E-cadherin is widely recognized as a tumor- and/or metastasis suppressor, with its activity as a cell-cell adhesion receptor is dependent on tightly coupled interactions with cytoplasmic cofactors p120-, α-, and β-catenins. However, how the catenins, particularly p120, influence tumor initiation and metastasis is not clearly established. Here, we use conditional p120 ablation along with the Apc1638 and ApcMin mouse models to directly examine the role of p120 in intestinal tumorigenesis. Surprisingly, limited p120 knockout in less than 10% of the intestinal epithelium increased tumor development by up to 10-fold. The tumors, however, did not exhibit the expected complete loss of p120 and were found instead to be monoallelic, suggesting that p120 functions as a haploinsufficient tumor suppressor. Indeed, further studies show loss of one p120 allele is sufficient to induce the changes observed in total tumor number and that these results were not due to interaction with p120s nuclear binding partner, Kaiso. Surprisingly, retention of the remaining p120 allele was evidently obligatory in adenomatous cells, as complete loss of p120 was selectively incompatible with tumor cell viability. To better appreciate the impact of p120 haploinsufficiency, we turned to a Sleeping Beauty mutagenesis system that enables semi-quantitative analyses of relative tumorigenic potency through mutation frequency. Surprisingly, p120 and α-catenin are among the highest ranked genes in this system, with E-cadherin close behind. Remarkably, p120, α-catenin, and E-cadherin all ranked within the top 4% of 919 identified mutations in the Apc-sensitized intestine. As loss of any of these three components destabilized the cadherin complex, monoallelic disruption at any of these loci likely impacts functionality and accelerates tumorigenesis. Thus, although E-cadherin is widely recognized as a metastasis suppressor in colorectal cancer, our data show that the integrity of the cadherin complex is critical at the earliest stages of tumor initiation and progression.

The branched actin regulator cortactin is a central element connecting signaling pathways with the actin cytoskeleton and plays a role in diverse cellular processes. Cortactin-mediated actin assembly is known to contribute to cancer aggressiveness by regulating secretion of molecules that promote cell motility, invasion, and tumor growth. To better understand the role of cortactin in protein secretion, I focused on molecular and cellular mechanisms underlying cortactin-mediated regulation of protein transport in this dissertation. I demonstrated that cortactin-mediated branched actin assembly regulates late endosomal/lysosomal compartment maturation and trafficking, and subsequent retrograde transport to the Golgi complex. In addition, I identified a novel molecular mechanism by which cortactin control of actin dynamics on late endosomes is regulated by the phosphoinositide PI(3,5)P2. Using a combination of biochemical and cellular imaging approaches I demonstrated that PI(3,5)P2 removes cortactin from late endosomal branched actin networks by competing with actin filaments for binding to cortactin. This interaction promotes net disassembly of branched actin networks at late endosomal membranes. Overall, these findings suggest that cortactin-PI(3,5)P2 interactions regulate actin dynamics on late endosomal membranes by promoting net actin turnover, thereby promoting late endosomal/lysosomal compartment maturation and trafficking.

Lung cancer remains the leading cause of cancer related deaths in the United States despite a significant number of advancements in the molecular diagnosis and treatment of this disease. Recent genome wide expression analyses of human lung cancer has identified a number of receptor tyrosine kinases (RTKs) as overexpressed and potentially representing molecular drivers of lung cancer. Among these RTKs identified was EPHA2, which is highly expressed in lung cancer correlating to poor clinical outcomes. The role of EPHA2 in lung cancer, specifically in distinct molecular subtypes, is largely unknown. In this study, we dissected the role of EPHA2 in a variety of molecular subtypes of lung cancer and discovered that KRAS and EGFR(T790M) mutant lung cancers were most vulnerable to inhibition of EPHA2 by either genetic or pharmacological methods. We demonstrated functional evidence in vivo that the EPHA2 receptor is required for tumor growth and survival in both Kras(G12D) and EGFR(L858R+T790M) transgenic mouse models. We also showed that in lung cancer EPHA2 controls cell viability through a PI3K/mTOR dependent mechanism regulating apoptosis. Additionally, we identified a novel, ATP competitive EPHA2 RTK inhibitor, ALW-II-41-27, which was capable of inhibiting KRAS and EGFR mutant lung cancer cell viability both in vitro and in vivo. Overall these findings provide genetic, functional, mechanistic, and pharmacologic evidence that EPHA2 promotes the progression and survival of lung tumors. In addition, these findings provide rationale for the development of EPHA2 targeted therapeutics for clinical use.

