No abstract

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-SCIDmice. 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.

Tumor organoids mimic the architecture and heterogeneity of in vivo tumors and enable studies of collective interactions between tumor cells as well as with their surrounding microenvironment. Although tumor organoids hold significant promise as cancer models, they are also more costly and labor-intensive to cultivate than traditional 2D cell culture. We sought to identify critical factors regulating organoid growth ex vivo, and to use these observations to develop a more efficient organoid expansion method. Using time-lapse imaging of mouse mammary tumor organoids in 3D culture, we observed that outgrowth potential varies nonlinearly with initial organoid size. Maximal outgrowth occurred in organoids with a starting size between~10 to 1000 cells. Based on these observations, we developed a suspension culture method that maintains organoids in the ideal size range, enabling expansion from 1 million to over 100 million cells in less than 2 weeks and less than 3 hours of hands-on time. Our method facilitates the rapid, cost-effective expansion of organoids for CRISPR based studies and other assays requiring a large amount of organoid starting material.

There is mounting evidence that tumor cells can migrate and metastasize as cell clusters. The presence of circulating tumor cell clusters is associated with early relapse and death in many cancer types. Experimentally, clustering of tumor cells greatly increases colonizing potential, but the molecular mechanisms explaining this phenomenon are largely unknown. We performed time‐lapse imaging and genome wide transcription studies in mouse and human breast tumor organoids. This revealed that clustering of tumor cells rapidly induces expression of multiple EGFR pathway genes and increased auto‐phosphorylation of EGFR. Interestingly, pEGFR colocalized with multiple EGFR ligands at cell‐cell contacts, suggesting that clusters' EGFR activation may be due to increased ligand binding. Consistent with this hypothesis, RNAi knockdowns of multiple individual ligands reduced tumor organoid growth by 40 to 95% of control. Downstream of receptor activation, expression of EGFR effector pERK was increased in clusters. The most significantly cluster‐upregulated transcription factor was the ERK‐activated oncogene Fra1. Fra1 knockdown reduced growth as did ERK inhibition. Using patient breast cancer samples, including primary and metastatic tumors, we found that Fra1 expression was highly upregulated in human tumor cell clusters, supporting the human disease relevance of this pathway. Taken together, our studies show that tumor cell clusters activate EGFR‐ERK‐Fra1 growth signaling at cell‐cell contacts. We propose that disrupting this pathway could reduce metastasis and extend survival in cancer patients.Support or Funding InformationI am grateful for funding support from the Burroughs Wellcome Fund, Department of Defense, Susan G. Komen, Breast Cancer Research Foundation, and the V Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Maintenance of cell polarity and tissue architecture are essential in preventing neoplasia. Three different protein complexes control cellular polarity, including the Par3/aPKC/Par6 complex, the Crumbs/Pals/Patj complex and the Scribble/Dlg/Lgl complex. Of these, only the loss of Lgl promotes massive tissue growth and cellular migration in Drosophila as well as altered cellular polarity. Humans have two well-conserved homologs, Hugl1 and Hugl2. Expression of Hugl1 is downregulated, lost, or mutated in many cancers, including colorectal, endometrial, hepatocellular carcinoma, malignant melanomas, and breast cancer. We have previously examined the role of Hugl1 and Hugl2 in breast epithelial cells and found that Hugl1 loss results in a failure of growth control, gain of mesenchymal phenotypes, and a loss of both apicobasal and planar polarity. We have now determined that these phenotypes represent a fundamental change in cellular differentiation and that loss of Hugl1 results in cellular transdifferentiation. Loss of Hugl1 expression in breast epithelial cells results in the induction of a mixed-phenotype population (composed of CD44high/CD49flow and CD44low/CD49fhigh), whose growth and migration are driven by the Epidermal Growth Factor Receptor (EGFR). These populations exhibit increased anchorage-independent growth and form soft agar colonies in an EGF-dependent manner, and these behaviors are selectively enhanced in the CD44high/CD49flow population. Hugl1 loss results in the mislocalization of EGFR, resulting in TAZ and SLUG nuclear translocation. Together, these data indicate that when Hugl1 is lost in normal epithelial cells, polarity loss is accompanied by a transdifferentiation and increased anchorage-independent growth and migration. Citation Format: Erin Greenwood, David Ebertz, Anthony Fabiano, Joyce A. Schroeder. Loss of Hugl1 induces an EGF-dependent cellular transdifferentiation. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A16.

Background: Invasion is a fundamental step in metastasis, the leading cause of breast cancer-related death. We have shown previously that primary breast tumors invade collectively as clusters of cancer cells (Cheung et al. Cell 2013). These clusters are led by a small subpopulation of cancer cells that highly express the basal epithelial protein cytokeratin 14 (K14) and other stem cell genes (Cheung et al. PNAS 2016). K14+ leader cells metastasize as clusters, reverting to a proliferative growth state upon colonization of a secondary site (Cheung and Ewald. Science 2016). The mechanisms regulating their transitions between collective invasion and growth states remains poorly understood. Understanding these mechanisms could produce new insights into tumor cell collectivity and uncover new ways to treat disseminated cell clusters that have already metastasized. Results: RNA-seq analysis of K14+ and K14- tumor cells revealed that K14+ tumor cells have increased expression of genes encoding the hemidesmosome, a major adhesion complex that plays an essential role in anchoring epithelial cells to the basement membrane. The highest upregulated hemidesmosome gene was collagen 17A1 (COL17A1), a transmembrane protein and structural component of the hemidesmosome that is involved in cell migration during wound healing and tissue regeneration. Consistent with these RNA-seq data, we observed by IF staining in vivo that K14 and COL17A1 are typically co-expressed in collective invasive strands at the invasive front of MMTV-PyMT mammary carcinomas. We next applied ex-vivo 3D culture assays to determine the function of COL17A1 in K14+ cell invasion and proliferation. In mammary tumor organoids we observed that COL17A1 is expressed in basal K14+ tumor cells. However, while the COL17A1 endodomain is uniformly expressed, the ectodomain is expressed in patches, indicating active ectodomain cleavage, a process that reduces cellular motility (Franzke et al. The EMBO Journal 2002). Surprisingly, loss of total COL17A1 expression by shRNA-knockdown significantly impairs tumor organoid growth ˜2 fold and prevents invasion in basement membrane rich matrigel. Further studies are underway to investigate the role of COL17A1 during collective invasion in other extracellular matrix environments. Conclusions: Taken together, our data shows that invasive leader cells express COL17A1 in 3D culture and in vivo and that this protein is required for tumor organoid invasion and growth. Therefore, therapeutically targeting COL17A1, a cell surface protein, could prevent both the migratory and proliferative phases of breast cancer metastasis. We are currently investigating how COL17A1 and its shed COL17A1 ectodomain enable leader cells to toggle between invasive and proliferative states. Citation Format: Greenwood E, Cane Gasull P, Moore B, Cheung K. The hemidesmosome protein collagen 17A1 is required for collective invasion and growth of mammary tumor organoids [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-01-14.

The properties of stem cells that participate in mammary gland branching morphogenesis remain contested. Reporting in Nature, Scheele et al. (2017) establish a model for post-pubertal mammary branching morphogenesis in which position-dependent, lineage-restricted stem cells undergo cell mixing in order to contribute to long-term growth.