Final Activity Report Summary - BREAST CANCER MODELS (Animals models for breast tumor formation)
To eradicate tumour progression and/or metastatic development, cancer cells need to be targeted directly by treatments, which may not be the case with current therapeutical drugs. The objective of the project was to establish animal models for cancer development in close relation with genomic alterations observed in tumour cells. The laboratory is investigating different types of cancer.
We decided to start by generating a transgenic mouse model that reproduces a fusion gene observed in a rare and aggressive case of myeloproliferative disorder. This gene results from a translocation of chromosome arm 6q to chromosome arm 8p, translocation t(6;8)(q27;p11). We first generated the plasmidic construct and verified its activity in vitro. We observed the expression of an active fusing protein in vitro. We obtained 65 founders by pronuclear injection but we were not able to obtain any founder that expresses the transgene. However, the construct is working and we were able to setup the conditions necessary for the genotyping by PCR that will be useful for future transgenic lines as the genotyping is done by detection of the luciferase gene.
We next searched for new potential oncogenes located in the amplified region of chromosome arm 8p. In human carcinomas, especially breast cancer, chromosome arm 8p is frequently involved in complex chromosomal rearrangements. However the structure of the alteration affecting chromosome 8 and the oncogenic gene located in this region are not well known. By array-CGH the laboratory identified 4 amplicons in the 8p11-12 region. Gene expression analysis of 123 samples using DNA micro arrays identified 14 genes significantly overexpressed in relation to amplification. Other laboratories identified in the same region a minimal amplification containing two potential oncogenes, FLJ14299 also known as ZNF703, and SPFH2. We compared the frequency of amplification and overexpression of the 14 genes in tumor cells lines (Sircoulomb et al. 2006). Two genes appeared to be the most frequently overexpressed: FGFR1 and ZNF703. ZNF703 is amplified and overexpressed in luminal cells.
We studied the oncogenic potentiality of ZNF703 and its functionality in cells before starting to elaborate any transgenic line. We were able to localised ZNF703 in the cytoplasm and nucleus. The ectopic overexpression of ZNF703 induces a nuclear localisation of the protein particularly in speckles. By yeast two hybrid screen we found an interaction with atrophin-1, which was confirmed in vivo in tumorigenic cell lines that overexpressed ZNF703. Co-localisation was visible by immunofluorescence in the nucleus with the ectopic overexpressed protein in MCF7 and in Cos cells. A partial co-localisation was observed with the endogenous protein in the nucleus and in the cytoplasm of the MDA-MB-134 cell line, but the specificity of the commercial antibody is doubtful. Atrophin-1 is a nuclear co-repressor but we do not have more information about the possible functionality of the interaction with ZNF703 in gene regulation.
We did not see any transformation potentiality of ZNF703 in NIH3T3 cells, Ba/F3 cells or REF primary fibroblast cells. ZNF703 does not seem to have a master function in regulation of the cell cycle. But ZNF703 seems to increase the tumour potentiality of MCF7 cells after orthotopic injection in the mammary gland. Our observation indicates that ZNF703 is not a master oncogenic gene but seems to have an important role in tumourigenic luminal cells. ZNF703 has been shown to be a downstream gene of the oestrogen receptor and atrophin-1 is a regulator of the EGFR in drosophila.
We decided to start by generating a transgenic mouse model that reproduces a fusion gene observed in a rare and aggressive case of myeloproliferative disorder. This gene results from a translocation of chromosome arm 6q to chromosome arm 8p, translocation t(6;8)(q27;p11). We first generated the plasmidic construct and verified its activity in vitro. We observed the expression of an active fusing protein in vitro. We obtained 65 founders by pronuclear injection but we were not able to obtain any founder that expresses the transgene. However, the construct is working and we were able to setup the conditions necessary for the genotyping by PCR that will be useful for future transgenic lines as the genotyping is done by detection of the luciferase gene.
We next searched for new potential oncogenes located in the amplified region of chromosome arm 8p. In human carcinomas, especially breast cancer, chromosome arm 8p is frequently involved in complex chromosomal rearrangements. However the structure of the alteration affecting chromosome 8 and the oncogenic gene located in this region are not well known. By array-CGH the laboratory identified 4 amplicons in the 8p11-12 region. Gene expression analysis of 123 samples using DNA micro arrays identified 14 genes significantly overexpressed in relation to amplification. Other laboratories identified in the same region a minimal amplification containing two potential oncogenes, FLJ14299 also known as ZNF703, and SPFH2. We compared the frequency of amplification and overexpression of the 14 genes in tumor cells lines (Sircoulomb et al. 2006). Two genes appeared to be the most frequently overexpressed: FGFR1 and ZNF703. ZNF703 is amplified and overexpressed in luminal cells.
We studied the oncogenic potentiality of ZNF703 and its functionality in cells before starting to elaborate any transgenic line. We were able to localised ZNF703 in the cytoplasm and nucleus. The ectopic overexpression of ZNF703 induces a nuclear localisation of the protein particularly in speckles. By yeast two hybrid screen we found an interaction with atrophin-1, which was confirmed in vivo in tumorigenic cell lines that overexpressed ZNF703. Co-localisation was visible by immunofluorescence in the nucleus with the ectopic overexpressed protein in MCF7 and in Cos cells. A partial co-localisation was observed with the endogenous protein in the nucleus and in the cytoplasm of the MDA-MB-134 cell line, but the specificity of the commercial antibody is doubtful. Atrophin-1 is a nuclear co-repressor but we do not have more information about the possible functionality of the interaction with ZNF703 in gene regulation.
We did not see any transformation potentiality of ZNF703 in NIH3T3 cells, Ba/F3 cells or REF primary fibroblast cells. ZNF703 does not seem to have a master function in regulation of the cell cycle. But ZNF703 seems to increase the tumour potentiality of MCF7 cells after orthotopic injection in the mammary gland. Our observation indicates that ZNF703 is not a master oncogenic gene but seems to have an important role in tumourigenic luminal cells. ZNF703 has been shown to be a downstream gene of the oestrogen receptor and atrophin-1 is a regulator of the EGFR in drosophila.