Ephrin ligands and their Eph receptor tyrosine kinases both are surface tethered proteins that control cell shape and movements through direct cell-cell contact. Their binding, and subsequent clustering, triggers bidirectional signalling pathways, with signals transduced from the receptor (forward) and the ligand (reverse), that regulate the behaviour of both Eph- and ephrin- expressing cells. Recent evidence suggests that reverse ephrin-B2 signalling controls endothelial cell sprout outgrowth and tip elongation, and smooth muscle cell shape changes and behaviour. In addition, misregulation of ephrin-B2 expression is observed in various tumour types and high expression of this ligand is correlated with increased tumour vascularisation and tendency to metastasise. To investigate how ephrin-B2 "reverse" signalling pathways direct changes during angiogenesis and how the expression level of ephrin ligands influences changes in cell behaviour and cell mot motility, I have used Human Umbilical Vein Endothelial Cells (HUVECs) overexpressing ephrin ligands as a model system.

CANCER BIOLOGY The role of mTORC2 in mammary morphogenesis and HER2-mediated tumorigenesis Meghan Melinda Morrison Dissertation under the direction of Professor Rebecca Cook The phosphatidyl inositol-3-kinase (PI3K)/Akt signaling pathway is aberrantly activated in nearly 60% of breast cancers, through HER2 amplification, PIK3CA mutation, PTEN inactivation, and other alterations. The mTOR complexes mTORC1 and mTORC2 operate as activators (mTORC2) or effectors (mTORC1) of Akt and control key cellular processes, including growth, survival and metabolism. While most studies of mTOR in breast cancer have focused on mTORC1, little is known about the distinct role of mTORC2 in breast cancer. As molecular pathways that are essential for normal development are often hijacked by cancer cells, we sought to test the requirement of Rictor/mTORC2 in untransformed mammary epithelial cells (MECs) and transformed MECs. We show that genetic disruption of Rictor decreased Akt phosphorylation, ductal length, secondary branching, cell motility, and cell survival, effects that were recapitulated with a pharmacological dual inhibitor of mTORC1/mTORC2, but not upon genetic disruption of mTORC1 function via Raptor deletion. Surprisingly, Akt re-activation was not sufficient to rescue cell survival, invasion, or branching of mTORC2-impaired MECs. However, PKCα-mediated signaling through the small GTPase Rac1 was necessary for mTORC2-dependent mammary epithelial development during puberty, revealing a novel role for Rictor- PKCα-Rac1 signaling axis in MEC survival and motility during branching morphogenesis. These data lead us to hypothesize that Rictor/mTORC2 drives PI3K/Akt-mediated cell survival of HER2-amplified breast cancer cells. RICTOR gene ablation in a transgenic mouse model of HER2-amplified breast cancer delayed tumorigenesis and decreased lung metastasis, Akt-S473 phosphorylation, cell growth and survival. In human HER2-amplified breast cancer cells, Rictor loss, but not Raptor loss, decreased Akt-S473 phosphorylation, reducing cell survival and motility/invasion. Interestingly, Rictor/mTORC2 loss or treatment with a dual mTORC1/2 inhibitor improved lapatinib-induced cell killing in parental and resistant tumor cells to a greater extent than mTORC1 inhibition. Akt re-activation rescued cell survival, but not motility/invasion, in Rictor-depleted cells. However, Rictor loss caused accumulation of the Rac inhibitor RhoGDI2, thus impairing Rac1-dependent invasion. We conclude that HER2-amplified breast cancers use Rictor/mTORC2 to drive Akt-mediated cell survival and Rac1-mediated cellular invasion. These studies support additional studies into mTORC2-specific inhibitors. Approved Rebecca Cook 6/26/15

Intestinal homeostasis relies on complex interactions between the intestinal epithelium, microbiota, and host immune system, all of which cooperate to maintain homeostasis in an environment colonized and challenged by an estimated 100 trillion bacteria. It is the disruption and dysregulation of these interactions that is currently thought to underlie intestinal pathologies such as inflammatory bowel disease (IBD) and colorectal cancer (CRC). The Myeloid translocation gene (MTG) family of transcriptional corepressors and the tight-junction associated protein Blood vessel epicardial substance (BVES) have been shown to be important regulators of the intestinal epithelium. I hypothesized that loss of MTG or BVES function play important roles in IBD and CRC. Using genetic mouse models, cell lines, and organoid cultures, we clarified the role MTGs and BVES play in intestinal pathologies by identifying their binding partners and what signaling pathways they regulate. Overall, the primary objective of my work was to further elucidate the functional role of MTGs and BVES in the intestinal epithelium and carcinogenesis.

