Author ORCID Identifier
0000-0002-6771-5356
Date of Award
Spring 2025
Document Type
Thesis (Ph.D.)
Department or Program
Biochemistry and Cell Biology
First Advisor
Arminja Kettenbach, PhD
Second Advisor
Michael D. Cole, PhD
Third Advisor
Todd W. Miller, PhD
Abstract
Proteomics and phosphoproteomics have emerged as powerful tools to elucidate the intricate molecular underpinnings that drive cancer development by quantifying changes in protein and post-translational modification abundances. Throughout my thesis work, I have advanced these techniques to map cancer signaling networks on a global scale. Among breast cancer subtypes, triple-negative (TNBC) remains the most challenging to treat, with poorer clinical outcomes attributed to tumor heterogeneity and yet unidentified, oncogenic driver proteins for therapeutic targeting. To identify potential targeted treatment strategies, we employed mass spectrometry-based quantitative proteomics to analyze 55 formalin-fixed paraffin- embedded TNBC tumor specimens. Unsupervised hierarchical clustering of relative protein abundances identified four distinct subtypes. Functional annotation analyses designated the subtypes as: Immune-active (IMA), Immune-suppressed (IMS), Luminal androgen receptor (LAR), and Mesenchymal (MES). These protein-based subtypes correlated strongly with several clinicopathological features, including significantly high and low tumor-infiltrating lymphocyte scores for the IMA and IMS subtypes, respectively. DNA methylation analysis for inferred cell-type deconvolution of the tumor microenvironment corroborated these findings. Finally, to establish a foundation for pre-clinical investigations into each subtype, we assigned representative cell line models.
To expand beyond proteomic analyses, my research also focused on dissecting the regulatory mechanisms underlying phosphorylation-dependent signaling in cancer cells. Protein phosphorylation is an essential regulatory mechanism of cell signaling that is deregulated in many cancers. In eukaryotic cells, most phosphosites are dephosphorylated by members of the Phosphoprotein Phosphatase (PPP) family. However, no selective PPP inhibitor exists to study their individual functions. Here, we utilized endogenous tagging of genomic loci with an auxin-inducible degron system to investigate specific PPP signaling in cancer. Using Protein Phosphatase 6 (PP6) as an example, we demonstrated how inducible protein degradation can be employed to discover dephosphorylation sites and elucidate PP6 biology. Upon auxin-induced degradation of PP6, we performed quantitative mass spectrometry-based proteomics and phosphoproteomics to identify PP6 substrates in colorectal cancer cells. Consistently, we identified candidate PP6-dependent phosphosites on proteins that mediate MAPK and Hippo signaling. Finally, we demonstrated that PP6 opposes the activation of LATS1 by dephosphorylating Threonine 35 site on MOB1, thereby inhibiting the tumor-suppressive activity of the Hippo pathway.
Original Citation
Mariano NC, Rusin SF, Nasa I, Kettenbach AN. Inducible Protein Degradation as a Strategy to Identify Phosphoprotein Phosphatase 6 Substrates in RAS-Mutant Colorectal Cancer Cells. Mol Cell Proteomics. 2023 Aug;22(8):100614. doi: 10.1016/j.mcpro.2023.100614. Epub 2023 Jun 30. PMID: 37392812; PMCID: PMC10400926.
Recommended Citation
Mariano, Natasha Chipman, "Proteomic-based strategies to determine aberrant signaling in cancer" (2025). Dartmouth College Ph.D Dissertations. 338.
https://digitalcommons.dartmouth.edu/dissertations/338
