelial-to-mesenchymal transition (EMT). We had earlier developed a SNAIL-p53 interaction inhibitor (GN-25) that was shown to suppress SNAIL function. In this report, using systems biology and pathway network analysis, we show that GN-25 could cause reversal of EMT leading to mesenchymal-to-epithelial transition (MET) in a well-recognized HMLE-SNAIL and Kras-HMLE-SNAIL models.
GN-25 induced MET was found to be consistent with growth inhibition, suppression of spheroid forming capacity and induction of apoptosis. Pathway network analysis of mRNA expression using microarrays from GN-25 treated Kras-HMLE-SNAIL cells showed an orchestrated global re-organization of EMT network genes. The expression signatures were validated at the protein level (down-regulation of mesenchymal markers such as TWIST1 and TWIST2 that was concurrent with up-regulation of epithelial marker E-Cadherin), and RNAi studies validated SNAIL dependent mechanism of action of the drug. Most importantly, GN-25 modulated many major transcription factors (TFs) such as inhibition of oncogenic TFs Myc, TBX2, NR3C1 and led to enhancement in the expression of tumor suppressor TFs such as SMAD7, DD1T3, CEBPA, HOXA5, TFEB, IRF1, IRF7 and XBP1, resulting in MET as well as cell death.
Our systems and network investigations provide convincing pre-clinical evidence in support of the clinical application of GN-25 for the reversal of EMT and thereby reducing cancer cell aggressiveness.
The epithelial-to-mesenchymal transition (EMT) and the reverse process, termed the mesenchymal-to-epithelial transition (MET), plays a central role in cancer progression and cell death. Cells undergoing EMT are characterized by their elongated morphology, inherent aggressiveness, propensity to maintain in long term cell culture conditions that is reminiscent of stem cell characteristics, which is also associated with resistance to standard chemotherapies and targeted therapies. Regimens designed to hit bulk of the tumor cells, in most cases do not eliminate these EMT sub-population of cells and this has been suggested to be the underlying cause for drug resistance and tumor recurrence. Therefore, targeted elimination of these EMT cells would be an important prerequisite for achieving optimal results for successful anti-cancer therapy.
SNAIL family of proteins have been shown to play an important role in the acquisition of malignant (aggressiveness) phenotype of epithelial tumors. SNAIL homologues are thought to act as transcriptional repressors and show a conserved function in mesoderm development from flies to mammals. Their role in de-lamination and migration is mediated by triggering the processes that leads to the acquisition of EMT by directly repressing the transcription of E-cadherin. Activation of SNAIL has been shown in pathological specimens at the invasive front of chemically induced mouse skin tumors, mammary, ovarian and in human breast carcinomas. A number of different signaling pathways such as TGF-β, BMP, FGF and Wnt signaling have been implicated in the induction of Snail family members during the process of EMT. Based on its critical role, targeted inhibition of SNAIL proteins has been investigated in pre-clinical setting as a therapeutics strategy to reverse EMT phenotype.
Even though many different cell models have been developed that mimic the genotypic and phenotypic characteristics of EMT; however, the Weinberg’s HMLE-SNAIL models stand out to be very useful among many others. This is in part due to the fact that EMT induction in HMLEs is driven by over-expression of a single mesenchymal driver i.e. SNAIL. These and related HMLE cells have been well characterized for their unique expression signatures that promote EMT in earlier studies. These cells have also been shown to form spheroids, maintain survival in long term cell culture condition, and carry markers of cancer stem-like cells (over-expression of Vimentin, ZEB1, TWIST 1 and TWIST 2 and down-regulation of epithelial markers E-Cadherin). Therefore, HMLEs could serve as an excellent tool for investigating perturbations induced by agents specific towards SNAIL (SNAIL inhibitors).
Previously, our group has developed a specific SNAIL-p53 interaction inhibitor GN-25, and this drug was originally designed to disrupt SNAIL-p53 interaction, thereby removing the p53-post-translational regulatory control and rescuing the cell surveillance functions of this master regulator. GN-25 is cancer cell specific and does not induce growth inhibition or apoptosis in normal immortalized cells. In the current study, we found that GN-25 could reverse the EMT phenotype and could also induce apoptosis in cancer cells, which prompted us to further investigate the mechanism of action of GN-25 against HMLE-SNAIL model. In this report, we performed network analysis using the HMLE-SNAIL models (EMT phenotypic cells induced by stable transfection with SNAIL) pre- and post-treatment with a SNAIL inhibitor GN-25. Our network analysis and biological validation showed that (a) EMT in HMLE-SNAIL arises through a complex crosstalk between different mesenchymal phenotype promoting networks of pathways, and (b) SNAIL inhibitor induces a coordinated set of perturbations that re-align the EMT networks with reversal to MET phenotype.
