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Title: miR-335-Oct4-Rb1: a new axis to control cell proliferation, tumorigenesis and self-renewal
Other Titles: miR-335-Oct4-Rb1: a new axis to control cancer cell proliferation and self-renewal
Authors: Scarola, Michele
Supervisore/Tutore: Benetti, Roberta
Issue Date: 22-Apr-2013
Publisher: Università degli studi di Trieste
The retinoblastoma tumor suppressor protein (pRb) belongs to a cellular pathway that is deregulated in several human tumors. A hallmark of the pRb pathway is its ability to control G1-S transition of the cell cycle and to prevent uncontrolled cell proliferation. Nevertheless, research in the last years identified multiple alternative cellular functions regulated by pRb including several aspects of stem cell biology, that in contrast to pRb role in cell cycle control, are poorly understood.
The work presented in this thesis aimed to investigate the post-transcriptional regulation of pRb expression and pRb function in the context of human cancer and stem cell biology. In the first part of this thesis work we evaluated the role of miR-335, a microRNA predicted to target RB1, to regulate pRb at post-transcriptional level and the impact of this regulation on pRb tumor suppressor function in cancer cells, focusing on the control of cell proliferation; in the second part we investigate the role of pRb in stem cells context studying the relationship between the control of cell-cycle progression and the control of self-renewal features. In particular we focused our research both on the regulation of its expression by evaluating the impact of miR-335 on pRb in the context of embryonic stem cells, and to link pluripotency transcription factor Oct4 with the regulation of the pRb pathway.
MicroRNAs are small non-coding RNAs critically involved in the post-transcriptional regulation of gene expression and influencing all biological processes, including tumorigenesis, stem cell self-renewal and differentiation. Although Rb proteins have a critical role in many cellular processes, information on microRNAs that control Rb family proteins at post-transcriptional levels are still very limited.
The initial aim of this project was to address a putative role for miR-335, a microRNA predicted to target mammalian RB1, in the control of pRb expression, cell proliferation and tumorigenesis. We were able to demonstrate that miR-335 is differentially expressed in human cancer cells and that it regulates the expression of human pRb by directly target a conserved sequence motif in its 3’UTR. High conservation of miR-335 and its RB1 target sequence in placental mammals, as well as differential expression in human cancer cell lines indicates an important role in cell cycle control. We found that the
impairment of the pRb pathway by miR-335 is paralleled by a significant upregulation of p53 levels and that the activation of the p53 pathway in the context of miR-335 overexpression impairs cell proliferation and neoplastic transformation. In line with this, reducing p53 levels is sufficient to drive hyperproliferation and increased transformation in the context of ectopically increased miR-335 levels.
In our study, we also showed that the activation of the p53 pathway during DNA damage significantly increases miR-335 levels and demonstrated that miR-335 mediates efficient cell cycle arrest on DNA damage in a positive feedback loop with p53. In conclusion, these results identify miR-335 as a potent regulator of pRb at posttranscriptional level, indicate that miR-335 helps control proliferation by balancing the activities of the pRb and p53 tumor suppressor pathway and that miR- 335 activation plays an important role in the induction of p53-dependent cell cycle arrest after DNA damage.
Recently it has becoming evident that cancer cells exhibit several traits that are also characteristic of embryonic stem cells. In particular, the constitutive hyperphosphorylation of pRb that ensures rapid and indefinite cell proliferation potential of ESCs recapitulates the impaired function of the pRb pathway present in virtually all tumors. In addition, self-renewal expression signature including the pluripotency transcription factors Oct4, Nanog and Sox2 were also found in several types of human cancer. Although these evidences support the idea that the acquisition of stem cell-like gene expression signature represents a key step in tumorigenesis, evidence for a functional link between pluripotency transcription modules and the control of aberrant function of tumor suppressor pathways is limited.
The second aim of this thesis work was to better characterize the role of pRb in stem cells context focusing on the regulation of its expression and function, connecting pluripotency transcription factor Oct4 with the Retinoblastoma tumor suppressor pathway.
We were able to demonstrate that Oct4 cooperates with pRb in the establishment of self-renewal program of mouse embryonic stem cells (mESCs) and that these two factors are under control of miR-335 that targets conserved sequence motifs in the 3’UTRs of Oct4 and Rb1.
We demonstrated that Oct4 is required to ensure the hyperphosphorylation of pRb, thereby permitting rapid cell proliferation of self-renewing mESCs. miR-335 plays a
central role in interrupting the self-renewal promoting Oct4-pRb axis at the onset of mESC differentiation by targeting the expression of both proteins at the post- transcriptional level. In particular, we found that in self-renewing mESCs, high Oct4 expression levels drive the expression of Nipp1 and Ccnf to inhibit the activity of the protein phosphatase type 1 (Pp1) thereby establishing pRb hyperphosphorylation as a key feature of rapidly cycling, pluripotent mESCs. Upon induction of differentiation, transcriptional repression of Oct4 in conjunction with targeting of Rb1 and Oct4 by miR-335 causes the collapse of the Oct4-Nipp1/Ccnf1-Pp1-pRb axis, leading to a rapid pRb dephosphorylation, the exit from self-renewal and the establishment of pRb regulated cell cycle profile of differentiated cells.
In conclusion, our results introduce a novel regulatory circuit, modulated by miR- 335, which connects the pluripotency transcription factor Oct4 with the pRb pathway to control mESC self-renewal and differentiation, and anticipate an important role for Oct4 in the inactivation of the Retinoblastoma tumor suppressor pathway in human cancers with embryonic stem cell gene expression signatures.
Ciclo di dottorato: XXV Ciclo
Keywords: Rb1Oct4miR-335cancerstem cells
Type: Doctoral
Language: en
Settore scientifico-disciplinare: BIO/11 BIOLOGIA MOLECOLARE
NBN: urn:nbn:it:units-11382
Appears in Collections:Scienze biologiche

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