Browsing by Subject "Transforming Growth Factor beta"
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Item Delineating the Mechanisms Through Which ZNF165 Supports Growth and Survival of Triple-Negative Breast Cancer(2021-05-01T05:00:00.000Z) Gibbs, Zane Alexander; Cobb, Melanie H.; D'Orso, Iván; Hon, Gary C.; Whitehurst, Angelique WrightCancer/testis (CT) antigens are proteins whose expression is normally restricted to germ cells yet aberrantly activated in tumors, where their functions remain relatively cryptic. The transcription factor ZNF165, a CT antigen frequently expressed in triple-negative breast cancer (TNBC), was previously identified as essential for the growth and survival of TNBC and sustained transforming growth factor beta (TGFβ) signaling. However, the mechanisms through which ZNF165 functions in this context to regulate both TGFβ signaling and survival were poorly understood. To investigate its mechanism of action, I first sought to determine how ZNF165 interfaces with the TGFβ pathway in TNBC using a combination of genomics and biochemical approaches. I found that ZNF165 associates with SMAD3, a key signal transducing factor in the TGFβ pathway, to modulate transcription of TGFβ-dependent genes and thereby promote growth and survival of human TNBC cells. Importantly, my data demonstrate that through functioning as a cofactor for SMAD3, ZNF165 is able to specify a TGFβ-dependent gene expression program that promotes oncogenic phenotypes while repressing tumor-suppressive functions. In addition, I identified the KRAB zinc finger protein, ZNF446, and its associated tripartite motif protein, TRIM27, as obligate components of the ZNF165-SMAD3 complex that also support tumor cell viability. I found that while ZNF446 is bound to chromatin with the ZNF165-SMAD3 complex, TRIM27 alone is necessary for ZNF165 transcriptional activity and is required for TNBC tumor growth in vivo using an orthotopic xenograft model in immunocompromised mice. Moreover, my data also suggest that ZNF165 is SUMOylated by TRIM27 to enhance its protein stability, providing further insight into the mechanisms that regulate ZNF165 activity in TNBC. Together, my findings indicate that aberrant expression of a testis-specific transcription factor is sufficient to co-opt somatic transcriptional machinery to drive a pro-tumorigenic gene expression program in TNBC.Item Fibulin-5 Promotes Pancreatic Tumor Growth through Inhibition of Integrin-induced ROS: Insights into Tumor-Matrix Signaling(2016-07-18) Topalovski, Mary; Terada, Lance; Cobb, Melanie H.; Abrams, John M.; Brekken, Rolf A.Elevated oxidative stress is an aberration seen in many solid tumors, and exploiting this biochemical difference has the potential to enhance the efficacy of anti-cancer agents. Homeostasis of reactive oxygen species (ROS) is important for normal cell function, but excessive production of ROS can result in cellular toxicity and therefore ROS levels must be balanced finely. Here, we highlight the relationship between the extracellular matrix and ROS production by reporting a novel function of the matricellular protein Fibulin-5 (Fbln5). We found that Fbln5 is abundantly expressed in mouse and human pancreatic cancer compared to normal pancreas. By employing genetically engineered mouse models of pancreatic ductal adenocarcinoma (PDA), we showed that mutation of the integrin-binding domain of Fbln5 led to decreased tumor growth, increased survival, and enhanced chemoresponse to standard PDA therapies. Through mechanistic investigations, we found that improved survival was due to increased levels of oxidative stress in Fbln5 mutant tumors. Furthermore, loss of the Fbln5-integrin interaction augmented fibronectin (FN) signaling, driving integrin-induced ROS production in a 5-lipooxygenase-dependent manner. These data indicate that Fbln5 promotes PDA progression by functioning as a molecular rheostat that modulates cell-ECM interactions to reduce ROS production and thus tip the balance in favor of tumor cell survival and treatment-refractory disease. The latter part of this thesis is focused on the underlying mechanism that leads to upregulation of Fbln5 in PDA. The deposition of ECM is a defining feature of PDA where ECM signaling can promote cancer cell survival and epithelial plasticity programs. ECM-mediated signaling is governed by expression of the ECM proteins, the presence of cell surface receptors and the expression and activity of matricellular proteins that function as extracellular adaptors to reduce ECM-cell interaction. As stated above, Fbln5 is a matricellular protein that blocks FN-integrin interaction and thus directly limits ECM-driven ROS production and supports PDA progression. Compared to