Epithelial-mesenchymal transition and tumor suppression are controlled by a reciprocal feedback loop between ZEB1 and Grainyhead-like-2

Abstract

Epithelial-mesenchymal transition (EMT) in carcinoma cells enhances malignant progression by promoting invasion and survival. EMT is induced by microenvironmental factors, including TGF-β and Wnt agonists, and by the E-box-binding transcription factors Twist, Snail, and ZEB. Grainyhead-like-2 (GRHL2), a member of the mammalian Grainyhead family of wound-healing regulatory transcription factors, suppresses EMT and restores sensitivity to anoikis by repressing ZEB1 expression and inhibiting TGF-β signaling. In this study, we elucidate the functional relationship between GRHL2 and ZEB1 in EMT/MET and tumor biology. At least three homeodomain proteins, Six1, LBX1, and HoxA5, transactivated the ZEB1 promoter, in the case of Six1, through direct protein-promoter interaction. GRHL2 altered the Six1-DNA complex, inhibiting this transactivation. Correspondingly, GRHL2 expression prevented tumor initiation in xenograft assays, sensitized breast cancer cells to paclitaxel, and suppressed the emergence of CD44(high)CD24(low) cells (defining the cancer stem cell phenotype in the cell type studied). GRHL2 was downregulated in recurrent mouse tumors that had evolved to an oncogene-independent, EMT-like state, supporting a role for GRHL2 downregulation in this phenotypic transition, modeling disease recurrence. The combination of TGF-β and Wnt activation repressed GRHL2 expression by direct interaction of ZEB1 with the GRHL2 promoter, inducing EMT. Together, our observations indicate that a reciprocal feedback loop between GRHL2 and ZEB1 controls epithelial versus mesenchymal phenotypes and EMT-driven tumor progression.

Figure 1. Homeodomain proteins LBX1 and Six1 activate ZEB1 expression and are inhibited by GRHL2 protein

(a) The region of interest from the ZEB1 promoter is conserved between various species: Ms: Mus musculus, Hs: Homo sapiens, Bp: Bos primigenius, Dr: Danio rerio. Bold=homeobox consensus sites; Italics=grainyhead consensus sites. (b). LBX1 and Six1 activate transcription from the ZEB1 promoter in a GRHL2-sensitive manner. The ZEB1 promoter-luciferase reporter with wild-type sequence (WT), the 3′ homeobox site mutated (MUT) or both the mutation of the latter site plus deletion of the 5′ homeobox binding site (DD) were co-transfected into MSP cells together with the indicated transcription factors; values represent the average of biological duplicates from one representative of two experiments. (c) Six1 induces the endogenous ZEB1 gene and EMT in unfractionated HMLE cells. HMLE cells expressing vector or FLAG-Six1 were analyzed for the indicated EMT markers (upper panel); or morphologic changes (lower panel).

Figure 2. GRHL2 and Six1 bind specifically to sequences in the ZEB1 promoter, and GRHL2 alters this complex

Wild-type (wt) or mutant (mut) oligonucleotides corresponding to the conserved sequence of interest from the ZEB1 promoter were used to analyze and demonstrate the specificity of GRHL2 protein (a) or Six1 (b) protein interactions. (c) GRHL2 positive control oligo (GRHL2PC), Six1 positive control oligo (Six1PC), oligo from the E-cadherin promoter which neither protein is predicted to bind (NC), and WT ZEB1 promoter oligo were used in an EMSA with the indicated combinations of GRHL2 and Six1 proteins. (d). Six1 protein interacts with the ZEB1 promoter in vivo. Chromatin from HMLE expressing either vector or FLAG-Six1 were used in a CHIP assay and analyzed by qRT-PCR using ZEB1 promoter primers or GAPDH control primers. Values are the average of biological duplicates from one representative of two experiments.

Figure 3. GRHL2 suppresses tumor initiation capacity and sensitizes breast cancer cells to chemotherapy-induced cytotoxicity

(a) GRHL2 suppresses tumor initiation (knockdown approach). Immunodeficient mice were injected orthotopically with HMLER cells expressing control shRNA vs. GRHL2 shRNA (one of two GRHL2 shRNAs shown previously to suppress EMT in this cell line (22)); individual tumor volumes at ten weeks are shown. (b) GRHL2 suppresses tumor initiation (over-expression approach). MDA-MB-231LN cells expressing GRHL2 (three individual clones, labeled G1, G2, G3) or MDA-MB-231LN cells with empty vector (vec) were assayed at the indicated time points. (Mice bearing vector control tumors were sacrificed between 4.5–7 weeks due to signs of tumor burden-related illness.) Odds ratios for tumor initiation were 0.0108 and 0.0558 for the early and late time point data, respectively. (c). GRHL2 is down-regulated during the transition from primary to recurrent phenotype in tetO-Wnt1 and tetO-neuNT mouse models: qRT-PCR on RNAs from independent tumors, normalized against β2-microglobulin, conducted in technical duplicate.

Figure 4. Wnt and TGF-β cooperate to down-regulate GRHL2

HMLE cells treated with the canonical Wnt pathway agonist BIO (a) or stably expressing β-catenin S33Y (b) were assayed for GRHL2 down-regulation in response to TGF-β treatment by Western blotting; each is one representative of two duplicate experiments. (c) HMLE cells that stably expressed Twist-ER protein were treated with or without 4-OHT (to activate Twist-ER) in the presence or absence of the TGF-β pathway antagonist BMP2, and cell lysates were assayed for GRHL2 and other indicated protein expression.

Figure 5. ZEB1 down-regulates the GRHL2 promoter

(a, b): si/shRNA mediated knockdown of ZEB1 partially alleviated GRHL2 down-regulation caused by 4-OHT-activated Twist-ER protein (a) or BIO plus TGF-β treatment (b). (c) HMLE stably expressing a doxycycline-inducible ZEB1 expression construct were induced with doxycycline and lysates were analyzed for ZEB1, GRHL2, E-cadherin and Akt by Western blotting. (d) The GRHL2 promoter in a luciferase reporter vector was co-transfected with Snail, Twist or ZEB1 expression vectors in HMLE cells or the promoter was transfected by itself into MSP cells; normalized luciferase activity values are shown. (e) HMLE-MSP transfected with control or ZEB1 siRNA, were transfected with the GRHL2 promoter-luciferase construct; normalized luciferase activity values are shown. Values are the average of biological duplicates from one representative of two experiments.

Figure 6. ZEB1 protein interacts directly with the GRHL2 promoter

(a): CHIP–seq data (Encode project;(55)) for ZEB1 reveal a potential binding site near the transcription start site. (b) Confirmation of CHIP-seq. Chromatin from HMLE expressing tet-ON ZEB1 were subjected to CHIP using ZEB1 antibody and analyzed by qRTPCR using the indicated primers. The average of biological duplicates from one representative of two experiments is shown.

Figure 7. A reciprocal feedback loop between Grainyhead-like-2 and ZEB1 controls EMT and tumor suppression

Details are explained in the text.