GN-25 induces growth inhibition in SNAIL-transduced HMLE cell line models
GN-25 suppresses spheroid formation in SNAIL transduced HMLE cells
We then investigated whether our drug could suppress the propensity of SNAIL-Transduced Kras-HMLEs to form spheroids using sphere-forming assay. As can be seen from of photomicrographs presented in Figure 2B (control, DMSO-treated spheres), all cells were able to grow as spheroids in 3D culture. However, upon 20 μM GN-25 treatment there was a marked disintegration of spheres in all three cell lines tested (Figure 2B left panels). These results clearly showed that SNAIL inhibitor GN-25 not only suppresses growth of HMLEs cells, but also reduces their ability to form spheroids in 3 D culture.
GN-25 induces apoptosis in HMLE EMT cell models
Effect of SNAI2 siRNA on GN-25 activity
As SNAIL is the primary target of GN-25, we investigated how the GN-25 drug compared with the effects of SNAIL-targeted siRNA on the induction of apoptosis. As can be seen from the results presented in Figure 3C, the apoptotic potential of GN-25 was similar to that of control siRNA. However, introduction of SNAI2-targeted siRNA when combined with GN-25 appears to enhance the degree of apoptosis. In Kras-HMLE cells (that have not been transduced with SNAIL), the siRNA treatment did not enhance the activity of the drug. These results provided the proof-of-concept showing that SNAIL protein is a direct target of GN-25. However, further in-depth analysis is needed to analyze what apparently constitute the broader effects of GN-25 treatment in order to understand the genes and functions that are deregulated during the reversal of EMT to MET after treatment with GN-25, and for which microarray and pathway modeling experiments were performed.
Systems-level analyses of gene expression level changes induced by GN-25 treatment specifically in SNAIL-transduced HMLE cell lines
Impact of GN-25 treatment on transcription factor programs in Kras-HMLE-SNAIL cells
Predicted activation state
p-value of overlap
Biological validation of network genes at expression level
Discussion and conclusion
Here we report, for the first time, the network analysis and biological validation of the EMT reversing perturbations induced by a SNAIL inhibitor GN-25 in the HMLE-based model system. Using systems level investigations, we showed that GN-25 induces MET and consequently growth inhibition and induction of apoptosis in SNAIL-transduced HMLE cells through coordinated suppression of EMT network genes. Our findings also highlight the role of a number of secondary players that cumulatively support GN-25’s mechanism of action. These findings demonstrate that the successful design of drugs against EMT should not only be focused on EMT specific genes but additional secondary networks that may require a promiscuous targeting by drugs that have pleiotropic mode of action.
EMT confers mesenchymal properties to epithelial cells, and this has been closely associated with aggressiveness of carcinoma cells. Emerging research shows that EMT programs are orchestrated not by one, but a set of pleiotropically acting transcription factors (TFs). The actions of these EMT-TFs enable the propensity for early steps of metastasis; local invasion and subsequent dissemination of carcinoma cells to distant sites. The genetic and epigenetic mechanisms that regulate the activation of EMT-TFs and the traits they induce are areas under intensive investigation. Such studies are expected to provide new opportunities for therapeutic intervention and may help to overcome tumor heterogeneity and therapeutic resistance.
The discovery and development of HMLE cell line models were facilitated through introduction of hTERT, which encodes the catalytic subunit of the human telomerase holoenzyme, as well the SV40 early region. Additionally, introduction of EMT promoting genes such as snail and twist in these models facilitated the reprogramming of the transduced and transformed HMLE cells to give rise to EMT phenotype. These developments have helped to molecularly, understand the basis of this complex phenomenon. This has, in turn, driven the research on pharmaceutical strategies that target reversal of EMT. Nevertheless, EMT is a complex process arising from the de-regulations in complex biological networks. These EMT biological networks cannot be investigated in isolation (using reductionist approaches), and thus it requires advanced, holistic and systems level analyses. This is especially needed in order to develop drugs that reverse EMT.
