Induction of E-cadherin in lung cancer and interaction with growth suppression by histone deacetylase inhibition

Abstract

Introduction: Loss of E-cadherin confers a poor prognosis in lung cancer patients and is associated with in vitro resistance to endothelial growth factor receptor inhibitors. Zinc finger E box-binding homeobox (ZEB)-1, the predominant transcriptional suppressor of E-cadherin in lung tumor lines, recruits histone deacetylases (HDACs) as co-repressors.

Methods: NSCLC cell lines were treated with HDAC inhibitors and analyzed for E-cadherin induction, growth inhibition and apoptosis. National Cancer Institute-H157 cells expressing ectopic E-cadherin were tested for tumorigenicity in murine xenografts.

Results: We found that treatment with MS-275, compared to vorinostat (SAHA), valproic acid or trichostatin A, was most effective in E-cadherin up-regulation and persistence in non-small cell lung cancers. As with other tumor types and HDAC inhibitors, MS-275 inhibited growth and induced apoptosis. Importantly, blocking E-cadherin induction by short hairpin RNA resulted in less inhibition by MS-275, implicating the epithelial to mesenchymal phenotype process as a contributing factor. In contrast to H460 and H661, H157 cells were resistant to E-cadherin up-regulation by HDAC inhibitors. However, E-cadherin was restored, in a synergistic manner, by combined knockdown of ZEB-1 and ZEB-2. In addition, H157 cells stably transfected with E-cadherin were markedly attenuated in their tumor forming ability. Lastly, combining MS-275 with the microtubule stabilizing agent, paclitaxel, or 17-(allylamino)-17-demethoxygeldanamycin, a heat shock protein 90 inhibitor, resulted in synergistic growth inhibition. Since MS-275 has no reported activity against HDAC6, which regulates both microtubule and heat shock protein 90 functions, other mechanisms of synergy are anticipated.

Conclusions: These results support the role of ZEB proteins and HDAC inhibitors in the pathogenesis and treatment of lung cancer.

Figure 1. Induction of E-cadherin in NSCLC cell lines by HDAC inhibition

(A) Time course of E-cadherin induction in H661 and H460 cells with TSA, VPA, MS-275 and vorinostat (VOR). The indicated concentrations of each agent were added to cultures at time zero and samples taken over four days; agents were present throughout the time course. Equal aliquots of protein lysates were immunoblotted for E-cadherin. Equivalent loading was verified using anti-β-actin (H661) or Coomassie blue staining (H460, Coom.). (B) Dose-response to MS-275 and vorinostat. Cultures of H661 and H460 cells were treated with the indicated concentrations of MS-275 or vorinostat for 24 hours and analyzed for E-cadherin levels and actin. (C) Persistence of E-cadherin induction. H661 cells were treated with TSA or MS-275 for 12 hours followed by wash-out with fresh medium at zero time. Samples were then harvested at the indicated time points and analyzed for E-cadherin and actin. (D) Time-course of acetylated histone H4 accumulation by HDAC inhibitors. H661 and H460 cells were treated with the indicated concentrations of TSA and VPA and samples analyzed for acetylated Histone H4 levels over time.

Figure 2. Growth inhibition following MS-275 treatment in NSCLC lines

MTT growth assays for three representative cell lines show the extreme responses among the panel of 12 NSCLCs tested. Cells were seeded into 96-well microtitre plates at between 2000 and 5000 per well. MS-275 was added from between 25 nM to 25 μM and treatment continued for 3 days. IC₅₀ values for the complete cell line panel are provided in Table 1.

Figure 3. E-cadherin induction contributes to anti-proliferative effects of HDAC inhibition

(A) NCI-H661 cells were stably transfected with a shRNA construct targeting CDH1 mRNA or a non-targeting control. Transfected cells were tested for E-cadherin protein levels by Western blot after 24h MS-275 treatment using the indicated doses. (B) E-cadherin knock-down increased resistance to MS-275. MTT assays were performed on control and CDH1 knock-down H661 cells. Values are the average of two independent experiments, each performed in triplicate. Growth was significantly restored in CDH1 knock-down cells compared to controls when treated with 4 μM MS-275 (p = 0.001, Tukey’s HSD test).

