Principal Investigator: Mauricio J. Reginato, Ph.D.
Institution: Drexel University College of Medicine, Philadelphia, PA
Principal Investigator: Lauren Ball, Ph.D.
Institution: Medical University of South Carolina, Charleston, SC
Role of O-GlcNAcome on Breast Cancer Initiating Cells
It is widely accepted that tumors are highly heterogeneous. There is a subpopulation of cells in a tumor, called tumor-initiating cell, that can be isolated and are able to self-renew, differentiate and form the bulk of the tumor. Many cancers don't respond to traditional chemotherapy or radiotherapy, and those that initially respond, often relapse. Conventional therapy only attacks proliferating cells, leaving behind a pool of resistant stem-like cells that are able to regenerate the whole tumor. Understanding mechanisms that regulate tumor- initiating activity will lead to designing and developing effective therapeutics. Our lab has demonstrated for the first time that the nutrient sensor O-GlcNAc transferase (OGT) regulates cancer-initiating cells in vitro and in vivo. Reducing OGT, genetically or pharmacologically, blocks mammosphere formation in vitro and reduced epithelial-mesechymal markers (EMT), cancer stem cell markers. Importantly, overexpression of OGT, in multiple breast cancer cells, increases cancer stem cell markers including NANOG and CD44, increases mammosphere formation in vitro and increases tumor initiation in vivo. In this proposal, we hope to uncover molecular mechanism by which OGT regulates tumor initiation, by in part, understanding OGT interactome and O- GlcNAcome in breast cancer tumor initiating cells. This information will allow us to identify novel therapeutic targets in treating cancer and reverse drug resistance. Based on our preliminary results, the central hypothesis of this application is that the nutrient sensor O-GlcNAc transferase plays a fundamental role in breast cancer initiating cells via, in part, stem cell factor regulation. Completion of these experiments will contribute to our understanding of how nutrient-sensing pathways connects at the molecular level to self-renewing cancer stem cells (CSCs) and providing a framework for understanding how cancer alterations in metabolic pathways regulate core self-renewal signaling that controls CSC maintenance.
In Aim #1, we will determine the molecular basis of OGT/O-GlcNAc regulation of stem cell factors. This aim will determine the molecular basis of OGT regulation of stem cell factors in breast cancer tumor initiating cells.
In Aim #2, we will Identify OGT interactome/O-GlcNAcome between between cancer cells and cancer stem cells. This aim will uncover a global regulation of how OGT and O-GlcNAcylation regulate breast cancer initiating cells and may uncover O-GlcNAcome associated with cancer stem cells and uncover molecular pathways linking glycosylation and tumor initiation. The final aim will evaluate the role of OGT in regulating tumor-initiating activity in vivo. Importantly, we will test novel OGT inhibitors in preclinical cancer models and test whether OGT targeting drugs as potential anti-tumor initiation cell therapeutic strategy against breast cancer growth and metastasis in vivo. These studies will further our understanding of how metabolic reprogramming in cancer cells connects at the molecular level to tumor initiating cells and will create mechanistic understanding of how nutrient sensor OGT can couple to cancer initiation pathways and establish OGT as therapeutic target for treatment of resistant cancers.
Public Health Relevance
Breast cancer is the one of the leading causes of cancer-related deaths among women despite advances in screening, diagnosis and therapies. Intratumoral cellular heterogeneity is considered a critical player of persistent tumor growth, metastasis and therapeutic resistance. This heterogeneity is driven, in part, by a self-renewing population of cancer stem-like cells or tumor-initiating cells. This proposal centers on the molecular understanding of how the nutrient sensor O-GlcNAcylation regulates tumor-initiation in conferring stem-like cell behaviors that drive tumor initiation, chemo-resistance and metastasis.
- Click-IT assay to identify O-GlcNAcylated proteins from lysates.
- In vivo cancer models: Mammary fat pad injections for breast cancer primary tumor growth, Intracardiac injection for metastasis model, intra-cranial injection model of breast cancer brain metastasis.
- Ex vivo/In vitro models: Ex vivo breast cancer brain metatasis brain slice model, In vitro three-dimensional breast cancer model, mammosphere culture models.
We have the following nano-LC-MS/MS instrumentation for quantitative proteomics and characterization of the sites of glycosylation:
- Orbitrap Fusion Lumos ETD/UVPD
- Orbitrap Elite ETD.