Cancer is the second leading cause of death in the United States. It is important to identify novel drivers of cancer in order to understand these diseases so we can improve patient prognosis. This dissertation focuses on two types of cancer, colorectal adenocarcinoma (CRC) and head and neck squamous cell carcinoma (HNSCC), and the discovery of novel regulators in each using different methods. In CRC, publically available data from The Cancer Genome Atlas (TCGA) was utilized. A bioinformatics analysis was used to connect protein expression data to clinical outcome, thus identifying proteins that are associated with poor prognosis in CRC. This analysis identified both known and novel regulators of CRC. Two of these molecules were further studied: insulin-like growth factor binding protein 2 (IGFBP2) and GATA3. These initial findings were validated by follow-up experiments. High levels of IGFBP2, a known but understudied molecule in CRC progression, were associated with poor prognosis. Using a tissue microarray (TMA) stained with IGFBP2, I found that IGFBP2-high staining patients had significantly decreased survival and recurrence-free survival. Low levels of GATA3, a transcription factor never associated with CRC, were associated with poor prognosis. With in vitro experiments using GATA3-manipulated cell lines, I showed that GATA3 overexpression decreases three-dimensional colony growth and invasiveness but not intrinsic proliferation rates. Cortactin is overexpressed through the 11q13 amplicon in 30-40% of HNSCC cases and controls a number of actin-based cellular phenotypes important for tumor progression. I found the cortactin SH3 domain was critical for Golgi morphology and in vivo tumor growth. Shank2, a cortactin SH3 binding protein that is also overexpressed through the 11q13 amplicon, has never been studied in any type of cancer. Using Shank2-manipulated cells, I found Shank2-knockdown decreased invasion, and preliminary evidence suggested a decrease in MMP secretion and invadopodia activity. Identification and characterization of these novel regulators, IGFBP2, GATA3, and Shank2, gives us mechanistic insight into the progression of CRC and HNSCC.

EGFR-mutant lung cancers are highly sensitive to EGFR tyrosine kinase inhibitors (TKIs; erlotinib/gefitinib/afatinib), but tumors develop drug resistance within 9-16 months. Resistance to gefitinib/erlotinib commonly occurs via a second-site EGFR mutation, T790M. Two strategies to overcome T790M+ resistance are mutant-specific EGFR TKIs, such as AZD9291, and dual inhibition of EGFR with afatinib plus the anti-EGFR antibody, cetuximab (A+C). Unfortunately, second-line acquired resistance to A+C and AZD9291, after first-line acquired resistance to erlotinib/gefitinib/afatinib, also occurs. To prevent/delay resistance to AZD9291, the combination of AZD9291 plus selumetinib (MEK1/2 inhibitor; AZD6244/ARRY-142886) is also currently being tested in a Phase I clinical trial (NCT02143466). The effects of sequential and combination treatment with various anti-EGFR agents on tumor evolution and drug resistance are largely unknown. In these studies, we modeled drug resistance pre-clinically to: 1. Assess the heterogeneity of mechanisms of first-line resistance to erlotinib and afatinib 2. Determine the optimum order of treatment with A+C vs. AZD9291 in the setting of T790M+ EGFR-mutant lung tumors 3. Elucidate mechanisms of first- and second-line acquired resistance to AZD9291 and 4. Elucidate mechanisms of resistance to AZD9291 plus selumetinib. Next-generation sequencing of genomic DNA from cell line models of resistance to erlotinib/afatinib revealed multiple potentially functional genomic changes within a given pool of resistant cells (including T790M). We also found that AZD9291 is more potent than A+C at inhibiting cell growth in the setting of T790M+ resistance to erlotinib. A+C-resistant cell lines remain sensitive to AZD9291, but AZD9291-resistant cell lines are cross-resistant to A+C. Resistance to AZD9291 is associated with dysregulation of MAPK signaling and can be overcome by addition of the MEK 1/2 inhibitor, selumetinib. Finally, AZD9291 plus selumetinib-resistant cell lines display increased baseline phospho-MEK/ERK and are sensitive to in vitro treatment with an ERK inhibitor, SCH772984 or alternative MEK inhibitor, trametinib. These studies provide a more comprehensive understanding of how EGFR-mutant tumors undergo rewiring of their signaling circuitry in response to single-agent EGFR- and combined EGFR+MEK-inhibition. This work, emphasizing a mechanistic understanding of the effects of therapies on tumor evolution, provides a framework for future clinical trials testing different treatment sequences.

