The HECT E3 ubiquitin ligase Nedd4-1 was previously shown to regulate diverse processes such as cell and animal growth, insulin signaling, and lysosomal trafficking. To further elucidate the cellular functions of Nedd4-1, Nedd4-1 knockout mouse embryonic fibroblasts were characterized relative to their wild type counterparts. Immunofluorescence experiments revealed an altered lysosomal distribution in the knockout cells, although their lysosomal proteolytic function appeared normal. Transmission Electron Microscopy revealed striking morphological differences, especially regarding the lysosome and endoplasmic reticulum of the knockout cells. Another aspect of my studies examined the interaction between Nedd4-1 and Angiomotin (p130-AMOT), which involves the same motifs required to sequester transcriptional co-activators YAP and TAZ in the cytoplasm. To test either a competitive or non-competitive mode of binding, co-immunoprecipitation experiments involving p130-AMOT, the Nedd4 proteins, and YAP or TAZ were performed, with results not supporting a competitive mode of interaction. Overall, my results demonstrate new Nedd4-1 cellular functions.

Indiana University-Purdue University Indianapolis (IUPUI) / Chronic inflammation is a major driver of tumor progression in over fifty percent of breast cancers. Tumors activate inflammatory processes by secreting factors that recruit and trigger inflammatory cells to release cytokines such as Interleukin 6 (IL-6). IL-6 stimulates the activity of signal transducers and activators of transcription 3 (STAT3), a transcription factor that has been extensively studied for its role in promoting breast cancer. Recently, downregulated HIPPO signaling was shown to drive the pro-growth effects of IL 6. Reduced HIPPO signaling allows for the nuclear translocation of transcriptional co-activator yes associated protein (YAP), implicating IL-6 in the co-activation of several transcription factors such as the TEADs that trigger pro growth programs. While IL-6/STAT3 stimulation has been shown to increase YAP activity, the mechanism driving this remains undocumented. The Angiomotins (Amots) are adapters of the HIPPO pathway that directly bind and regulate YAP activity. Molecular characterization of Amot transcriptional regulation unexpectedly revealed a single promoter controlling the expression of its two major isoforms: Amot 130 and Amot 80. Through immunofluorescent analysis, this study found that total Amot levels were elevated across multiple breast tumor subtypes and highest in samples with increased presence of stromal inflammatory cells. Further, the induction of total Amot expression by IL 6 was found to be essential for YAP dependent growth of breast cancer cells. The activation of Amot transcription by IL-6 was found to be through Specificity Protein 1 (Sp1), a transcription factor that is activated by STAT3. This work connects the activation of YAP1 by IL-6/STAT3 through the elevation of Amot expression by Sp1. Taken together, this explains a new avenue whereby breast cancer cells acquire enhanced oncogenic properties in response to inflammatory signaling.

Background During development, endothelial cells acquire mesenchymal-like properties to migrate and facilitate normal vascular formation. This process of transformation is known as endothelial to mesenchymal transition (EndMT) and has also been implicated in diseases like vascular pathologies contributing to endothelial inflammation, atherosclerosis and tumour angiogenesis. The Angiomotin family of scaffold proteins play a role in transducing mechanical force at cell junctions. Of this family, Angiomotin-Like 2 (AmotL2) localises to endothelial cell junctions and was recently found to play a role in regulating endothelial cell mechanosensing and inflammation. Methods/Materials Primary human endothelial cell lines (HUVEC) were cultured and manipulated in vitro to investigate the role of AmotL2 in EndMT. Lentiviral short hairpin RNA interference was employed in AmotL2-loss-of-function studies, (produced using HEK - Human Embryonic Kidney - cells) to generate knockdown(kd) cells. Western blotting (WB) was used to assess AmotL2 depletion and changes in protein expression of key EndMT markers. qPCR was performed to look at the same at a transcriptional level. Immunofluorescent staining and confocal imaging were performed to validate WB and qPCR results as well as to study protein localisation. Results AmotL2 was found to regulate Snail1 and N-cadherin at both protein and mRNA levels. Morphological findings displayed the AmotL2kd cells to be elongated, deviating from the regular cobblestone morphology observed in control cells. An increase in scaffold protein levels was observed in the AmotL2 kd samples. Similar results were seen in qPCR data where increased mRNA expression was observed in the AmotL2 kd samples for the same targets. On analysis of IF image data, more nuclear staining was observed in the kd samples. qPCR analysis done on samples treated with TGF-β, exhibited an increase in mRNA expression of targets involved in the EndMT pathway in the treatment samples against the controls. Conclusion The results suggest that AmotL2 plays a role in EndMT by affecting the transcription factors and proteins involved in the pathway, which leads to changing morphology and behaviour of the cells. Looking into more targets involved in EndMT may give us a better understanding of how this process leads to diseases like atherosclerosis and tumour angiogenesis.