While Weinberg’s EMT cell line models have been the subject of individual-set of differentially expressed (DE) gene analyses using the t-test and the F-test, these are still insufficient knowledge to interrogate the EMT phenomena, which is in part due to the presence of additional genes that do not meet the DE criteria. Such analysis cannot extract EMT-specific characterization of mesenchymal pathway genes; i.e. identifying the distinguishing set of mesenchymal patterns in the entire co-expressed gene groups that may be specific to EMT only. Additionally, to date there are no drug related studies reported that showed alterations in broader effects on EMT signaling or functional networks. Here, we showed a network-based differential analysis model for analyzing the topological differences between two gene networks constructed from the expression data from GN-25 treated cells. We selected Kras-HMLE-SNAIL over HMLE-SNAIL for our network analysis since earlier it was shown that Kras transforms HMLE cells and drives SNAIL expression through the activation of Gli. Supporting this notion, Morel and colleagues have shown that sequential retroviral-mediated expression of the telomerase catalytic subunit (giving rise to HMEC/hTERT cells), SV40 large T and small t antigens (HMLE cells) and an oncogenic allele of H-Ras, H-RasV12 (HMLER cells) accelerates EMT. This allowed us to ask two questions: (1) what are the networks modulated in response to our SNAIL inhibitor, and (2) is there a compensatory influence of oncogenic Ras on GN-25 activity.
Our in-depth network analyses provided insights for multiple factors that are involved in what can be considered on-target drug predictions (e.g., the functional up-regulation of certain transcription factors resulted from GN-25 treatment) consistent with what is known about EMT. The analysis also presented broader secondary downstream or pleiotropic effects of the drug that may enhance its effectiveness in reversing EMT. Further, the analysis points to pathways where de novo or acquired resistance mechanisms may be the important route. These preliminary investigations demonstrate that drug design guided solely by presumed targets and differentially expressed genes may not be successful in reversing EMT due to the presence of multiple factors that function together to reinforce the phenotype. However, as shown by our network results, agents such as GN-25, with far-reaching effects (i.e. with inherent network pharmacology properties), can better serve the purpose in reversing EMT phenotype by not only directly targeting an assumed target and differentially expressed genes, but also secondary yet important signaling pathways or functional networks. In conclusion, our network investigations provided convincing pre-clinical rationale in support of the clinical application of GN-25 and related agents for the treatment of EMT cells in order to overcome therapeutics resistance of aggressive and metastatic cancers.
Cell lines and culture conditions, and research reagents
SNAIL-transduced HMLEs (HMLE-SNAIL, Kras-HMLE and Kras-HMLE-SNAIL) were generously provided by Dr. Robert Weinberg, Whitehead Institute, Massachusetts. SNAIL inhibitor GN-25 was developed as documented previously. Quercetin; an indirect inhibitor of SNAIL was purchased from SIGMA (St Louis USA). Primary antibodies for SNAIL, Vimentin, TWIST1 and TWIST2 were purchased from Cell Signaling (Danvers, MA). All the secondary antibodies were obtained from Sigma (St. Louis, MO).
Cell growth inhibition by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT)
SNAIL-transduced HMLE cells were seeded at a density of 5 × 103 cells per well in 96-well micro-titer culture plates. After overnight incubation, medium was removed and replaced with fresh medium containing GN-25 at indicated concentrations (0–25 μM) diluted from a 10 mM stock or Quercetin (used as positive control at 20 μM). After 72 hours of incubation, MTT assay was performed by adding 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Sigma (St. Louis, MO) solution (5 mg/mL in PBS) to each well and incubated further for 2 hours. Upon termination, the supernatant was aspirated and the MTT formazan formed by metabolically viable cells was dissolved in 100 μL of isopropanol. The plates were gently rocked for 30 minutes on a gyratory shaker, and absorbance was measured at 595 nm using a plate reader (TECAN, Durham, NC).
Sphere formation/disintegration assay
Briefly, single-cell suspensions of HMLE-SNAIL, Kras-HMLE and K-ras-HMLE-SNAIL were plated on ultra–low adherent wells of 6-well plates (Corning) at 1,000 cells per well in sphere formation medium (1:1 DMEM/F12 medium supplemented with B-27 and N-2; Invitrogen). After 7 days, the spheres were collected by centrifugation (300 xg, 5 minutes) and counted. The proportion of sphere-generating cells was calculated by dividing the number of spheres by the number of cells seeded. Single-cell suspensions of spheres were plated at 500 cells per well in the sphere formation medium. After 1 or 3 weeks of incubation with GN-25, secondary spheres were harvested for counting as described above. For sphere disintegration assay, 1,000 cells per well on ultra–low adherent wells of 6-well plate were incubated for a total of 10 days following 5 days of drug treatment, and the cells were harvested as described previously. The spheres were collected by centrifugation and counted under a microscope as described above.
