Thyroid cancer is the most common endocrine malignancy and his global incidence has rapidly increased in last decades. Despite the major part of thyroid cancer is represented by well differentiated hystotypes, the acquisition of additional mutations, such as p53 and β-catenin ones, causes loss of differentiation and confer high malignancy. Current treatment for undifferentiated thyroid cancers is regarded as almost ineffective, with a median survival of 3- 4 months, somewhat better in localized and worse in metastatic disease. SP600125 is a multi-kinase inhibitor that has recently been shown to be a promising anticancer drug. In the last five years it has been proved able to induce endoreduplication and subsequent polyploidization, but there are contrasting results about its intracellular actions and there is no evidence on the specific mechanism of action. Moreover, in 2012, SP600125 has been found to be the most effective against p53 deficient cells among more than 300 screened compounds. However, opposite results have also been obtained depending on cell type, concentration and time of incubation. In the current study the effects of micromolar doses of SP600125 have been characterized in six thyroid cancer cell lines with different p53 status. The results show that at low concentrations SP600125 dramatically reduces the proliferation of p53 mutated cells, with lesser effects on p53 null and no effects on the wild-type ones. In p53 mutated cells it has been proved able to induce p53 nuclear translocation and phosphorylation at serine 15; this modification resulted to be responsible of increased levels of p21. Importantly other considerable novel effects have been revealed. Firstly, SP600125 caused alterations of microtubule dynamics in p53 mutated cells, with increase of acetylation levels and loss of Microtubule Organizing Center (MTOC)-periphery organization. These effects were accompanied by alterations in cellular morphology and in late endosome/lysosome trafficking. Endoreduplication and alteration of microtubule dynamics finally resulted in aberrant mitosis and cell death. The second mechanism involves alteration of cellular motility: different kinases involved in this process are affected by SP600125 treatment and β-catenin remains at the intercellular junctions in affected cells,consistent with a failure of cell detachment (a figure consistent with inhibition of cell migration/motility). Microtubule alterations concomitantly also account for motility alterations. Both tubulin and β-catenin variations are due to HDAC6 activity alterations. This enzyme regulates the levels of acetylation of these proteins and is profoundly inhibited following SP600125 in p53 deficient cells. Alteration of HDAC6 activity is hypothesized to be the result of SP600125 direct inhibition of ROCK2, an upstream kinase regulator of HDAC6 activity. In conclusion SP600125 is a promising drug particularly active on p53-mutated cancers at concentrations unable to affect normal cell viability. The effects of SP600125 action include arrest of tumor growth, and importantly induction of tumor cell death and metastatic diffusion inhibition.

P53 AND MICROTUBULES TARGETING AS A NOVEL STRATEGY WITH POTENTIAL FOR TREATMENT OF AGGRESSIVE POORLY DIFFERENTIATED THYROID CANCER / E.s. Grassi ; relatore: L. Persani ; coordinatore: A. Gianni. - : . DIPARTIMENTO DI BIOTECNOLOGIE MEDICHE E MEDICINA TRASLAZIONALE, 2014 Feb 19. ((26. ciclo, Anno Accademico 2013. [10.13130/grassi-elisa-stellaria_phd2014-02-19].

P53 AND MICROTUBULES TARGETING AS A NOVEL STRATEGY WITH POTENTIAL FOR TREATMENT OF AGGRESSIVE POORLY DIFFERENTIATED THYROID CANCER.

E.S. Grassi
2014-02-19

Abstract

Thyroid cancer is the most common endocrine malignancy and his global incidence has rapidly increased in last decades. Despite the major part of thyroid cancer is represented by well differentiated hystotypes, the acquisition of additional mutations, such as p53 and β-catenin ones, causes loss of differentiation and confer high malignancy. Current treatment for undifferentiated thyroid cancers is regarded as almost ineffective, with a median survival of 3- 4 months, somewhat better in localized and worse in metastatic disease. SP600125 is a multi-kinase inhibitor that has recently been shown to be a promising anticancer drug. In the last five years it has been proved able to induce endoreduplication and subsequent polyploidization, but there are contrasting results about its intracellular actions and there is no evidence on the specific mechanism of action. Moreover, in 2012, SP600125 has been found to be the most effective against p53 deficient cells among more than 300 screened compounds. However, opposite results have also been obtained depending on cell type, concentration and time of incubation. In the current study the effects of micromolar doses of SP600125 have been characterized in six thyroid cancer cell lines with different p53 status. The results show that at low concentrations SP600125 dramatically reduces the proliferation of p53 mutated cells, with lesser effects on p53 null and no effects on the wild-type ones. In p53 mutated cells it has been proved able to induce p53 nuclear translocation and phosphorylation at serine 15; this modification resulted to be responsible of increased levels of p21. Importantly other considerable novel effects have been revealed. Firstly, SP600125 caused alterations of microtubule dynamics in p53 mutated cells, with increase of acetylation levels and loss of Microtubule Organizing Center (MTOC)-periphery organization. These effects were accompanied by alterations in cellular morphology and in late endosome/lysosome trafficking. Endoreduplication and alteration of microtubule dynamics finally resulted in aberrant mitosis and cell death. The second mechanism involves alteration of cellular motility: different kinases involved in this process are affected by SP600125 treatment and β-catenin remains at the intercellular junctions in affected cells,consistent with a failure of cell detachment (a figure consistent with inhibition of cell migration/motility). Microtubule alterations concomitantly also account for motility alterations. Both tubulin and β-catenin variations are due to HDAC6 activity alterations. This enzyme regulates the levels of acetylation of these proteins and is profoundly inhibited following SP600125 in p53 deficient cells. Alteration of HDAC6 activity is hypothesized to be the result of SP600125 direct inhibition of ROCK2, an upstream kinase regulator of HDAC6 activity. In conclusion SP600125 is a promising drug particularly active on p53-mutated cancers at concentrations unable to affect normal cell viability. The effects of SP600125 action include arrest of tumor growth, and importantly induction of tumor cell death and metastatic diffusion inhibition.
PERSANI, LUCA
GIANNI, ALESSANDRO
thyroid ; cancer ; p53 ; microtubules ; endoreduplication ; SP600125
Settore MED/06 - Oncologia Medica
Settore MED/13 - Endocrinologia
Settore BIO/13 - Biologia Applicata
Settore BIO/14 - Farmacologia
P53 AND MICROTUBULES TARGETING AS A NOVEL STRATEGY WITH POTENTIAL FOR TREATMENT OF AGGRESSIVE POORLY DIFFERENTIATED THYROID CANCER / E.s. Grassi ; relatore: L. Persani ; coordinatore: A. Gianni. - : . DIPARTIMENTO DI BIOTECNOLOGIE MEDICHE E MEDICINA TRASLAZIONALE, 2014 Feb 19. ((26. ciclo, Anno Accademico 2013. [10.13130/grassi-elisa-stellaria_phd2014-02-19].
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/232400
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