Cancer is characterized by abnormal regulation of cell growth, a process that ultimately depends on the correct expression and regulation of a large number of genes by transcription factors that can act as oncoproteins and tumor suppressors. We developed an inducible transformation model in which a transient inflammatory signal causes a stable non-transformed human breast epithelial cell line to undergo an epigenetic switch to a stable transformed state that includes a population of cancer stem cells (CSCs). The epigenetic switch is mediated by an inflammatory feedback loop consisting of transcription factors, miRNAs, and target genes that are oncogenes or tumor suppressors; this pathway is relevant for many forms of human cancer. We showed that CSCs and their non-stem cancer cell counterparts in the transformed population are not epigenetically distinct, but rather exist in a dynamic equilibrium involving interleukin 6 and an integrated transcriptional regulatory circuit that acts as a bistable switch. Lastly, we demonstrated that 3 transcriptional co-activator (-catenin, YAP/TAZ, S100A9/A8) that, respectively, are the ultimate targets of the Wnt, Hippo, and calcium signaling pathways, are critical for transformation. The central goal of this proposal is to elucidate, on a whole-genome scale, the transcriptional regulatory circuits involved in cellular transformation and CSC formation, neither of which have been investigated in this fashion. First, we will perform genetic (loss of function via siRNA or CRISPR followed by RNA-seq) and ChIP-seq experiments on candidate transcription factors we have already identified to determine where the factors bind in the genome and what genes they regulate. The results will be integrated into transcriptional regulatory circuits. Second, we will identify direct and indirec targets of co-activators (-catenin, YAP/TAZ, S100A9/A8) and integrate the results with those of the DNA-binding transcription factors. In addition, we identified WDR77 and the arginine methylase PRMT5 as interacting with -catenin as well as components of the polyadenylation machinery as interacting with YAP/TAZ; we will examine the molecular mechanisms by which these interacting proteins mediate their effects on gene expression and transformation. Third, using novel conceptual approaches based on mRNA profiles mediated by oncogenic and non-oncogenic protein derivatives or by different signaling molecules, we will identify oncogenically relevant targets. As a complement, we will use our high-throughput transformation assay to perform genome-scale genetic screens for genes important for transformation. The identified genes will be integrated into the transcriptional circuitry elucidated in aims 1 and 2. Fourth, the regulatory circuits derived from these results will be validated in other cancer cell types and by gene expression patterns in cancer patient samples. In summary, this tightly integrated set of genetic and functional genomic experiments on an inducible model of transformation and CSC formation will shed new light on fundamental issues in cancer progression at the molecular level, and new pathways and targets for therapy might be identified.

Public Health Relevance

Cancer occurs in a step-wise and progressive fashion from non-transformed to transformed to cancer stem cells, and it is characterized by abnormal regulation of cell growth, a process that ultimately depends on the correct regulation of many genes. This proposal uses genetic and functional genomic methodologies to elucidate, in a comprehensive manner, the gene regulatory circuitry involved in the processes of cellular transformation and cancer stem cells. The results will shed new light on fundamental issues in cancer progression at the molecular level, and potential new pathways and targets for therapy might be identified.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA107486-09A1
Application #
9103822
Study Section
Molecular Genetics B Study Section (MGB)
Program Officer
Fingerman, Ian M
Project Start
2004-05-25
Project End
2021-04-30
Budget Start
2016-05-24
Budget End
2017-04-30
Support Year
9
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Harvard Medical School
Department
Biochemistry
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
Gameiro, Paulo A; Struhl, Kevin (2018) Nutrient Deprivation Elicits a Transcriptional and Translational Inflammatory Response Coupled to Decreased Protein Synthesis. Cell Rep 24:1415-1424
Li, Ben B; Qian, Changli; Gameiro, Paulo A et al. (2018) Targeted profiling of RNA translation reveals mTOR-4EBP1/2-independent translation regulation of mRNAs encoding ribosomal proteins. Proc Natl Acad Sci U S A 115:E9325-E9332
Henry, Whitney S; Hendrickson, David G; Beca, Francisco et al. (2016) LINC00520 is induced by Src, STAT3, and PI3K and plays a functional role in breast cancer. Oncotarget 7:81981-81994
Ji, Zhe; Song, Ruisheng; Huang, Hailiang et al. (2016) Transcriptome-scale RNase-footprinting of RNA-protein complexes. Nat Biotechnol 34:410-3
Rotem, Asaf; Janzer, Andreas; Izar, Benjamin et al. (2015) Alternative to the soft-agar assay that permits high-throughput drug and genetic screens for cellular transformation. Proc Natl Acad Sci U S A 112:5708-13
Jin, Yi; Geisberg, Joseph V; Moqtaderi, Zarmik et al. (2015) Mapping 3' mRNA isoforms on a genomic scale. Curr Protoc Mol Biol 110:4.23.1-17
Ji, Zhe; Song, Ruisheng; Regev, Aviv et al. (2015) Many lncRNAs, 5'UTRs, and pseudogenes are translated and some are likely to express functional proteins. Elife 4:e08890
Janzer, Andreas; German, Natalie J; Gonzalez-Herrera, Karina N et al. (2014) Metformin and phenformin deplete tricarboxylic acid cycle and glycolytic intermediates during cell transformation and NTPs in cancer stem cells. Proc Natl Acad Sci U S A 111:10574-9
Hirsch, Heather A; Iliopoulos, Dimitrios; Struhl, Kevin (2013) Metformin inhibits the inflammatory response associated with cellular transformation and cancer stem cell growth. Proc Natl Acad Sci U S A 110:972-7
Polytarchou, Christos; Iliopoulos, Dimitrios; Struhl, Kevin (2012) An integrated transcriptional regulatory circuit that reinforces the breast cancer stem cell state. Proc Natl Acad Sci U S A 109:14470-5

Showing the most recent 10 out of 22 publications