Quantification of apoptosis by histone DNA ELISA and annexin V FITC assay
Cell Apoptosis was detected using Annexin V FITC (Biovision Danvers MA) and Histone DNA ELISA Detection Kit (Roche, Life Sciences) according to the manufacturer's protocol. HMLE cells were seeded at a density of 50,000 cells per well in six-well plates in 5 ml of corresponding media. 24 hrs after seeding the cells were exposed to GN-25 at different concentrations for 72 hrs. At the end of treatment period cells were trypsinized and equal numbers were stained with Annexin V and Propidium Iodide. The stained cells were analyzed using a Becton Dickinson flow cytometer at the Karmanos Cancer Institute Flow cytometry core. The second apoptosis assay quantifies histone-complexed DNA fragments (mono- and oligonucleosomes) from the cytoplasm of cells after the induction of apoptosis or when released from necrotic cells. Since the assay does not require pre-labeling of cells, it can detect internucleosomal degradation of genomic DNA during apoptosis. All procedures were performed according to our previously published protocol.
Immunofluorescence and confocal microscopy
Cells were grown on glass chamber slides and exposed to GN-25 at indicated concentrations for 24 hrs. In another set of experiments, at the end of the treatment the cells were fixed with 10% paraformaldehyde for 20 min. The fixed slides was blocked in TBST and probed with primary and secondary antibody according to our previously published methods. The slides were dried and mounting medium was added to it and covered with a coverslip and were analyzed under an inverted fluorescent microscope. A total of three independent experiments were performed.
Western blot analysis
HMLE-SNAIL and Kras-HMLE-SNAIL cells were grown in 100 mm petri-dishes over night to ~70% confluence. Next day, cells were exposed to indicated concentrations of GN-25 for 24 hrs followed by extraction of protein for western blot analysis. Preparation of cellular lysates, protein concentration determination and SDS-PAGE analysis was done as described previously.
siRNA and transfection
To study the effect of SNAIL silencing on activity of GN-25, we utilized siRNA silencing technology. SNAI2 siRNA and control siRNA were obtained from Santa Cruz Biotechnology. Cells were transfected with either control siRNA or SNAIL siRNA for 24 hrs using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. All procedures have been standardized and published previously. After the siRNA treatment period, cells were further treated with GN-25 (at IC50 concentration) in 96-well plates for MTT and 6-well plates for Annexin V FITC assays, respectively. Knock-down efficiency was evaluated by western blot analysis.
Microarrays and gene expression data analysis
K-ras-HMLE-SNAIL cells were plated so that they reached 75% confluence after 3 days. At this point, cultures were treated with or without GN-25 (15 μM). Total RNA was isolated from four sets of parallel plated culture plates, treated with or without GN-25, at 24 hours after the addition of inhibitor. Media was changed the day after plating and at the start of treatment. Total RNA quantity and quality was determined by analysis using the NanoDrop 1000 and Agilent Bioanalyzer (Agilent Technologies). All analyzed samples had RIN scores ≥ 7. Whole-genome expression levels were analyzed by a two-color microarray-based approach. A treated and untreated sample for one cell line was combined and hybridized to Agilent 4x44k human arrays and scanned with the Agilent G2505B microarray scanner system. Data quality was assessed, and data were processed by Agilent Feature Extraction software that produced expression data measures including LogRatio expression levels, LogRatio Error and P Value LogRatio. Features included in further analysis were annotated, gene-level that passed a p Value LogRatio cut-off ≤ 0.001. ANOVA analysis and multi-test correction (Benjamin-Hochberg p ≤ 0.05) was done using Partek software to compare the 2 sets of 4 two-color arrayed replicates (the 2 sets were Kras-HMLE and Kras-HMLE-SNAIL cell lines), to identify gene expression level changes (≥2 fold-change) that were uniquely affected by GN-25 treatment in the context of SNAIL-transduced Kras-HMLE cell line compared to GN-25 effects on the expression in the Kras-HMLE cell line (ANOVA p ≤ 0.001). The net result was a set at 2,737 genes, and corresponding HMLE-SNAIL expression ratios (GN-25-treated versus vehicle control) that were examined in follow-up systems-level analyses.
Availability of supporting data
The supporting microarray data is publicly available and can be freely accessed at the following link:http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE44397.
We thank Dr. Robert Weinberg for generously providing the key research reagents (HMLE-SNAIL paired cells) for this study. Grant Support: NIH Grant support 1 R21 CA175974 01 to RM is acknowledged.
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