Development of inducible transgenic mouse models for melanoma.
PhD thesis, University of Glasgow.
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Despite many studies on pathology and aetiology during the past decades, the molecular mechanism(s) of melanoma development remains largely unknown. Therefore the purpose of this project was to establish a transgenic mouse model able to investigate the molecular mechanism(s) of melanoma aetiology mediated by N-Ras and PTEN genes. To achieve this, an inducible gene switch approach was employed exploiting the Cre/loxP recombinase system. This approach has the advantage of avoiding embryonic lethality and helps to minimise disease(s) in other tissue(s) that may interfere with animal viability. It was envisaged that this inducible system would establish a model that accurately mimic the development of melanoma in humans. A disadvantage being that the tyrosinase-based promoter was only responsive to the inducer when melanocytes were proliferating.
Initially, regulator vectors were created by sub-cloning Cre under the control of a melanocyte-specific promoter either enhanced tyrosinase (EICre) or tyrosinase related protein 2 (Trp2Cre). A Cre responsive, target N-Raslys61 transgene was also cloned, where expression was induced by Cre ablation of ‘Stop' cassette (cmv.stop.N-Raslys61) together with a report target transgene (cmv.stop.EGFP) to aid in expression characterisation.
The functional activity and gene-switch specificity of these constructs were subsequently confirmed employing co-transfection of regulator and report target into B16 melanoma cells, but to confirm their activity in primary melanocytes, melanocyte culture conditions had to be defined for optimum growth and transfection as there is no optimized commercial medium available for murine melanocyte culture unlike for human melanocyte. In this study therefore, murine primary melanocyte culture method (50/50) has been defined, which exploited keratinocytes for initial melanocytes growth support as a feed layer. The other advantages of this 50/50 medium were that pigmented cells grew without spontaneous transformation and gave the higher transfection efficiency compared to media exploited by other groups. Using primary melanocytes cultured in 50/50 medium, transgene construction and identification of regulator expression were performed by RT-PCR in vitro prior to in vivo analysis thereby avoiding unnecessary breeding.
When concerns had arisen regarding an unexpected lack of melanoma phenotype in vivo particularly in addition of PTEN loss, this culture protocol supplied a successful test of oncogenic potential of N-Raslys61 and PTEN loss in vitro, where N-Raslys61 expression transformed melanocytes and PTEN loss promoted N-Raslys61 to give more aggressive cells. However a functional redundancy was identified, as transformed colonies were not immortalized and eventually senesced, possibly due to their opposing gene functions being on the same signalling pathway; i.e. PTEN fails to provide additional genetic aberrant pathway(s) for the cross-talk with Ras signalling necessary to form malignant tumours.
The in vivo experiments commenced by crossing transgenic expressers of EICre regulator with target cmv.stop.N-Raslys61, to generate bigenic EICre/N-Ras mice. Treatment with Ru486 initially apparently failed to exhibit an abnormal phenotype, despite confirmation of N-Raslys61 expression following hair plucking to initiate the hair cycle and anagen melanocytes proliferation, therefore PTENflx/flx mutation was introduced. Unexpectedly, a similar result was obtained following treatment of EICre/PTENflx/flx/N-Raslys61 and EICre/PTENflx/flx in the test of whether PTEN functional loss promoted N-Raslys61 tumourigenesis. However with time, at 12-15 months (systemic) Ru486 treatment, phenotypes of enlarged eyes and harderian gland adenomas were obtained in N-Raslys61 expressing mice, whilst PTEN loss did not produce additional melanocytic phenotype.
This confirmation of in vivo activity prompted a more careful analysis of treated mouse skin that discovered the appearance of white hair at treated sites which gave a subtle grey appearance to the coat colour compared to age matched untreated littermates or non-transgenic controls. Subsequent analysis found that melanocyte apoptosis was induced by N-Raslys61 mediated by caspsase-3, and this may explain the lack of melanomas. This new finding implied the existence of a cell defence system to protect mice from oncogenic expression, as a general feature or to overcome specific mutations that have the potential to induce melanoma. Furthermore, the same apoptotic pathway mediated by caspase-3 was mounted against PTEN functional loss. This implied a potential surveillance mechanism to compensate for PTEN function loss and also verified in vivo, the functional redundancy in melanocytes between these two genes observed in vitro, as it may be that until the appropriate anti-apoptotic pathway overcomes this sentinel mechanism, PTEN loss synergism with N-Raslys61 is insufficient for melanoma tumourigenesis.
Due to the lack of melanoma, given the well characterized effects of the microenvironment in melanoma development, this study assessed the consequences of keratinocytes disruption. This was achieved employing a keratinocyte-specific K14Cre regulator transgenic line, expressed in proliferative basal cells, hair follicles and stem cells. In Ru486-treated tetragenic compound K14Cre/EICre/cmv.stop.N-Raslys61/PTENflx/flx mice pigmented papillomas were produced. This identified a melanocyte survival loop generated by microenvironment disruption that enabled anagen melanocytes to escape apoptosis during papillomagenesis. Furthermore, the mechanism involved elevated Kit/SCF expression in papillomas. The co-localisation of Kit and TRP-2 positive melanocytes in papilloma basal layers revealed that a Kit/SCF paracrine survival loop resulted in melanocyte survival. These results clearly demonstrated melanocyte cooperation with its immediate microenvironment consistent with the requirement for proliferative keratinocyte support of primary murine melanocyte cultures. Furthermore, these pigmented papillomas, may represent a model relevant to development of human seborrheic keratoses, which are pigmented benign lesions similar to papilloma, and neither nevi nor melanoma (4-6). These murine data suggest that these lesions may arise where papilloma formation occurs alongside anagen and the Kit/SCF paracrine survival loop creates an environment in papillomas sufficient to incorporate the survival and proliferation of anagen melanocytes, although the further studies are necessary to confirm this.
To date, most transgenic melanoma models employ H-Ras, with only recent development of a relevant N-Ras model where constitutive, but not inducible, N-Raslys61 expression throughout embryogenesis eventually gave a hyperplastic melanocyte phenotype which is consistent with the report of N-Ras mutation common in congenital nevi but less in acquired nevi (9-13). As with H-Ras models, it appears that the CDKN2A locus deficiency is necessary for melanoma aetiology. In this study, unlike CDKN2A, PTEN loss failed to promote N-Ras melanoma tumourigenesis. This is possibly due to regulating the same signalling pathways, creating a functional redundancy, and the same susceptibility to apoptosis from newly identified potential compensatory surveillance systems. These results show the necessity of cross-talk between multiple genetic pathways to achieve malignant tumour formation and also the advantage of an inducible gene-switch approach to identify useful compensatory systems by allowing addition/deletion of many different interesting genes. Taking the insights from this study further, logically the introduction of p16/p19 deficient mice and/or other melanocyte/melanoma development related genes (specifically not on Ras signalling pathway, e.g. MC1R pathways) would provide an up-to-date, superior mouse model able to mimic molecular aetiology of human melanoma to investigate the functions and mechanisms of other genes such as MITF, B-Raf, MC1R etc involved in the development of human melanoma.
||transgenic, melanoma, inducible, modelling, PTEN gene, N-Ras gene, Kit, SCF, Papilloma, skin
||R Medicine > RL Dermatology
||College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
||Greenhalgh, Dr. David A.
|Date of Award:
Mr denggao yao
||Copyright of this thesis is held by the author.
||18 Feb 2009
||10 Dec 2012 13:19
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