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Impact of early centrosome deregulation in malignant transformation
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Resumo(s)
Centrosome amplification (CA) is a hallmark of human cancers and is therefore a potential
feature to explore for prognosis and therapy. However, the poor understanding of its origin and impact
has limited its use in the clinic. Here, we used Barrett’s esophagus (BE) tumorigenesis as a human cancer
model to test if CA has a contributory role in tumorigenesis and progression.
Barrett’s esophagus is a premalignant condition and its neoplastic progression is a process with
well-characterized and defined steps that go from metaplasia (premalignant condition - BE) to dysplasia
(low- or high-grade intraepithelial neoplasia), adenocarcinoma (invasive neoplasia) and metastasis.
Given that CA arises in BE metaplasia and that its incidence significantly increases from metaplasia to
dysplasia, we posed the hypothesis that CA may play an important role in the acquisition of malignant
properties. If this hypothesis is correct, then an increase of CA in metaplasia and/or a decrease of CA in
dysplasia would be sufficient to respectively promote or reduce malignant properties such as
invasiveness potential.
To test these hypotheses, we first tested two different methods to reduce the CA levels in
dysplasia cells: depletion of important molecules in the centrosome duplication cycle - PLK4, SAS6 and
STIL - by siRNA, and inhibition of PLK4 activity using the specific chemical inhibitor centrinone-B.
We then assessed changes in migration and invasive potential using 2D migration and invasion assays
and 3D cell cultures. Both strategies were effective in reducing CA in dysplasia cells but as the treatment
with the inhibitor acts directly on the protein's activity level, its effects were more quickly detected.
Importantly, by providing the opportunity to reduce CA levels by affecting different molecules, the
siRNA approach allowed us to confirm if the effects in the invasiveness properties of dysplasia cells is
in fact a consequence of the reduction in the number of centrioles, and not just due to the dysfunction of
specific molecules. Indeed, while results obtained with 2D migration and invasion assay were mostly
inconclusive and further optimization is needed, we found that reduction of CA levels, with both
approaches, reduced the invasiveness capacity of dysplasia cells in 3D cultures. Our findings therefore
support a role for CA in promoting the invasiveness capacity in dysplasia, and provided important clues
into their mechanisms that will be explored in the future. We then tested if an increase in CA is sufficient
to induce invasion in metaplasia cells. To do this we first depleted p53 in metaplasia cells by siRNA,
already known to increase CA in these cells, and then assessed their behavior using 3D cell cultures.
Interestingly, our preliminary tests did not reveal any phenotypic differences in metaplasia cells with
increased CA when compared to the controls. These findings indicate that increasing CA levels in
metaplasia may not be sufficient, by itself, to induce an invasive capacity in metaplasia cells. In the
future, testing different approaches to induce CA in these cells, as well as assessing their impact in cell
migration and invasion using other assays, will be important to confirm this.
By showing that CA contributes to the invasiveness potential in dysplasia, our results reveal the
importance of CA in the acquisition of malignant properties in BE progression. These findings may
contribute to new clinical tools, namely using CA as a biomarker of progression. Given widespread
occurrence of CA in human tumors, our results may be extended to other cancers where CA is prevalent.
Descrição
Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2022
Palavras-chave
Cancro humano Esófago de Barrett Amplificação do centrossoma Invasão Progressão do tumor Teses de mestrado - 2022