Molecular Oncology

Work team

Research project

FUNCTIONAL CHARACTERIZATION OF ZEB1 IN METASTATIC AND DIFFERENTIATING CELLS

Breast and prostate cancer metastasis are the second leading cause of cancer deaths in women and men, respectively. Characterizing genes that regulate the growth and metastatic ability of these cancers may identify novel biomarkers to help clinicians guide current treatments, and may offer new targets for therapy. To acquire the invasive abilities, epithelial cancer cells must undergo several phenotypic changes termed epithelial-mesenchymal transition (EMT)(1). Several master gene regulatory programs have recently been discovered to play key roles in cancer progression and EMT which can be activated by diverse cytokine signals, including TGFβ, Wnt, and tyrosine kinase receptor signaling. These external signals regulate transcription factors such as ZEB1, Snail, and Twist (1, 2) by integrative molecular mechanisms still not well understood.

Zinc finger E-box-binding homeobox (ZEB1) controls a diverse array of processes such as mesoderm-derived cell differentiation, proliferation and senescence (3,4). ZEB1 can act in the TGFβ pathway as a Smad-binding factor, and is strongly involved in EMT during both normal development and disease (5,6). At a molecular level, ZEB1 is mainly characterized as a transcriptional repressor of genes implicated in EMT (E-cadherin among others)(4). However, little is known about how the regulation of ZEB1 is integrated with other signaling pathways. It is very important to understand the basic biology of cancer to target the particular pathways involved.

Previous work of the Cabanillas’s lab showed that ZEB1 phosphorylation induced by protein kinase C (PKC) and MEK/ERK modifies transcriptional activity of this transcription factor in normal epithelial cells (7,8). We have also recently identified a small N-terminal fragment of ZEB1 (N-ZEB1) which changes its subcellular location from the nucleus to the cytoplasm upon cells activation by PKC. Pretreatment of the cells with a pan-PKC inhibitor reverted the effect of PMA/Ionomycin (PKC activator) on ZEB1 nuclear location. On the other hand, activators or inhibitors of other signaling kinases did not change ZEB1 nuclear expression. N-ZEB1 is able to induce EMT on NMuMG murine normal epithelial breast cells independently of TGFβ since is able to repress E-cadherin expression, to reorganize actin cytoskeleton, and to increase expression of MMP2, which favors the invasive abilities of these cells (9).

PKC is a family of serine-threonine kinases that comprises three groups of isozymes with unique biochemical properties: classical/conventional or calcium-dependent PKCs (cPKCs) α, β, ϒ; novel or calcium-independent PKCs (nPKCs) δ, ε, η, θ; and atypical PKCs (aPKCs) ζ, λ. It is well established that PKCs control mitogenicity, cell survival, and invasion. Despite their high homology and similar substrate specificity in vitro, PKC isozymes possess distinctive functional selectivity due to their characteristic intracellular localization and differential access to substrates(10).

Our general hypothesis is that ZEB1 is regulated at different levels, post-transcriptional, post traslacional, by external or internal factors, which in a deregulated status (cancer) would induce an anomalous response of ZEB1 with the subsequent activation of metastatic programs such as EMT. We hypothesize that phosphorylation may be a very effective mechanism for the regulation of the biological actions of ZEB1.

If our hypothesis is true, it will have profound implications in developmental processes and in cancer invasion.

The specific aims of this project are as follows:

S.A.1. To investigate post translational mechanisms of ZEB1 regulation in normal and in breast cancer cells and its relevance in EMT and cancer progression. We will analyze the involvement of intracellular signals in phosphorylation of ZEB1 by gain- and lost-of-function experiments.

S.A.2. To determine whether the signaling pathways act either directly modifying ZEB1 or indirectly through phosphorylation of the proteins that form ZEB1 interactoma (CtBP1, CtBP2, p/CAF /p300).

S.A.3. To determine the biological relevance of the changes in cellular location of ZEB1 by different activators of signaling pathways. In this sense, we will study mechanisms of degradation of the protein such as ubiquitynation and SUMOylation.

S.A.4. To identify the PKC isoforms involved in ZEB1 phosphorylation and their (direct or indirect) mechanism/s of action, using gain- and loss-of-function experiments in benign and malignant breast and prostate cancer cell lines and in vivo approaches.

S.A.5 To investigate the eventual cross-talk among selected signaling pathways (ZEB1 regulators) and TGFβ on EMT.

Publications

  1. Nieto, MA. (2011). The ins and outs of the epithelial to mesenchymal transition in health and disease. Annu Rev Cell DevBiol, 27: 347-76.
  2. Roxanis, I. (2013). Occurrence and significance of epithelial-mesenchymal transition in breast cancer. J. Clin. Pathol. 66: 517 –21.
  3. Sanchez-Tillo, E., Liu,Y., O de Barrios, L., Siles, L., Fanlo, L., Cuatrecasas, M., Darling,DS., Dean,DC, Castells,A and Postigo, A. (2012). EMT-activating transcription factors in cancer: beyond EMT and tumor invasiveness. Cell Mol Life Sci 69(20): 3429-56.
  4. Vandewalle, C., Van Roy, F. & Berx, G. (2009). The role of the ZEB family of transcription factors in development and disease. Cell Mol Life Sci.:1-15.
  5. Sanchez-Tillo, E., Siles, L., O de Barrios, L., Cuatrecasas, M., Vaquero, E.C., Castells, A. and Postigo, A. (2011). Expanding roles of ZEB factors in tumorigenesis and tumor progression. Am J Cancer Res 1(7): 897-912.
  6. Scheel, C & Weinberg, RS. (2012). Cancer stem cells and epithelial-mesenchymal transition: concepts and molecular links. Semin Cancer Biol 22(5-6): 396-403.
  7. C Llorens*, G Lorenzatti*, N Cavallo, MV Vaglienti, A Perrone, A Carenbauer, DS Darling & AM Cabanillas. “Phosphorylation Regulates Functions of ZEB1 Transcription Factor”. J Cell Physiol 2016 en prensa. doi: 10.1002/jcp.25338. * Ambos autores contribuyeron como primeros autores.
  8. Llorens MC, Cavallo N, Perrone AP, Cabanillas AM. (2013). Participación de las Vías de Señalización en la Fosforilación del Dominio Amino Terminal de ZEB1y su Localización Subcelular. Medicina 73 (Supl III): 4.
  9. Llorens MC; Cavallo NL; Cabanillas AM. (2014). Identificación y caracterización de un fragmento del dominio amino terminal de ZEB1 que induce la Transición Epitelio Mesenquimatosa (EMT). Medicina. 74 (supl. III): 133.
  10. Griner, EM., & Kazanietz, MG. (2007). Protein kinase C and other diacylglycerol effectors in cancer. Nat. Rev. Cancer 7, 281–294.