{"id":{"repo_id":"buffalo","oai_identifier":"oai:ubir.buffalo.edu:10477/86836"},"canonical_url":"https://search.dev.ndltd.org/etd/buffalo/oai:ubir.buffalo.edu:10477/86836","repository":{"repo_id":"buffalo","name":"Buffalo","base_url":"https://ubir.buffalo.edu/oai/request"},"display":{"title":"Regulation of the Hippo Signaling Pathway in Mammary Epithelial Cells","abstract":"Ph.D.","abstract_html":"Ph.D.","abstract_has_math":false,"creators":["Mussell, Ashley; 0000-0002-8260-6879"],"institution":"State University of New York at Buffalo","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["Zhang, Jianmin","Roswell Park"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2025,"date_issued":"2025-02-25T23:23:16Z","date_published":"2025-02-25T23:23:16Z","updated_at":"2026-07-27T19:05:37Z","subjects":["cellular biology","genetics","molecular biology"],"languages":["eng"],"rights":["Users of works found in University at Buffalo Institutional Repository (UBIR) are responsible for identifying and contacting the copyright owner for permission to reuse. University at Buffalo Libraries do not manage rights for copyright-protected works and cannot assist with permissions.","Copyright retained by author."],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/10477/86836","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Zhang, Jianmin","Roswell Park"]},{"key":"dc:creator","label":"Author","values":["Mussell, Ashley; 0000-0002-8260-6879"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2025-02-25T23:23:16Z","2020","2020-08-04 11:08:35"]},{"key":"dc:publisher","label":"Institution","values":["State University of New York at Buffalo"]},{"key":"dc:type","label":"Dc Type","values":["Text","Dissertation"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["cellular biology","genetics","molecular biology"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["Users of works found in University at Buffalo Institutional Repository (UBIR) are responsible for identifying and contacting the copyright owner for permission to reuse. University at Buffalo Libraries do not manage rights for copyright-protected works and cannot assist with permissions.","Copyright retained by author."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["http://hdl.handle.net/10477/86836"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Ph.D.","The Hippo signaling pathway is an evolutionarily conserved pathway originally discovered in Drosophila melanogaster and later found to have human orthologues. Regulation of this pathway is tightly controlled throughout development and in maintaining tissue homeostasis. Upstream regulators and external signaling nodes that converge upon this pathway to control Hippo gene activity has been extensively studied in both normal development and disease. Upon external stimuli from GPCRs, cell adhesion proteins, and various other signaling pathways a kinase cascade occurs. MST1/2 are phosphorylated and activated, resulting in the interaction with scaffold protein Sav1. This complex then induces the phosphorylation of LATS1/2, leading to activation and interaction with PTPN14 and KIBRA. LATS1/2 phosphorylates the key effector proteins of the pathway, YAP/TAZ, leading to their cytoplasmic retention or proteasomal degradation. Dysregulation of upstream regulators leaves YAP/TAZ in an un-phosphorylated state and allows their translocation to the nucleus. YAP/TAZ act as transcriptional co-activators of the TEAD family of transcription factors. The YAP-TAZ-TEAD complex drives transcription of genes associated with cell proliferation, cell cycle progression, epithelial to mesenchymal transition, metastasis, and stemness. While this pathway has been well established in cancer there are still many questions that need to be answered. KIBRA is a member of the WW domain-containing protein family and has recently been reported to be an upstream protein in the Hippo signaling pathway. The clinical significance of KIBRA deregulation and the underlying mechanisms by which KIBRA regulates breast cancer (BC) initiation and progression remain poorly understood. Here, we report that KIBRA knockdown in mammary epithelial cells induced epithelial-to-mesenchymal transition (EMT) and increased cell migration and tumorigenic potential. Mechanistically, we observed that inhibiting KIBRA induced growth factor-independent cell proliferation in 2D and 3D culture due to the secretion of amphiregulin (AREG), an epidermal growth factor receptor (EGFR) ligand. Also, we show that AREG activation in KIBRA-knockdown cells is dependent on the transcriptional coactivator YAP1. Significantly, decreased expression of KIBRA is correlated with recurrence and reduced BC patient survival. In summary, this study elucidates the molecular events that underpin the role of KIBRA in BC. As a result, our work provides biological insight into the role of KIBRA as a critical regulator of YAP1-mediated oncogenic growth and may have clinical potential for facilitating patient stratification and identifying novel therapeutic approaches for BC patients. USP1 is a deubiquitinating enzyme that allows for the removal of ubiquitin from target substrates, leading to increased protein stability or altered cellular trafficking. The role of USP1 has been well established in DNA damage response in multiple pathways including the Fanconi Anemia pathway, trans-lesion synthesis, and homologous recombination, however, how it regulates other genes is still under investigation. Herein we describe how loss of USP1 can alter TAZ protein stability leading to reduced proliferation. Mechanistically, we show that loss of USP1 reduces TAZ protein levels in both non-transformed and transformed mammary epithelial cells, without altering TAZ mRNA levels. Inhibition of the proteasome can rescue TAZ loss in the absence of USP1. Also, treatment of stable USP1 knockdown cells with cycloheximide showed a reduction in the half-life of TAZ proteins. Using ubiquitin-based immunoprecipitation assays we show that loss of USP1 can increase TAZ ubiquitination and this ubiquitin modification may occur at lysine 45 and 46 within TAZ. We further mapped the interaction domain within TAZ that confers its complexing with USP1. Significantly, USP1 and TAZ were shown to co-occur in Triple-Negative breast cancer patients and can reduce relapse free survival. Our data has shown a novel role of USP1 in controlling TAZ protein levels that may give insight into new treatment options for triple negative breast cancer patients.","**To request an accessible version of the file(s) associated with this item, contact library@buffalo.edu. Please include the item's persistent URL [http://hdl.handle.net/. . .] in your request.