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. In the previous granting period, we developed an experimental model involving a Src-inducible breast epithelial line (MCF-10A) that makes it possible to kinetically follow the process of cellular transformation, including the formation of cancer stem cells and mammospheres. Furthermore, we discovered an epigenetic switch between non-transformed and transformed cells mediated by a positive feedback loop that involves the inflammatory transcription factor NF-:B, Lin28 microRNA regulator, Let-7 microRNA, and interleukin 6. The central goal of this proposal is to elucidate the transcriptional regulatory circuits and underlying molecular mechanisms involved in the processes of cellular transformation and the formation of cancer stem cells. First, using molecular biological approaches, we will functionally dissect the regulatory pathway (i.e. the ordered series of steps involving transcription factors, direct target genes, microRNAs, and microRNA targets) by which an inflammatory signal leads to cellular transformation. We will address the relationship between the inflammatory response and transformation in the fibroblast model and other non-transformed cells lines as well as mechanistic analysis of promoters/enhancers of key target genes (e.g. Lin28 and IL6) in order to understand why and how the epigenetic switch occurs in MCF-10A, but not in other cells. In addition, having shown that miR-335 microRNA behaves similarly to Let-7 in many respects, we will perform similar mechanistic experiments to determine how miR-335 (and its targets tenascin C and Sox4 transcription factor) contributes to the process linking inflammation to cellular transformation. Second, we will obtain whole-genome profiles of mRNAs, microRNAs, transcription factor binding sites and chromatin features (via ChIP-sequencing), as well will genetically test transcription factors for their effects on gene expression genome-wide and on transformation. The resulting description will provide a whole-genome molecular view of the transformation process, identify direct targets of transcription factors relevant for transformation, provide critical information that will be followed up with detailed mechanistic experiments. Third, using the same logic and experimental approaches, we will identify genes, microRNAs, and regulatory pathways involved in the generation of cancer stem cells and the formation of mammospheres. Key genes and pathways implicated in these processes will be subjected to genetic and other mechanistic experiments to elucidate transcriptional circuitry. As an example, we will mechanistically pursue our recent finding that the miR-200 microRNA family is down-regulated in cancer stem cells and directly targets SUZ12, a component of the polycomb complex. Overall, this tightly integrated set of functional genomic and directed mechanistic experiments on isogenic models of cellular transformation will help elucidate transcriptional regulatory circuits involved in human cancer.

Public Health Relevance

Cancer is a process that ultimately depends on the correct expression and regulation of a large number of genes and microRNAs by transcription factors that can act as oncoproteins and tumor suppressors. Cancer is linked to inflammatory and metabolic diseases, and we discovered a gene regulatory pathway activated by an inflammatory signal that mediates an epigenetic switch from normal to cancerous cells. By performing a tightly integrated set of functional genomic and directed mechanistic experiments on isogenic models of cellular transformation, we will elucidate transcriptional regulatory circuits involved in human cancer, which will have an impact on the basic understanding of the disease with implications for treatment.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA107486-06
Application #
8212179
Study Section
Special Emphasis Panel (ZRG1-GGG-H (02))
Program Officer
Mietz, Judy
Project Start
2006-05-18
Project End
2015-01-31
Budget Start
2012-02-01
Budget End
2013-01-31
Support Year
6
Fiscal Year
2012
Total Cost
$705,118
Indirect Cost
$287,102
Name
Harvard University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
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
Iliopoulos, Dimitrios; Hirsch, Heather A; Struhl, Kevin (2011) Metformin decreases the dose of chemotherapy for prolonging tumor remission in mouse xenografts involving multiple cancer cell types. Cancer Res 71:3196-201
Iliopoulos, Dimitrios; Rotem, Asaf; Struhl, Kevin (2011) Inhibition of miR-193a expression by Max and RXRýý activates K-Ras and PLAU to mediate distinct aspects of cellular transformation. Cancer Res 71:5144-53
Polytarchou, Christos; Iliopoulos, Dimitrios; Hatziapostolou, Maria et al. (2011) Akt2 regulates all Akt isoforms and promotes resistance to hypoxia through induction of miR-21 upon oxygen deprivation. Cancer Res 71:4720-31
Iliopoulos, Dimitrios; Hirsch, Heather A; Wang, Guannan et al. (2011) Inducible formation of breast cancer stem cells and their dynamic equilibrium with non-stem cancer cells via IL6 secretion. Proc Natl Acad Sci U S A 108:1397-402
Iliopoulos, Dimitrios; Jaeger, Savina A; Hirsch, Heather A et al. (2010) STAT3 activation of miR-21 and miR-181b-1 via PTEN and CYLD are part of the epigenetic switch linking inflammation to cancer. Mol Cell 39:493-506
Hirsch, Heather A; Iliopoulos, Dimitrios; Joshi, Amita et al. (2010) A transcriptional signature and common gene networks link cancer with lipid metabolism and diverse human diseases. Cancer Cell 17:348-61
Iliopoulos, Dimitrios; Lindahl-Allen, Marianne; Polytarchou, Christos et al. (2010) Loss of miR-200 inhibition of Suz12 leads to polycomb-mediated repression required for the formation and maintenance of cancer stem cells. Mol Cell 39:761-72
Iliopoulos, Dimitrios; Polytarchou, Christos; Hatziapostolou, Maria et al. (2009) MicroRNAs differentially regulated by Akt isoforms control EMT and stem cell renewal in cancer cells. Sci Signal 2:ra62

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