normal pancreatic tissue, Fbln5 is expressed abundantly in the stroma of PDA; however, the mechanisms underlying the stimulation of Fbln5 expression in PDA are undefined. Using in vitro and in vivo approaches, we report that hypoxia triggers Fbln5 expression in a transforming growth factor β (TGF-β)- and PI3K-dependent manner. Pharmacologic inhibition of TGF-β receptor (TGF-βR), PI3K, or protein kinase B (AKT) was found to block hypoxia-induced Fbln5 expression in mouse embryonic fibroblasts and 3T3 fibroblasts. Moreover, tumor-associated fibroblasts from mouse PDA were also responsive to TGF-βR and PI3K/Akt inhibition with regard to suppression of Fbln5. In genetically engineered mouse models of PDA, therapy-induced hypoxia elevated Fbln5 expression while pharmacologic inhibition of TGF-β signaling reduced Fbln5 expression. These findings offer insight into the signaling axis that induces Fbln5 expression in PDA and a potential strategy to block its production.Item Oncogene-Induced Signaling Heterogeneity in Lung Cancer(2015-07-22) Deb, Dhruba; Garcia, Christine K.; White, Michael A.; Cobb, Melanie H.; Altschuler, Steven J.; Wu, Lani; Minna, John D.Lung cancer causes the maximum number of cancer related deaths worldwide. In recent years, the cancer genome atlas (TCGA) initiative has identified 138 frequently occurring driver oncogenes and tumor suppressor genes in lung cancer. Currently, only 15 of these genes can be targeted therapeutically. Study of downstream signaling alterations of these oncogenes and tumor suppressor genes may identify novel therapeutic targets. Although studies on genetic heterogeneity in subclonal populations within one tumor using deep sequencing and multiple sectioning have gained popularity recently, the signaling heterogeneity within tumor cells with identical genetic changes remain poorly understood. Hence, I focus on TP53, KRas and C-Myc as they are among the most frequently occurring oncogenic alterations in lung adenocarcinoma. The downstream signaling changes of these genes may be different from one cell to another. Here, I develop high throughput approaches to study alterations of 6 major signaling readouts - phospho-Erk1/2, phospho-Stat3, Smad2/3, β-catenin, P65, and Foxo1 and quantitatively analyze thousands of cells with defined set of genetic changes. I ask - Can I utilize oncogene-induced signaling alterations in single cells to identify novel targetable vulnerabilities? Using single-cell image analysis I show that the genetically transformed HBECs with all 3 oncogenic changes (TP53, KRas and C-Myc) show significant signaling heterogeneity. They exhibit downregulated Smad2/3 signaling in single cells. Next, using a dominant negative construct, I confirm that this phenotype is partially reversible by the removal of C-Myc oncogenic stress. I further observe that the transformed HBECs exhibit upregulated Stat3 signaling in single cells. In addition, the Stat3 inhibitor Stattic causes more cell death in transformed HBECs. Interestingly, our single-cell image analysis suggests that Stat3 upregulation and Smad2/3 downregulation are mutually exclusive. Hence, Stattic will not be able to target the Smad2/3 downregulated cells. To target Smad2/3 downregulated cells, I identify Bcl6, a downstream target of Smad2/3, and I show that Bcl6 is a novel targetable vulnerability in transformed HBECs. I observe that C-Myc and Bcl6 gene expressions are strongly correlated in cell populations as well as in single-cell level. I further show that Bcl6 can be a targetable vulnerability in a subset of c-Myc addicted non-small cell lung cancers. I conclude that single-cell analysis of driver oncogenes and their downstream signaling can identify novel targetable vulnerabilities.Item Quantitative Single-Cell Imaging Reveals Insulation of Morphogenic Signal Transduction(2014-07-24) Coster, Adam D.; Alto, Neal; Altschuler, Steven J.; Wu, Lani; Ranganathan, Rama; Amatruda, James F.How cells integrate external cues in order to make behavioral decisions is a central problem of cell biology. In development and in tissue-homeostasis, cell-fate decisions are made by the integration of multiple morphogenic signals, but how cells convert such combinations of signals into distinct behaviors is not well understood. A major complication is our incomplete knowledge of which signal properties encode the information that cells use for decision-making. A further complication is that the static networks we use to describe cellular signaling pathways are likely to be overly-complex; the true signaling network, in a given cellular context and at a particular point in time, may be much simpler. Using a rigorous and quantitative single-cell imaging approach, I find that such