This competing renewal focuses on the characterization of an intracellular receptor for the phorbol ester tumor promoters and the second messenger diacylglycerol (DAG), ?2-chimaerin, and its relevance in the etiology of breast cancer. With the support of this RO1 we unambiguously established the concept of "divergence in DAG signaling", which implies that DAG and phorbol ester tumor promoters can trigger responses via direct activation of effectors that do not belong to the protein kinase C (PKC) family, considered until not so long ago the only intracellular receptors for DAG and phorbol esters. ?2-chimaerin possesses 3 well-defined regions: a N-terminal SH2 domain of unknown function (present only in a2- and ?2-chimaerins), a C1 domain (responsible for DAG and phorbol ester binding), and a C-terminal GTPase Activating Protein (GAP) domain that has the ability to accelerate the hydrolysis of GTP from the small G-protein Rac, leading to Rac inactivation. We established that ?2-chimaerin is a direct effector of the EGF receptor via the DAG pathway, and that activation of ?2-chimaerin serves as a "brake" that limits the activation of Rac in response to tyrosine kinase receptor stimulation. Moreover, we determined that in breast cancer cells ?2-chimaerin negatively modulates Rac1-mediated responses, including actin cytoskeleton reorganization, migration, invasion, proliferation, cell cycle progression, and cyclin D1 induction in response to ErbB receptor ligands, including EGF and heregulin. Remarkably, preliminary studies revealed that ?2-chimaerin is down-regulated in human breast cancer cells and specimens, suggesting a potential tumor suppressor role for this DAG-regulated Rac-GAP. As an extension of our previous work we now propose 4 aims.
In Aim 1 we will determine the relevance of ?-chimaerin in breast cancer progression using xenografts and also 1ith knock-out and knock-in mice for a hyperactive ?2-chimaerin mutant (I130A allele).
In Aim 2 we will analyze the expression of ?2-chimaerin in a panel of human mammary tumors and their matched normal counterparts in order to validate our preliminary evidence that ?2-chimaerin is down-regulated in breast cancer. We will also focus on promoter methylation as the potential cause for this down-regulation.
In Aim 3 we will assess the regulation of ?2-chimaerin activity and function by phosphorylation, as data revealed a PKC phosphorylation potentially modulates the Rac-GAP activity of ?2-chimaerin. Lastly, in Aim 4 we will identify and validate potential proteins that interact with ?2-chimaerin via the SH2 domain and determine the functional consequences of such interactons. The identification of a DAG/phorbol ester receptor unrelated to the PKC family that negatively regulates responses associated with the progression of breast cancer, and probably other neoplasias, not only has fundamental connotations in cancer signaling, but it may also reveal novel aspects of the etiology of cancer and have important therapeutic implications.

Public Health Relevance

In this proposal we will characterize a receptor for the phorbol ester tumor promoters and the lipid second messenger diacylglycerol (DAG) named ?2-chimaerin. We found that this protein negatively regulates important cellular functions involved in tumorigenesis and metastasis, and that it is down-regulated in breast cancer, suggesting a potential relationship with breast cancer etiology. This proposal will investigate the relevance of ?2-chimaerin in breast cancer progression as well as key regulatory mechanisms that control its activation and function.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA074197-15
Application #
8242852
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Johnson, Ronald L
Project Start
1997-09-30
Project End
2013-03-31
Budget Start
2012-04-24
Budget End
2013-03-31
Support Year
15
Fiscal Year
2012
Total Cost
$315,567
Indirect Cost
$115,207
Name
University of Pennsylvania
Department
Pharmacology
Type
Schools of Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Zubeldia-Brenner, Lautaro; Gutierrez-Uzquiza, Alvaro; Barrio-Real, Laura et al. (2014) ?3-chimaerin, a novel member of the chimaerin Rac-GAP family. Mol Biol Rep 41:2067-76
Gutierrez-Uzquiza, Alvaro; Colon-Gonzalez, Francheska; Leonard, Thomas A et al. (2013) Coordinated activation of the Rac-GAP *2-chimaerin by an atypical proline-rich domain and diacylglycerol. Nat Commun 4:1849
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Caino, M Cecilia; Lopez-Haber, Cynthia; Kissil, Joseph L et al. (2012) Non-small cell lung carcinoma cell motility, rac activation and metastatic dissemination are mediated by protein kinase C epsilon. PLoS One 7:e31714
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Wang, Hongbin; Kazanietz, Marcelo G (2010) p23/Tmp21 differentially targets the Rac-GAP beta2-chimaerin and protein kinase C via their C1 domains. Mol Biol Cell 21:1398-408
Notcovich, Cintia; Diez, Federico; Tubio, Maria Rosario et al. (2010) Histamine acting on H1 receptor promotes inhibition of proliferation via PLC, RAC, and JNK-dependent pathways. Exp Cell Res 316:401-11
Griner, Erin M; Caino, M Cecilia; Soledad Sosa, Maria et al. (2010) A novel cross-talk in diacylglycerol signaling: the Rac-GAP beta2-chimaerin is negatively regulated by protein kinase Cdelta-mediated phosphorylation. J Biol Chem 285:16931-41
Sosa, Maria Soledad; Lopez-Haber, Cynthia; Yang, Chengfeng et al. (2010) Identification of the Rac-GEF P-Rex1 as an essential mediator of ErbB signaling in breast cancer. Mol Cell 40:877-92
Sosa, Maria Soledad; Lewin, Nancy E; Choi, Sung-Hee et al. (2009) Biochemical characterization of hyperactive beta2-chimaerin mutants revealed an enhanced exposure of C1 and Rac-GAP domains. Biochemistry 48:8171-8

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