We have previously demonstrated that Llgl1 loss results in a gain of mesenchymal phenotypes and a loss of apicobasal and planar polarity. We now demonstrate that these changes represent a fundamental shift in cellular phenotype. Llgl1 regulates the expression of multiple cell identity markers, including CD44, CD49f, and CD24, and the nuclear translocation of TAZ and Slug. Cells lacking Llgl1 form mammospheres, where survival and transplantability is dependent upon the Epidermal Growth Factor Receptor (EGFR). Additionally, Llgl1 loss allows cells to grow in soft-agar and maintain prolonged survival as orthotopic transplants in NOD-SCID mice. Lineage tracing and wound healing experiments demonstrate that mammosphere survival is due to enhanced EGF-dependent migration. The loss of Llgl1 drives EGFR mislocalization and an EGFR mislocalization point mutation (P667A) drives these same phenotypes, including activation of AKT and TAZ nuclear translocation. Together, these data indicate that the loss of Llgl1 results in EGFR mislocalization, promoting pre-neoplastic changes.

The Epidermal Growth Factor Receptor (EGFR) is associated with a variety of cancers, including brain, lung, cervix, renal and breast. It is part of a family of receptors known as the ErbB receptors (ErbB1/EGFR, ErbB2/HER2, ErbB3/HER3, and ErbB4/HER4), transmembrane proteins found on epithelial cells responsible for a multitude of signaling events. In cancers, EGFR is frequently mutated or improperly expressed, upregulated in more than 50 percent of basal-like cancers. Mutations commonly promote constitutive activation or increase receptor recycling. In basal-like breast cancers such as triple negative breast cancer (TNBC), named for the lack of hormone receptors (estrogen and progesterone) and the HER2 receptor, EGFR is highly upregulated and associated with a variety of oncogenic activity, including increased proliferation and migration, and inhibition of cell death. Changes in these pathways are predicated on altered trafficking and activation of EGFR, events driven by variation in stimuli and interacting partners, such as other ErbB family members or oncogenic adaptor proteins such as MUC1, a member of the mucin family. In TNBC, upon stimulus with epidermal growth factor (EGF), EGFR colocalizes with MUC1 in intracellular vesicles distributed throughout the cytoplasm. These intracellular vesicles are associated with early endosomes, as indicated by the presence of early endosome antigen 1 (EEA1). Association with MUC1 prolongs the presence of EGFR in these vesicles, as EGFR's stay is significantly reduced in cells lacking MUC1. Retention in these vesicles by MUC1 inhibits trafficking of EGFR to the lysosome for degradation and is also associated with an increase in EGF-dependent migratory ability. Introduction of late endosome inhibitors (thereby preventing lysosomal targeting) increases migration in the absence of MUC1, the same effect as in the presence of MUC1. Further, inhibition of retrograde trafficking significantly decreases the rate of migration and changes cellular distribution of filopodia corresponding to migratory ability in MUC1-containing cells. Taken together, these data indicate that MUC1 is responsible for altering EGFR trafficking by retaining EGFR in EEA1-positive vesicles for prolonged periods, allowing for increased signal transduction through retrograde trafficking of EGFR and structural reorganization promoting a migratory phenotype. Loss of the polarity protein Llgl1 is associated with alterations in EGFR trafficking, promoting highly diffuse EGFR distribution throughout the cytoplasm versus along basolateral membranes. These changes in trafficking are also associated with increases in AKT and dual-phosphorylated-ERK signal transduction, both downstream targets of activated EGFR. Altering localization of EGFR to other membranes and intracellular vesicles without inducing polarity loss through a point mutation at amino acid 667 was found to also upregulate the AKT pathway. Mislocalization driven by polarity loss or point mutation in the basolateral targeting domain is sufficient to increase migration speeds of non-cancerous epithelial cell lines in vitro. This increased oncogenic activity is likely attributed to increased nuclear localization of the transcription factor TAZ (transcription co-activator with a PDZ-binding domain), whose nuclear translocation is associated with increased stem-like properties such as migration and survival. Together, these data reveal the oncogenic potential caused by alterations in EGFR trafficking that occur when polarity is lost or EGFR is improperly associated with proteins that promote changes to canonical EGFR localization and degradation, such as MUC1.

Cell polarity is an important regulator of cellular processes and is vital in helping to prevent metaplasia and tumorigenesis. There are three many polarity complexes that regulate and maintain epithelial cellular polarity. The Par and Crumbs complexes locate to the apical membrane of the cell, while the Scribble complex is located basolaterally. Of the Scribble complex components, the polarity protein Hugl1, also known as Mgl1 in mice, is especially important in helping to maintain apical basolateral and planar polarity, and is lost in multiple types of cancer. When Hugl1 expression is lost in epithelial cells, it results in a mesenchymal phenotype. We now show that the loss of Hugl1 fundamentally shifts the cellular phenotype and specifically alters EGFR trafficking and signaling. Loss of Hugl1 results in the nuclear translocation of Taz and Slug, increased migration, and the mislocalization of EGFR (Epidermal Growth Factor Receptor), driving cellular growth. Hugl1 regulates the expression of multiple cell identity markers and its loss results in stem cell characteristics, including the increased expression of CD44, and a decrease of CD49f and CD24 expression. The loss of Hugl1 also results in increased growth in soft-agar and prolonged survival when transplanted into NOD-SCID mice; its loss also results in EGF-dependent migration which aids in increasing mammosphere survival. Furthermore, isolated EGFR mislocalization via a point mutation (P667A) also drives these same phenotypes, including activation of Akt and Taz nuclear translocation, indicating the importance of Hugl1 in the regulation of EGFR localization and its signaling. In mice, the loss of total Mgl1 is lethal within days of birth due to hydrocephaly and results in the formation of rosette like structures in the brain that are reminiscent of neuroectodermal tumors. We designed a targeted Mgl1 knockout in the mammary epithelial cells using the Cre/Lox system to evaluate the effects of Mgl1 loss in murine mammary gland development and tumorigenesis. The loss of Mgl1 expression in mice inhibits ductal outgrowth, increases side branching and epithelial layers, and results in the mislocalization of EGFR. While overt mammary tumors did not develop, some individuals did develop hyperplastic nodules that could progress into cancer. The knockdown of Hugl1 in vitro and Mgl1 in vivo reveal how the loss of polarity and presence of Hugl1 results in cancer stem cell characteristics, increased migration, and abnormal signaling due to the mislocalization of EGFR. While these changes result in metaplasia and a potential pre-cancerous state, the loss of Hugl1 alone is not enough to drive the cancer progression, indicating that other mutations or factors are necessary for the development of breast cancer. Because of the key role polarity plays in the prevention of breast cancer development we investigated if the addition of Hugl1 back into breast cancer cells could revert the cancerous cells to a normal epithelial phenotype. Most of the breast cancer cells transfected with Hugl1 expression did not survive, indicating that the re-expression of polarity regulators forces cancer cells to die. The small percentage of cells that did survive re-expression of Hugl1 had retarded growth in soft agar and a decrease in EGFR expression. Together, these data indicate that Hugl1 expression and EGFR activity are closely related and that Hugl1 is required for the proper localization and signaling of EGFR. When Hugl1 is lost, EGFR is mislocalized and fails to be degraded properly, promoting pre-neoplastic changes.