simplicity is present in the integration between Wnt and Transforming Growth Factor Beta (TGFB), which are key developmental pathways. Surprisingly, this insulation extends to the integration of signals within the TGFB superfamily, which are expected to compete for shared components and so interfere with one another during signal transduction. My results thus add clarity to and simplify our understanding of how cells integrate information from the Wnt and TGFB pathways, and further suggest that insulation of signal transduction may be a common feature of morphogenic pathways.Item A Quantitative Study of Parathyroid (1-34) and Bone Morphogenetic Protein-2 on Spinal Fusion Outcomes in a Rabbit Model of Lumbar Dorsolateral Intertransverse Process Arthrodesis(2013-01-22) Hwang, Lee; Santiago-Dieppa, David; Lina, John; Liauw, Jason; Witham, TimothyINTRODUCTION: Lumbar spinal fusion is a common neurosurgical procedure. Over 250,000 lumbar spinal fusion operations are performed annually in the U.S. and, by far, dorsolateral intertransverse process arthrodesis (DIPA) is the most common type of fusion technique performed in the lumbar spine. Unfortunately, the rate of non-fusion (pseudoarthrosis) has been reported to be as high as 35%. Pseudoarthrosis is also one known cause of Failed Back Surgery Syndrome (FBSS), which is characterized by chronic back pain that is often unbearable and debilitating to the patient. In the following study, our goal was to explore methods of improving the rate of fusion by first establishing an accurate animal model - more specifically the New Zealand White rabbit model, which has a pseudoarthrosis rate nearly identical to that of humans as well as similar spinal anatomy. We also aimed to assess the effects of osteoinductive agents PTH (1-34) and rhBMP-2 on bone turnover after DIPA spinal fusion in the rabbit model. We hypothesize that the use of both PTH (1-34) and rhBMP-2 has a synergistic effect on stiffness and composition of the bone fusion mass. METHODS: To elucidate the potential relationship between PTH (1-34) and rhBMP-2, 24 NZW rabbits will be assigned to each of the following 4 groups: spinal fusion with iliac crest autograft and saline-based injections (control); spinal fusion with rhBMP-2 matrix alone; spinal fusion with iliac crest autograft and PTH (1-34) injections alone; spinal fusion with rhBMP-2 matrix and PTH (1-34) injections. Each specimen will undergo manual palpation, radiographic analysis, four-point non-destructive biomechanical testing, and histological analysis. PRELIMINARY RESULTS: Based on the CT reconstructions, the spine fusion rates for the control group (n = 9) and the PTH (1-34) group (n = 5) were 44% and 60%, respectively. Although manual palpation is used as the standard measure of fusion, radiographic analysis proved more effective in differentiating fused masses based on bone, not fibrous tissue. In both the PTH (1-34) and control groups, lateral bending in both the right and left orientations proved to be the stiffest testing orientation. CONCLUSIONS: Preliminary data suggests that PTH (1-34) may enhance both the composition and mechanical properties of L5-L6 fusion in rabbits. As demonstrated by biomechanical testing, the enhanced bone formation in the PTH (1-34) group may be responsible for an increase in stiffness. We expect that data from the additional two groups will allow for further investigation of individual efficacies as well as any synergistic effects of PTH (1-34) and rhBMP-2.Item Regulation of the Tumor Suppressor ARF by TGFβ in Human Cancer Cells(2016-11-29) Hooks, Jared Cole; Brekken, Rolf A.; Shay, Jerry W.; Scaglioni, Pier Paolo; Skapek, Stephen X.Since its discovery, p14ARF (p19Arf in mice) has been shown to be an important regulator of the cell cycle, carrying out this function through p53-dependent and independent mechanisms. ARF expression has recently been shown by the Skapek lab to be induced by TGFβ in mouse embryonic fibroblast and HeLa cells. TGFβ itself has been well characterized for its role as a "dual-edged sword," acting as a tumor suppressor or oncogene depending on the context. Though preliminary work has looked at the basic connections between TGFβ pathway components and their necessity for induction of ARF, many open questions remain, especially in translating findings from mouse to human cells. For my dissertation research, I have chosen to further investigate the regulation of TGFβ-driven induction of ARF. The knowledge gained through understanding TGFβ-dependent regulation of ARF is important in understanding disease progression and could also provide new avenues for cancer treatment through restoration of the TGFβ pathway to harness the tumor suppressive effects of p14ARF.