A mouse model to study inducible oncogene cooperation in vivo

Modica, Teresa Maria Elisa (2012). A mouse model to study inducible oncogene cooperation in vivo. PhD thesis The Open University.

DOI: https://doi.org/10.21954/ou.ro.0000d3da

Abstract

The current model for cancer development envisions cells under going a series of genetic mutations and/or alterations which result in their inability to respond normally to intracellular and extracellular signals that control proliferation, differentiation and death. The number of required genetic alterations varies for different types of cancer and it is likely that further changes occur during its progression to increased malignancy.

Thus, cancer is not a static disease but during the development and progression of tumour, multiple changes occur in two kinds of genes: oncogenes and tumour suppressor genes.

Oncogene-products can be classified as growth factors, growth factor receptors, Ras oncoproteins, cytoplasmic protein kinases, transcription factors, anti-apoptotic proteins.

In particular, the ras oncogene family includes three members: N-ras, K-ras, H-ras. In non-transformed cells, Ras protein, belonging to G-protein family, transduces growth signals from external to the internal environment. In fact, when activated, Ras exchanges GDP with GTP and this allosteric change allows binding of Ras effector molecules and transduction of signalling cascades.R as activity is required for cell cycle progression.

In cancer it has been observed that this oncogene is constitutively activated by mutations and induces the cell to enter into cell-cycle also in the absence of growth signals.

Among the transcription factors, a gene involved in many tumours is myc. This transcription factor plays a key role in cell proliferation as its target proteins include many positive regulators of the cell-cycle. In tumour cells the protein product of this oncogene is overexpressed.

The cooperationb etweenm ultiple oncogenesa nd/orl oss of tumour suppressors from different functional classes is necessary for transformation to proceed.

In fact, it was observed that, although overexpression of a single oncogene does not transform wild-type mouse embryonic fibroblasts, combinations of myc and H-ras^VAL12, can induce cellular transformation and the cells expressing both oncogenes displayeda markedp roliferative advantage.

In thyroid, neoplastic transformation generates several different histotypes of tumours, ranging from poorly aggressive and well-differentiated, to highly malignant and undifferentiated anaplastic cancers.

The aim of my thesis was to study the tumorigenesis induced by oncogenes and the oncogene cooperation in vivo during the gradual passage from a poorly aggressive to a much more aggressive tumour.

To this end a mouse model expressing the two oncogenes H-ras^VAL12 and c-myc (referred as ras and myc) in a tissue-specific as well as in a conditional manner was generated.

For this purpose, the coding sequences of the two oncogenes were fused in a bicistronic construct and an IRES (Internal Ribosome Entry Sequence or Site) was inserted between them, to ensure the expression of the second oncogene. The construct was inserted under the control of the promoter of the ubiquitously expressed genes ROSA26 and Eeflal.

In order to express these oncogenes in a tissue-specific manner, the transcription of the two oncogenesis preventedb y a STOP sequencef lanked by two LoxP sites. Such a STOP sequence can be removed by Cre recombinase protein.

The transgenic mice were crossed with mice expressing Cre in a tissue-specific manner. Two strains of transgenic mice expressing Cre in thyroid cells were used:

1. transgenic mice for TgCre, in which Cre is expressed under the control of Tg promoter after the development of the thyroid;

2. Pax8Cre, in which the Cre sequence is inserted in the Pax8 locus and is expressed during the early stages of the thyroid development.

In such a manner the oncogenes were expressed only in thyroid cells, but were still inactive.

In particular, ras was fused to the mutated ligand binding domain of the estradiol receptor that is sensitive to tamoxifen and not to endogenous estradiol; while mycwas fused to the mutated ligand binding domain of the progesterone receptor (hPR891) that is sensitive to RU486 and not to endogenous progesterone.

With thesef usedo ncogeneist is possible to activate only Ras( with tamoxifen) or only Myc (with RU486) or both (providing both tamoxifen and RU486).

Moreover the activity of two oncogenes might be used to immortalize mouse cell lines in culture.