Figure 4. ZEB-1 and -2 inhibit E-cadherin in H157 cells in a HDAC class I/II independent manner

(A) Six NSCLC cell lines with negative or low E-cadherin expression were treated for 24h with TSA (0.33 μM) or MS-275 (5 μM). Protein lysates (10 μg) were analyzed for induced E-cadherin levels on Western blots. E-cadherin remained undetectable in H157, H1299 and H1703 cells after HDACi treatment. E-cadherin precursor (135 kDa) was observed only in H2122 (mature E-cadherin migrates at 120 kDa). (B) Synergistic induction of E-cadherin message by joint knockdown of ZEB-1 and -2. H157 cells were mock-transfected (mock), or transfected with control, ZEB-1 or ZEB-2-specific siRNAs, alone and in combination. Cells were harvested 48h post-transfection and processed for RNA and protein. ZEB-1, ZEB-2 and CDH1 were analyzed by realtime RT-PCR; bars are plotted as log₁₀ of expression relative to GAPDH. (C) Protein samples from similarly treated cells were analyzed for ZEB-1 and E- cadherin on Western blots. ZEB-1-specific siRNA achieved substantial knockdown of protein (b, lane 3) but no further knockdown was observed upon addition of a second anti-ZEB-1 siRNA (b+c, lane 4). Knockdown of ZEB-2 was verified by realtime PCR since available anti-ZEB-2 antibodies were non-specific. E-cadherin was detectably induced only in cells knocked down for both ZEB-1 and -2 (lane 6).

Figure 5. E-cadherin expression blocks tumorigenicity of NCI-H157 cells

(A) NCI-H157 cells were stably co-transfected with pEGFP-C1 and an E-cadherin expression construct (independent clones in lanes 3 and 4, pBATEM) or pEGFP-C1 alone (lane 2). Western blot for E-cadherin verified expression (top) while tubulin provided a loading control (bottom). (B) Orthotopic tumorigenicity assay. Ten million cells (H157/GFP controls or H157/E-cadherin transfectants) were introduced intra-tracheally into the left lung of irradiated athymic nude rats (6 per group), and tumor growth monitored for 60 days. H157 cells stably transfected with EGFP-C1 formed rapidly growing, metastatic tumors by 42 days post-inoculation (dark arrowhead). The E6 transfectant at 123 days had formed a typically small tumor (white arrowhead) in the left lung without metastasis. (C) Kaplan-Meier survival plots are shown for animals injected with the indicated transfectants (6 animals per group).

Figure 6. MS-275 induces apoptosis in NSCLC cell lines

(A–D) The indicated NSCLC cell lines were incubated with medium (controls), MS-275 (10 μM) or TSA (0.33 μM), as indicated, for 48 h. The percentage of apoptotic cells was determined by flow cytometry using dual staining with YO-PRO1 and propidium iodide. (A, C) Flow analysis of untreated control cultures for H661 (A) and H290 (C). (B, D) Flow analysis of H661 cells after treatment with TSA (B) and H290 cells after treatment with MS-275 (D). (E and F) MS-275 activates caspase-3 in sensitive NSCLC cell lines. (E, F) The moderately sensitive line H647 and the resistant line A549, were treated with MS-275 at different doses (E) or times (F) and examined for biochemical changes indicative of apoptosis. In (E), cultures were treated for 36 hours with the indicated doses of MS-275 (μM); in (F), cultures were treated with 5 μM MS-275 for the indicated times. Lysates wereanalyzed by Western blot for activated caspase 3 and poly ADP ribose polymerase (PARP) cleavage. Tubulin provided loading controls. (G) Bcl-2 protein levels were measured in 14 NSCLC lines, as indicated. Signals from a shorter exposure (not shown) were densitized and normalized to the level obtained in A549 to yield relative expression (Rel Exp). A Coomassie stained gel (Coom.) provided a loading control.