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Principal Investigator: Deniz Nesli Dolcen
Organization: STANFORD UNIVERSITY
Fiscal Year: 2024
Award: $97,265
Funding agency: National Cancer Institute
PROJECT SUMMARY
My lab discovered that NNMT is a direct GR transcriptional target gene in TNBC. I then observed relatively
high NNMT expression in several aggressive patient-derived TNBC cell lines. NNMT consumes the universal
methyl donor S-adenosyl methionine (SAM) for methylation of nicotinamide. High NNMT activity depletes SAM;
as a result, methyltransferase targets are hypomethylated in cells with high NNMT expression. NNMT-induced
DNA and histone hypomethylation have been shown to result in oncogenic gene expression in cancer cells but
NNMT mechanism of action in TNBC biology remains unclear. A link between NNMT expression and mRNA
hypomethylation has not previously been established as a mechanism contributing to cancer progression. N6-
methyladenosine (m6A) is an abundant and reversible RNA modification in eukaryotes. Our collaborator Dr.
Chuan He discovered that m6A-binding proteins mediate translational regulation by altering stability and
translational efficiency of m6A-modifed mRNAs. Importantly, altered m6A mRNA methylation is implicated in the
progression of several human cancers via causing changes in post-transcriptional gene expression of cancer
pathways. To our knowledge, I am the first to characterize the m6A methylome of a patient-derived TNBC cell
line model (MDA-MB-231): ~ 7000 m6A-modified transcripts are significantly enriched for pathways involved in
cellular stress response, cell death and cell survival. In addition, I have data suggesting that NNMT activity in
the MDA-MB-231 TNBC cell line results in 1) reduced m6A modification of mRNAs regulating key cancer
pathways and 2) increased in vivo tumor-growth. In my dissertation research, I am testing the hypothesis that
NNMT activity in TNBC cells results in 1) reduced m6A mRNA modification associated with altered protein
expression of pathways mediating cellular stress response and 2) cancer stem cell-like traits associated with
survival, metastatic potential and increased in vivo tumor-forming capacity.
During my postdoctoral research, I aim to test whether epitranscriptomic gene expression regulates
dynamic cellular phenotypes including adaptation to the changing microenvironment. I will first characterize the
actively transcribed genes with polymerase ChIPseq and perform whole proteome quantification with mass
spectrometry in cells exposed to distinct microenvironmental stressors (e.g. nutrient deprivation, hypoxia). I will
then determine whether differential transcription of genes correlate with protein expression in different cellular
states. If there is not a strong correlation, I will perform individual siRNA knockdown of all known m6A-
regulatory genes and determine the effect on protein expression. I will then utilize patient-derived xenograft
mouse models and the Sprague Dowley rat model of spontaneous breast cancer to determine whether the
m6A-regulatory proteins are differentially expressed in distinct tumor regions with single-cell RNA sequencing.
