Grainyhead genes are involved in wound healing and developmental neural tube closure. In light of the high degree of similarity between the epithelial-mesenchymal transitions (EMT) occurring in wound healing processes and the cancer stem cell-like compartment of tumors, including TGF-β-dependence, we investigated the role of the Grainyhead gene, Grainyhead-Like-2 (GRHL2) in oncogenic EMT. GRHL2 was down-regulated specifically in the claudin-low subclass breast tumors and in basal-B subclass breast cancer cell lines. GRHL2 suppressed TGF-β-induced, Twist-induced or spontaneous EMT, enhanced anoikis-sensitivity, and suppressed mammosphere generation in mammary epithelial cells. These effects were mediated in part by suppression of ZEB1 expression via direct repression of the ZEB1 promoter. GRHL2 also inhibited Smad-mediated transcription and it upregulated mir200b/c as well as the TGF-β receptor antagonist, BMP2. Lastly, ectopic expression of GRHL2 in MDA-MB-231 breast cancer cells triggered a mesenchymal-to-epithelial transition and restored sensitivity to anoikis. Taken together, our findings define a major role for GRHL2 in the suppression of oncogenic EMT in breast cancer cells.

A hallmark of Alzheimer's disease (AD) is the rearrangement of the β-amyloid (Aβ) peptide to a non-native conformation that promotes the formation of toxic, nanoscale aggregates. Recent studies have pointed to the role of sample preparation in creating polymorphic fibrillar species. One of many potential pathways for Aβ toxicity may be modulation of lipid membrane function on cellular surfaces. There are several mutations clustered around the central hydrophobic core of Aβ near the α-secretase cleavage site (E22G Arctic mutation, E22K Italian mutation, D23N Iowa mutation, and A21G Flemish mutation). These point mutations are associated with hereditary diseases ranging from almost pure cerebral amyloid angiopathy (CAA) to typical Alzheimer's disease pathology with plaques and tangles. We investigated how these point mutations alter Aβ aggregation in the presence of supported lipid membranes comprised of total brain lipid extract. Brain lipid extract bilayers were used as a physiologically relevant model of a neuronal cell surface. Intact lipid bilayers were exposed to predominantly monomeric preparations of Wild Type or different mutant forms of Aβ, and atomic force microscopy was used to monitor aggregate formation and morphology as well as bilayer integrity over a 12 hour period. The goal of this study was to determine how point mutations in Aβ, which alter peptide charge and hydrophobic character, influence interactions between Aβ and the lipid surface. While fibril morphology did not appear to be significantly altered when mutants were prepped similarly and incubated under free solution conditions, aggregation in the lipid membranes resulted in a variety of polymorphic aggregates in a mutation dependent manner. The mutant peptides also had a variable ability to disrupt bilayer integrity.

The mammalian homologs of the D. melanogaster Grainyhead gene, Grainyhead-like 1-3 (GRHL1, GRHL2 and GRHL3), are transcription factors implicated in wound healing, tubulogenesis and cancer. Their induced target genes encode diverse epithelial cell adhesion molecules, while mesenchymal genes involved in cell migration and invasion are repressed. Moreover, GRHL2 suppresses the oncogenic epithelial-mesencyhmal transition, thereby acting as a tumor suppressor. Mechanisms, some involving established cancer-related signaling/transcription factor pathways (for example, Wnt, TGF-β, mir200, ZEB1, OVOL2, p63 and p300) and translational implications of the Grainyhead proteins in cancer are discussed in this review article.

Resistance to anoikis is a pre-requisite for tumor metastasis. The epithelial-to-mesenchymal transition (EMT) allows tumor cells to evade anoikis. The wound healing regulatory transcription factor Grainyhead-like 2 (GRHL2) suppresses/reverses EMT, accompanied by suppression of the cancer stem cell (CSC) phenotype and by re-sensitization to anoikis. Here, the effects of GRHL2 upon intracellular metabolism in the context of reversion of the EMT/CSC phenotype, with a view toward understanding how these effects promote anoikis sensitivity were investigated. EMT enhanced mitochondrial oxidative metabolism. While this was accompanied by higher accumulation of superoxide, the overall level of Reactive Oxygen Species (ROS) declined, due to decreased hydrogen peroxide. Glutamate Dehydrogenase 1 (GLUD1) expression increased in EMT, and this increase, via the product α-ketoglutarate (α-KG), was important for suppressing hydrogen peroxide and protecting against anoikis. GRHL2 suppressed GLUD1 gene expression, decreased α-KG, increased ROS and sensitized cells to anoikis. Implications These results demonstrate a mechanistic role for GRHL2 in promoting anoikis through metabolic alterations.

GRHL2 suppresses EMT to give a default epithelial phenotype. GRHL2 inhibits this process through the histone acetyltransferase coactivator p300, repressing the partial EMT and preventing induction of MMPs. The results demonstrate novel roles for p300 and GRHL2 in promoting or suppressing EMT in morphogenesis and tumor progression.