Characterization of Novel SMAD4 Protein-Protein Interactions in Pancreatic Cancer Restricted; Files Only
Ouyang, Wukun (Spring 2024)
Abstract
SMAD4 is a critical adaptor protein in the essential TGFβ signaling pathway, and regulates cell cycle, cell proliferation, apoptosis, and metastasis. Loss-of-function mutation of SMAD4 have been frequently identified in various tumor lineages, such as pancreatic and colon cancer, and predict poor patient survival. However, how SMAD4 mutations contribute to cancer progression and therapeutic response remain elusive. The emerging TGFb-independent SMAD4 biology presents a daunting challenge for therapeutic translation and urges interrogation of the hidden molecular mechanisms underlying the intricate SMAD4 biology. Herein, in Chapter I, I have discovered >30 novel high-confidence SMAD4 binding partners. Form this novel SMAD4 interactome, new SMAD4 TGFβ independent immune-regulatory mechanism is discovered, by studying one of the novel PPI, SMAD4-NFATC1, using binding-deficient variants, gene knockdowns, pharmacological interventions and more. In summary, this high-confidence SMAD4 OncoPPI network not only provided a valuable resource of novel SMAD4 interactions that informed potential context-dependent SMAD4 biology, but also shed light on potential SMAD4-loss collateral vulnerabilities for therapeutic innovation. Next, TGFβ pathway plays crucial roles in the establishment of an immunosuppressive tumor microenvironment, making anti-TGFβ agents a significant area of interest in cancer immunotherapy. However, the clinical translation of current anti-TGFβ agents that target upstream cytokines and receptors remains challenging. Therefore, the development of small molecule inhibitors specifically targeting SMAD4, would offer an alternative approach for anti-TGF-β signaling. In Chapter II, I present the development of a multiplexed TR-FRET assay in an ultrahigh-throughput screening (uHTS) format. This assay enables simultaneous monitoring of the protein-protein interaction (PPI) between SMAD4 and SMAD3, as well as the protein-DNA interaction (PDI) between SMADs and their consensus DNA binding motif. This sensitive multiplexed TR-FRET assay allows the dynamic analysis of the SMAD4-SMAD3- DNA complex at single amino acid resolution. Through a pilot screening of an FDA-approved and bioactive compound library, I identified gambogic acid and gambogenic acid as potential hit compounds. These proof-of-concept findings underscore the utility of my optimized multiplexed TR-FRET platform for large- scale screening to discover small molecule inhibitors that target the SMAD4-SMAD3-DNA complex as novel anti-TGFβ signaling agents.
Table of Contents
Table of Contents (Chapter I)
INTRODUCTION..........................................................................................................................1
Pancreatic Cancer Genomics ......................................................................................................1
Pancreatic Cancer Treatments and Challenges ..........................................................................1
TGFβ and SMAD4 in Pancreatic Cancer ...................................................................................2
Scope of the Thesis ......................................................................................................................3
RESULTS........................................................................................................................................5
Identification of 37 novel SMAD4 binding partners ....................................................................5
Discovery of novel binding partners’ preferred SMAD4 interacting domain .............................7
Focused systematic validation of SMAD4-NFATC1PPI .............................................................8
SMAD4 and NFATC1 binds to each other via each other’s DNA binding domains ...................9
SMAD4 inhibits NFATC1’s upregulation of STAT3 expression ................................................10
DISCUSSIONS .............................................................................................................................13
FIGURES AND LEGENDS ........................................................................................................15
MATERIALS AND METHODS.................................................................................................23
GST pulldown, semi-immunoprecipitation, and co-immunoprecipitation (co-IP) assay...........23
Time-resolved fluorescence resonance energy transfer (TR-FRET) assay ................................23
Luciferase reporter assay...........................................................................................................24
Western blotting ........................................................................................................................25
Venus Protein-fragment Complementation Assay (Venus-PCA) ...............................................25
shRNA Knockdown ....................................................................................................................26 REFERENCES.............................................................................................................................26
Table of Contents (Chapter II)
INTRODUCTION........................................................................................................................30 RESULTS......................................................................................................................................32
Design of the multiplexed SMAD4-SMAD3-DNA TR-FRET assay ..........................................32
Development of the multiplexed SMAD4-SMAD3-DNA TR-FRET assay .................................33
The assay is sensitive for detection of SMAD4-SMAD3-DNA complex dynamics.....................34
Assay miniaturization into a 1536-well plate to enable uHTS ..................................................35
Discovery of small-molecule SMAD4-SMAD3-DNA complex inhibitors .................................36
Identification of gambogic and gambogenic acid as SMAD4-SMAD3 PPI inhibitors .............37
DISCUSSION ...............................................................................................................................39
FIGURES AND LEGENDS ........................................................................................................42
MATERIALS AND METHODS.................................................................................................52
Cell Culture ...............................................................................................................................52
Molecular cloning and mutagenesis .........................................................................................53
Multiplexed Time-resolved fluorescence resonance energy transfer (TR-FRET) assay ...........55
uHTS TR-FRET screening for small molecule PPI inhibitor discovery ...................................56
GST pull-down assay .................................................................................................................56
Western blot ...............................................................................................................................57
SMAD binding element (SBE) luciferase reporter assay ..........................................................57
REFERENCES.............................................................................................................................58
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File download under embargo until 22 May 2030 | 2024-04-12 16:59:44 -0400 | File download under embargo until 22 May 2030 |
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