Molecular mechanisms controlling metastasis in breast cancer are not well understood. During invasion of the extracellular matrix (ECM) cancer cells alternate between fast, amoeboid, and slow mesenchymal types of movement. This plasticity in movement is essential for efficient invasion, but it is not well understood how it is controlled. We have found that the RhoGEF Net1A is an important regulator of invasion plasticity in breast cancer cells, stimulating amoeboid and suppressing mesenchymal movement. Net1A regulates this process by controlling RhoA and deacetylase function to impact cytoskeletal organization and gene expression. Net1A is exceptional among RhoGEFs in that it is sequestered in the nucleus to prevent aberrant RhoA activation, and it is clear that subcellular localization is a key determinant of the oncogenic potential of Net1 isoforms. Recently we have shown that Rac1 activation downstream of integrin or growth factor receptor activation stimulates Net1A relocalization to the plasma membrane. We have also found that Net1A relocalization is maintained by site-specific acetylation, and that the actions of specific deacetylases are critical to suppressing Net1A activity towards RhoA. Moreover, we have observed that Net1A reciprocally controls the activity of specific deacetylases to regulate cytoskeletal organization and expression of key genes associated with cell invasion, such as MT1-MMP. These findings support our hypothesis that that Net1A functions as a nodal point to temporally and spatially regulate RhoA activation and deacetylase activity which underlie breast cancer cell motility, invasion and metastasis. In this proposal we will use a combination of cell-based and in vivo assays to understand how Net1A localization is regulated to drive RhoA activation and reciprocally control deacetylase activity during ECM invasion. We will also determine how Net1 isoforms contribute to mammary gland tumorigenesis and metastasis in vivo.
In Aim 1 we will demonstrate how EGFR and HER2 activation stimulates Net1A export from the nucleus to stimulate cell motility and ECM invasion. We will demonstrate how Net1A acetylation affects its function and identify the deacetylases that regulate Net1A.
In Aim 2 we will determine how Net1A controls HDAC and Sirtuin function to regulate acetylation of cytoskeletal organizing proteins and gene expression.
In Aim 3 we will identify the molecular basis for how genetic deletion of Net1 affects mammary gland development in mice, and determine whether Net1 is required for HER2/Neu driven mammary gland tumorigenesis and metastasis. Completion of these aims will provide a mechanistic understanding of how Net1 isoforms contribute to breast cancer cell motility and invasive potential, and will provide novel targets for intervention in metastatic disease.

Public Health Relevance

RhoA activation and deacetylase function are essential for breast cancer cell motility and metastasis, but mechanisms controlling their activation in breast tumors are not well defined. Our work will address this major gap in our understanding by defining how the RhoGEF Net1 is regulated to control these activities to impact breast cancer cell invasion and metastasis. This will provide a deeper understanding of the fundamental mechanisms driving breast cancer metastasis and offer new therapeutic strategies for breast cancer treatment.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA172129-04
Application #
9232085
Study Section
Tumor Progression and Metastasis Study Section (TPM)
Program Officer
Ault, Grace S
Project Start
2014-04-01
Project End
2019-02-28
Budget Start
2017-03-01
Budget End
2018-02-28
Support Year
4
Fiscal Year
2017
Total Cost
$283,860
Indirect Cost
$97,110
Name
University of Texas Health Science Center Houston
Department
Biology
Type
Schools of Medicine
DUNS #
800771594
City
Houston
State
TX
Country
United States
Zip Code
77030
Zuo, Yan; Ulu, Arzu; Chang, Jeffrey T et al. (2018) Contributions of the RhoA guanine nucleotide exchange factor Net1 to polyoma middle T antigen-mediated mammary gland tumorigenesis and metastasis. Breast Cancer Res 20:41
Ulu, Arzu; Oh, Wonkyung; Zuo, Yan et al. (2018) Stress-activated MAPKs and CRM1 regulate the subcellular localization of Net1A to control cell motility and invasion. J Cell Sci 131:
Ulu, Arzu; Frost, Jeffrey A (2018) Regulation of RhoA activation and cell motility by c-Jun N-terminal kinases and Net1. Small GTPases :1-7
van der Hoeven, Dharini; Cho, Kwang-Jin; Zhou, Yong et al. (2017) Sphingomyelin metabolism is a regulator of KRAS function. Mol Cell Biol :
Zuo, Yan; d'Aigle, John; Chauhan, Anjali et al. (2017) Genetic deletion of the Rho GEF Net1 impairs mouse macrophage motility and actin cytoskeletal organization. Small GTPases :1-8
Wu, Zizhen; Li, Lin; Xie, Fuhua et al. (2017) Activation of KCNQ Channels Suppresses Spontaneous Activity in Dorsal Root Ganglion Neurons and Reduces Chronic Pain after Spinal Cord Injury. J Neurotrauma 34:1260-1270
Zuo, Yan; Oh, Wonkyung; Ulu, Arzu et al. (2016) Minireview: Mouse Models of Rho GTPase Function in Mammary Gland Development, Tumorigenesis, and Metastasis. Mol Endocrinol 30:278-89
Ulu, Arzu; Frost, Jeffrey A (2016) Regulation of RhoA activation and cytoskeletal organization by acetylation. Small GTPases 7:76-81
Song, Eun Hyeon; Oh, Wonkyung; Ulu, Arzu et al. (2015) Acetylation of the RhoA GEF Net1A controls its subcellular localization and activity. J Cell Sci 128:913-22
Zuo, Yan; Berdeaux, Rebecca; Frost, Jeffrey A (2014) The RhoGEF Net1 is required for normal mammary gland development. Mol Endocrinol 28:1948-60

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