Terms: <3-Pyridinecarboxamide><Ademetionine><AdoMet><Binding Proteins><Biological><Breast Cancer><Breast Cancer Cell><Breast Cancer Model><Breast Cancer cell line><Breast tumor cell line><Breast tumor model><Cancer Biology><Cancer Model><Cancer Treatment><CancerModel><Cancers><Cell Body><Cell Culture Techniques><Cell Death><Cell Survival><Cell Viability><Cells><Cellular Stress><Cellular Stress Response><ChIP Sequencing><ChIP-seq><ChIPseq><Common Rat Strains><Consumption><DNA><DNA Modification><DNA Modification Process><Data><Deoxyribonucleic Acid><Development><EC 2.1.1><Epigenetic><Epigenetic Change><Epigenetic Mechanism><Epigenetic Process><Eukaryota><Eukaryote><Excretory function><Exhibits><Exposure to><Gene Action Regulation><Gene Expression><Gene Expression Regulation><Gene Regulation><Gene Regulation Process><Gene Transcription><Genes><Genetic><Genetic Transcription><Histones><Human><Hypoxia><Hypoxic><In Vitro><Individual><Ligand Binding Protein><Ligand Binding Protein Gene><Link><MDA MB 231><MDA-231><MDA-MB231><Malignant Breast Neoplasm><Malignant Cell><Malignant Neoplasm Therapy><Malignant Neoplasm Treatment><Malignant Neoplasms><Malignant Tumor><Mass Photometry/Spectrum Analysis><Mass Spectrometry><Mass Spectroscopy><Mass Spectrum><Mass Spectrum Analyses><Mass Spectrum Analysis><Mediating><Messenger RNA><Metastasis><Metastasize><Metastatic Lesion><Metastatic Mass><Metastatic Neoplasm><Metastatic Tumor><Methylation><Methyltransferase><Mice><Mice Mammals><Modeling><Modern Man><Modernization><Modification><Murine><Mus><Neoplasm Metastasis><Niacinamide><Nicotinamide><Nicotinamide N-Methyltransferase><Nicotinamidum><Nicotinic acid amide><Nicotylamide><Non-Polyadenylated RNA><Oncogenic><Oxygen Deficiency><PDX model><Pathology><Pathway interactions><Patient derived xenograft><Patients><Pellagra-Preventing Factor><Phenotype><Physiology><Play><Polymerase><Postdoc><Postdoctoral Fellow><Process><Protein Binding><Proteome><R-Series Research Projects><R01 Mechanism><R01 Program><RNA><RNA Expression><RNA Gene Products><RNA methylation><Rat><Rats Mammals><Rattus><Regulator Genes><Regulatory Protein><Relapse><Research><Research Associate><Research Grants><Research Project Grants><Research Projects><Ribonucleic Acid><Role><S-Adenosylhomocysteine><S-Adenosylmethionine><S-adenosyl methionine><S-adenosyl-methionine><SAMe><SYS-TX><Secondary Neoplasm><Secondary Tumor><Short interfering RNA><Small Interfering RNA><Sprague-Dawley Rats><Stem Cell like><Stimulus><Systemic Therapy><TNBC><Testing><Therapeutic Agents><Transcript><Transcription><Transcriptional Regulatory Elements><Translational Regulation><Urine><Vitamin B 3><Vitamin B3><Vitamin PP><anti-cancer therapy><biologic><bound protein><breast tumor cell><cancer cell><cancer gene expression><cancer metastasis><cancer microenvironment><cancer progenitor><cancer progenitor cells><cancer progression><cancer stem cell><cancer therapy><cancer-directed therapy><cell culture><cell cultures><cell stress><chromatin immunoprecipitation-sequencing><developmental><differential expression><differentially expressed><epigenetically><epitranscriptomics><excretion><genetic regulatory protein><histone modification><in vivo><knock-down><knockdown><mRNA><malignancy><malignant breast tumor><malignant progenitor><malignant stem cell><mammary cancer model><mammary tumor model><methylase><methylome><mouse model><murine model><necrocytosis><neoplasm progression><neoplasm/cancer><neoplastic progression><nicotinamide methylase><nicotinamide methyltransferase><nicotinamide-S-adenosylmethionine methyltransferase><novel><nutrient deprivation><nutritional deprivation><pathway><patient derived xenograft model><pharmacologic><post-doc><post-doctoral><post-doctoral trainee><posttranscriptional><prevent><preventing><protein expression><regulatory gene><regulatory gene product><research associates><response><s-adenosyl-l-methionine><scRNA-seq><siRNA><single cell RNA-seq><single cell RNAseq><single cell expression profiling><single cell transcriptomic profiling><single-cell RNA sequencing><social role><stem cell characteristics><stemness><stressor><trait><trans acting element><transcriptional differences><transmethylase><triple-negative breast cancer><triple-negative invasive breast carcinoma><tumor><tumor cell metastasis><tumor growth><tumor microenvironment><tumor progression>