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ESR Project 14:  TRIB3-mediated regulation of the invasiveness of cancer cells and of its interplay with the tumor microenvironment: consequences on tumor susceptibility, progression and metastatic capacity

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Jack Day

Prostate cancer (PCa) is the most common cancer among men, with an estimated 221,000 new diagnoses in the USA in 2015 (Peisch et al., 2016) and 1 in 39 men dying of the disease (cancer.org). PCa begins when healthy cells in the prostate gland undergo mutation (or a series of mutations) that allows them to grow uncontrollably, forming a tumour. Some cells can break away from the main tumour into the bloodstream or lymphatic system and find residence in other tissues, forming metastases and becoming more malignant, resulting in a decreased chance of survival.

The tumour suppressor Phosphatase and Tensin Homolog (PTEN) is considered one of the main negative regulators of the Phosphatidyl Inositol 3-Kinase (PI3K) pathway and is reduced or absent in approximately 70% of PCa´s (Trotman et al., 2003). Under normal conditions, PI3K activates Akt indirectly by generating PIP3 at the plasma membrane, recruiting Akt, through its PH domain, where it is phosphorylated by PDK1 at Thr308. This pathway is negatively regulated at the PI3K level by PTEN through dephosphorylation of PIP3. However, in cells where PTEN is either lost or reduced, enhanced Akt phosphorylation results in hyperphosphorylation and inactivation of the transcription factor FOXO3, resulting in enhanced tumourigenesis through the inhibition of apoptosis (Salazar et al., 2015a). It should be noted that there are other downstream targets of Akt (such as GSK3 and BAD), however, their role in prostate cancer development and progression has yet to be elucidated.

The Tribbles proteins (Tribs) are a family of 3 pseudokinases (Trib1-3) (Kiss-Toth et al., 2004). They each have a kinase domain which lacks critical residues required for its catalytic activity. Trib3 has onco-suppressive functionality (by facilitating apoptosis) that acts by inhibiting Akt activation at Ser473 by the mammalian Target of Rapamycin Complex 2 (mTORC2), also activated by PI3K, and is frequently lost in prostate cancers in conjunction with loss of PTEN (Salazar et al., 2015b). It has been found previously that loss of Trib3 enhances tumourigenesis by enhancing the incidence of malignant lesions and tumour growth, but is not sufficient alone to accelerate tumour growth (Salazar et al., 2015b)

The major aims of this PhD project at Complutense Universidad de Madrid (UCM) and Instituto de Investigaciónes Sanitarias San Carlos (IdISSC) are to:

-Establish the effect of Trib3 in the regulation of metastatic capacity of prostate cancer and to disseminate the mechanism of regulation.

-Investigate the role of Trib3 in the modification of the tumour microenvironment (stroma) and tumour susceptibility to progression.

-Study the role of Trib3 on the activation of fibroblasts (Cancer Associated fibroblasts, CAFs) and the transformation of epithelial cells in prostate.

In order to achieve these aims, in vitro and in vivo models will be used. For in vitro experiments, prostate cancer cell lines (±PTEN/±Trib3), transient Trib3 knockout prostate cancer cell lines, and the aforementioned CAFs, will be used while prostate-specific PTEN and Trib3 double knockout mice will be used for in vivo experiments. In vitro assays will include scratch assay, transwell migration assay, adhesion assays, and trans-endothelial migration assay in two-dimensional (2D) and 3D matrices that mimic the stroma. In vivo assays will monitor invasion, angiogenesis, inflammation, and proliferation. In many cases this will be by subcutaneous injection of cells into the flanks of the mice. In addition, there is the potential for collaborations within the TRAIN consortium to investigate the interplay of Trib3 in prostate cancer and the immune system, adipose tissue, and Trib1.

This study will be highly valuable to the understanding of prostate cancer progression, especially in human prostate cancers that are Trib3 deficient. It is hoped that this study will facilitate the identification of new druggable targets involved in prostate cancer progression and metastasis. Such findings can aid in the design of novel therapeutics for use in the clinic. In addition, this project has the potential to identify biomarkers to provide greater diagnostic potential and earlier detection of prostate cancer.

References:

cancer.org. 2017. Key Statistics for Prostate Cancer. [ONLINE] Available at:https://www.cancer.org/cancer/prostate-cancer/about/key-statistics.html. [Accessed 17 April 2017]

Kiss-Toth E, Bagstaff SM, Sung HY, Jozsa V, Dempsey C, Caunt JC, Oxley KM, Wyllie DH, Polgar T, Harte M, O´Neill LAJ, Qwarnstrom EE, Dower SK (2004) Human Tribble, a protein family controlling mitogen-activated protein kinase cascades, J Bio Chem, 279(41): 42703-42708

Peish SF, van Blarigan EL, Chan JM, Stampfer MJ, Kenfield SA (2016) Prostate cancer progression and mortality: a review of diet and lifestyle factors, World J Urol, doi:10.1007/s00345-016-1914-3

Salazar M, Lorente M, García-Taboada E, Pérez Gómez E, Dávila D, Zúñiga-García P, María Flores J, Rodríguez A, Hegedus Z, Mosén-Ansorena D, Aransay AM, Hernández-Tiedra S, López-Valero I, Quintanilla M, Sánchez C, Iovanna JL, Dusetti N, Guzmán M, Francis SE, Carracedo A, Kiss-Toth E, Velasco G (2015a) TRIB3 suppresses tumorigenesis by controlling mTORC2/AKT/ FOXO signaling, Molecular & Cellular Oncology, 2(3): e980134

Salazar M, Lorente M, García-Taboada E, Pérez Gómez E, Dávila D, Zúñiga-García P, María Flores J, Rodríguez A, Hegedus Z, Mosén-Ansorena D, Aransay AM, Hernández-Tiedra S, López-Valero I, Quintanilla M, Sánchez C, Iovanna JL, Dusetti N, Guzmán M, Francis SE, Carracedo A, Kiss-Toth E, Velasco G (2015b) Loss of Tribbles pseudokinase-3 promotes Akt-driven tumorigenesis via FOXO activation, Cell Death Differ, 22(1): 131-144

Trotman LC, Niki M, Dotan ZA, Koutcher JA, Di Cristofano A, Xiao A, Khoo AS, Roy-Burman P, Greenberg NM, Van Dyke T, Cordon-Cardo C, Paolo Pandolfi P (2003) Pten dose dictates cancer progression in the prostate, PLoS Bio, 1(3): 385-396