**"]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Regulation of the Hippo Signaling Pathway in Mammary Epithelial Cells"]}]}],"canonical_facts":{"dc:contributor":["Zhang, Jianmin","Roswell Park"],"dc:creator":["Mussell, Ashley; 0000-0002-8260-6879"],"dc:date":["2025-02-25T23:23:16Z","2020","2020-08-04 11:08:35"],"dc:description":["Ph.D.","The Hippo signaling pathway is an evolutionarily conserved pathway originally discovered in Drosophila melanogaster and later found to have human orthologues. Regulation of this pathway is tightly controlled throughout development and in maintaining tissue homeostasis. Upstream regulators and external signaling nodes that converge upon this pathway to control Hippo gene activity has been extensively studied in both normal development and disease. Upon external stimuli from GPCRs, cell adhesion proteins, and various other signaling pathways a kinase cascade occurs. MST1/2 are phosphorylated and activated, resulting in the interaction with scaffold protein Sav1. This complex then induces the phosphorylation of LATS1/2, leading to activation and interaction with PTPN14 and KIBRA. LATS1/2 phosphorylates the key effector proteins of the pathway, YAP/TAZ, leading to their cytoplasmic retention or proteasomal degradation. Dysregulation of upstream regulators leaves YAP/TAZ in an un-phosphorylated state and allows their translocation to the nucleus. YAP/TAZ act as transcriptional co-activators of the TEAD family of transcription factors. The YAP-TAZ-TEAD complex drives transcription of genes associated with cell proliferation, cell cycle progression, epithelial to mesenchymal transition, metastasis, and stemness. While this pathway has been well established in cancer there are still many questions that need to be answered. KIBRA is a member of the WW domain-containing protein family and has recently been reported to be an upstream protein in the Hippo signaling pathway. The clinical significance of KIBRA deregulation and the underlying mechanisms by which KIBRA regulates breast cancer (BC) initiation and progression remain poorly understood. Here, we report that KIBRA knockdown in mammary epithelial cells induced epithelial-to-mesenchymal transition (EMT) and increased cell migration and tumorigenic potential. Mechanistically, we observed that inhibiting KIBRA induced growth factor-independent cell proliferation in 2D and 3D culture due to the secretion of amphiregulin (AREG), an epidermal growth factor receptor (EGFR) ligand. Also, we show that AREG activation in KIBRA-knockdown cells is dependent on the transcriptional coactivator YAP1. Significantly, decreased expression of KIBRA is correlated with recurrence and reduced BC patient survival. In summary, this study elucidates the molecular events that underpin the role of KIBRA in BC. As a result, our work provides biological insight into the role of KIBRA as a critical regulator of YAP1-mediated oncogenic growth and may have clinical potential for facilitating patient stratification and identifying novel therapeutic approaches for BC patients. USP1 is a deubiquitinating enzyme that allows for the removal of ubiquitin from target substrates, leading to increased protein stability or altered cellular trafficking. The role of USP1 has been well established in DNA damage response in multiple pathways including the Fanconi Anemia pathway, trans-lesion synthesis, and homologous recombination, however, how it regulates other genes is still under investigation. Herein we describe how loss of USP1 can alter TAZ protein stability leading to reduced proliferation. Mechanistically, we show that loss of USP1 reduces TAZ protein levels in both non-transformed and transformed mammary epithelial cells, without altering TAZ mRNA levels. Inhibition of the proteasome can rescue TAZ loss in the absence of USP1. Also, treatment of stable USP1 knockdown cells with cycloheximide showed a reduction in the half-life of TAZ proteins. Using ubiquitin-based immunoprecipitation assays we show that loss of USP1 can increase TAZ ubiquitination and this ubiquitin modification may occur at lysine 45 and 46 within TAZ. We further mapped the interaction domain within TAZ that confers its complexing with USP1. Significantly, USP1 and TAZ were shown to co-occur in Triple-Negative breast cancer patients and can reduce relapse free survival. Our data has shown a novel role of USP1 in controlling TAZ protein levels that may give insight into new treatment options for triple negative breast cancer patients.","**To request an accessible version of the file(s) associated with this item, contact library@buffalo.edu. Please include the item's persistent URL [http://hdl.handle.net/. . .] in your request.**"],"dc:format":["application/pdf"],"dc:identifier":["http://hdl.handle.net/10477/86836"],"dc:language":["eng"],"dc:publisher":["State University of New York at Buffalo"],"dc:rights":["Users of works found in University at Buffalo Institutional Repository (UBIR) are responsible for identifying and contacting the copyright owner for permission to reuse. University at Buffalo Libraries do not manage rights for copyright-protected works and cannot assist with permissions.","Copyright retained by author."],"dc:subject":["cellular biology","genetics","molecular biology"],"dc:title":["Regulation of the Hippo Signaling Pathway in Mammary Epithelial Cells"],"dc:type":["Text","Dissertation"]},"updated_at":"2026-07-27T19:05:37Z"}