Indiana University-Purdue University Indianapolis (IUPUI) / While breast cancer is the second most commonly diagnosed cancer worldwide, its causes and natural history are not well defined. The female mammary organ is unique in that it does not reach full maturity until the lactation cycle following pregnancy. This cycle entails extensive growth and reorganization of the primitive epithelial ductal network. Following lactation, these same epithelial cells undergo an equally extensive program of apoptosis and involution. The mammary gland's sensitivity to pro-growth and pro-apoptotic signals may partly explain its proclivity to develop cancers. For epithelial cells to become transformed they must lose intracellular organization known as polarity as differentiated epithelial tissues are refractory to aberrant growth. One essential component of epithelial to mesenchymal transition is the intrinsic capacity of cells to repurpose polarity constituents to promote growth. Recently, a novel mechanism of organ size control has been shown to repurpose the apical junctional associated protein Yap into the nucleus where it functions as a transcriptional coactivator promoting growth and dedifferentiation. The focus of my work has been on a family of adaptor proteins termed Amots that have been shown to scaffold Yap and inhibit growth signaling. Specifically, I have shown that the 80KDa form of Amot, termed Amot80, acts as a dominant negative to the other Amot proteins to promote cell growth while reducing cell differentiation. Amot80 was found to promote the prolonged activation of MAPK signaling. Further, Amot80 expression was also found to enhance the transcriptional activity of Yap. This effect likely underlies the ability of Amot80 to drive disorganized overgrowth of MCF10A cells grown in Matrigel̈™. Overall, these data suggest a mechanism whereby the balance of Amot proteins controls the equilibrium between growth and differentiation within mammary epithelial tissues.

Indiana University-Purdue University Indianapolis (IUPUI) / Mammary ductal epithelial cell growth is controlled by microenvironmental signals in serum under both normal physiological settings and during breast cancer progression. Importantly, the effects of several of these microenvironmental signals are mediated by the activities of the tumor suppressor protein kinases of the Hippo pathway. Canonically, Hippo protein kinases inhibit cellular growth through the phosphorylation and inactivation of the oncogenic transcriptional co-activator Yes-Associated Protein (YAP). This study defines an alternative mechanism whereby Hippo protein kinases induce growth arrest via the phosphorylation of the long isoform of Angiomotin (Amot130). Specifically, serum starvation is found to activate the Hippo protein kinase, Large Tumor Suppressor (LATS), which phosphorylates the adapter protein Amot130 at serine-175. Importantly, wild-type Amot130 potently inhibits mammary epithelial cell growth, unlike the Amot130 serine-175 to alanine mutant, which cannot be phosphorylated at this residue. The growth-arrested phenotype of Amot130 is likely a result of its mechanistic response to LATS signaling. Specifically, LATS activity promotes the association of Amot130 with the ubiquitin ligase Atrophin-1 Interacting Protein 4 (AIP4). As a consequence, the Amot130-AIP4 complex amplifies LATS tumor suppressive signaling by stabilizing LATS protein steady state levels via preventing AIP4-targeted degradation of LATS. Additionally, AIP4 binding to Amot130 leads to the ubiquitination and stabilization of Amot130. In turn, the Amot130-AIP4 complex signals the ubiquitination and degradation of YAP. This inhibition of YAP activity by Amot130 requires both AIP4 and the ability of Amot130 to be phosphorylated by LATS. Together, these findings significantly modify the current view that the phosphorylation of YAP by Hippo protein kinases is sufficient for YAP inhibition and cellular growth arrest. Based upon these results, the inhibition of cellular growth in the absence of serum more accurately involves the stabilization of Amot130 and LATS, which together inhibit YAP activity and mammary epithelial cell growth.