A study has been made on the expression of cellular oncogenes <I>c-myc </I>and <I>c-fos</I> in rate skeletal muscle both <I>in vitro</I> in cell lines and primary culture and <I>in vivo </I>during development and after the induction of two models of hypertrophy. Using the technique of Northern hybridisation, results showed that the pattern of expression of <I>c-myc </I>and <I>c-fos </I>mRNA in primary myoblast cells and in established myoblast and fibroblast cells in culture were similar; stimulation of quiescent cells to proliferate induced expression from the <I>c-fos </I>gene within 30 minutes and the <I>c-myc </I>gene by 2 hours. The findings demonstrate that <I>c-myc</I> and <I>c-fos </I>mRNA levels are increased in terminally differentiated rat skeletal muscle both <I>in vitro </I>and <I>in vivo</I>. They confirm work which showed that <I>c-myc </I>is not exclusively connected with cell proliferation (Endo and Nadal-Ginard 1986) and extend previous work into the increased expression of <I>c-myc </I>and <I>c-fos </I>mRNA in cardiac muscle induced to hypertrophy (Mulvagh <I>et al., </I>1987; Izumo <I>et al</I>., 1988; Komuro <I>et al., </I>1988). The possible location of the elevated expression within the muscle after hypertrophy and the relative contributions from an inflammatory response, satellite cells and myofibres are discussed. Elevation of <I>c-myc </I>and <I>c-fos</I> mRNA in hypertrophy induced both surgically by tenotomy and pharmocologically with clenbuterol argue against a significant contribution from infiltrating fibroblasts, while from the literature, the activation of satellite cells appears to occur later. It is concluded that the elevated expression of <I>c-myc </I>in skeletal muscle is probably located within the myofibres and may be linked to mechanisms of cell enlargement. However further confirmation with <I>in situ </I>hybridisation and immunohistochemistry is required.

TNFR2 can activate NR-_{<span style='font-family:Symbol'>k}B independently of TNFR1 although to a lesser degree.  It was also demonstrated that caspases and NF-_{<span style='font-family:Symbol'>k}B do not interact directly as caspase inhibition could not prevent NF-_{<span style='font-family:Symbol'>k}B activation.  The interaction between caspases and MAP kinases in HeLa-TNFR2 cells was also investigated.  It was shown that JNK activation in response to TNF was attenuated following caspase inhibition while p42/44 MAP kinase and p38 MAP kinase activation were unaffected by caspase inhibition (Littlejohn et al, 2002). Further to these studies TNF receptor associated cytoplasmic adaptor proteins, in particular TRAF1, TRAF2 and RIP, were investigated with a view to determining the molecular switch between life and death in HeLa-TNFR2 cells.  It was established using immunoprecipitation that TNFR2 could associate with RIP, as could TRAF1 and TRAF2. However, on the whole this technique gave limited information and further experiments are required to determine more conclusively the interactions taking place between the adaptor proteins, TNFR1 and TNFR2. In order to investigate the molecular switch more effectively, TF-1 cells were used.  It was already known that TF-1 cells proliferate in response to TNF it cultured in the absence of GMCSF however, if cultured in the presence of GMCSF then TNF stimulation induces apoptotic cell death.  This model of TNF induced life versus death demonstrated that caspase mediated RIP cleavage only occurs in the death scenario.  RIP degradation is accompanied by reduced NF-_{<span style='font-family:Symbol'>k}B activation suggesting that NF-_{<span style='font-family:Symbol'>k}B activation is indeed a protective mechanism. Although many different avenues have been investigated the main conclusions that can be drawn from these studies are that TNF can regulate NF-_{<span style='font-family:Symbol'>k}B via TNFR1 and TNFR2 and that competent NF-_{<span style='font-family:Symbol'>k}B activation is required to protect cancer cells from TNF induced death.  Perhaps if it was possible to inhibit NF-_{<span style='font-family:Symbol'>k}B then TNF could be used effectively as a cancer killing agent.