Final Report Summary - BREAST CANCER ORIGIN (Analysis of the cellular origin of breast cancer) Executive Summary: The main aim of this project was to address the cellular origin of breast cancer using cutting-edge genetic approaches to delete genes in specific cell populations. There are two outstanding questions in relation to breast cancer: 1) Do specific cancer-causing genes (oncogenes) target preferentially different types of cells within the breast and 2) is it the initiating genetic mutation or the type of cell which becomes transformed that is the main factor in determining the tumour type generated? In order to address these questions, one of the approaches we are utilising is a transgenic mouse model expressing an inducible Diphtheria Toxin Receptor (iDTR). Specific populations of cells will be killed upon administration of Diphtheria Toxin (DT) since mice do not normally express the receptor for this toxin. Therefore, only DTR-expressing cells will be ablated by DT treatment. We are selectively depleting the basal sub-population of cells in vivo. This allows a more relevant recapitulation of human breast tumourigenesis. Another aim was to generate mammary tumours from single-cell oncogenic events. This is how we believe breast cancer arises and using such a model much more accurately recapitulates what occurs in humans. We have investigated the use of slippage cassette consisting of a dinucleotide repeat tract (up to 30 repeats of the DNA sequence CA) which is highly susceptible to mutations as a result of slippage of DNA polymerase on this tract. We have shown that this works in mammary gland in vivo. However, the efficiency was not optimal for our cancer studies and this is being further developed. We have identified genes that perturb mammary tissue architecture and cellular composition. Unexpectedly, we have also genetically ablated lineages and have focussed some time in characterising these changes further as this could impact on tumourigenesis if the cell of origin has been ablated. A previously uncharacterized gene Roma (Regulator of Mammary Alveologenesis) as a master regulator of differentiation of cells to the milk-producing lineage. We generated a Roma null/reporter mouse to allow us to investigate the function of Roma in mammary gland development. Surprisingly, Gata-3 (which is a good prognostic factor for breast cancer) becomes dispensable in the absence of Roma. This results in rescue of lactation failure which occurs in Gata3 deficient mice. We noticed that the ‘rescued’ Gata3 null alveolar cells were morphologically abnormal with an elongated shape and luminally displaced nuclei suggestive of mild epithelial dysplasia which could be predisposing for tumourigenesis. Double knockout cells have two nuclei, suggesting that they have failed to undergo cell division, and have markers of the DNA damage response such as the appearance of foci of H2AX followed by recruitment of 53BP1 to gammaH2AX foci. These exciting results suggest that Roma regulates the DNA damage response and is a novel tumour suppressor.We have also shown that the transcription factor Stat3, which if often aberrantly active in cancer cells, is required for mammary stem cells to maintain their full differentiation potential. This is an important discovery as it is likely that Stat3 is required for stem cell function in breast cancer stem cells. The main aim of this project was to address the cellular origin of breast cancer using cutting-edge genetic approaches to delete genes in specific cell populations. There are two outstanding questions in relation to breast cancer: 1) Do specific cancer-causing genes (oncogenes) target preferentially different types of cells within the breast and 2) is it the initiating genetic mutation or the type of cell which becomes transformed that is the main factor in determining the tumour type generated? In order to address these questions, one of the approaches we are utilising is a transgenic mouse model expressing an inducible Diphtheria Toxin Receptor (iDTR). Specific populations of cells will be killed upon administration of Diphtheria Toxin (DT) since mice do not normally express the receptor for this toxin. Therefore, only DTR-expressing cells will be ablated by DT treatment. We are selectively depleting the basal sub-population of cells in vivo. This allows a more relevant recapitulation of human breast tumourigenesis. Another aim was to generate mammary tumours from single-cell oncogenic events. This is how we believe breast cancer arises and using such a model much more accurately recapitulates what occurs in humans. We have investigated the use of slippage cassette consisting of a dinucleotide repeat tract (up to 30 repeats of the DNA sequence CA) which is highly susceptible to mutations as a result of slippage of DNA polymerase on this tract. We have shown that this works in mammary gland in vivo. However, the efficiency was not optimal for our cancer studies and this is being further developed. We have identified genes that perturb mammary tissue architecture and cellular composition. Unexpectedly, we have also genetically ablated lineages and have focussed some time in characterising these changes further as this could impact on tumourigenesis if the cell of origin has been ablated. A previously uncharacterized gene Roma (Regulator of Mammary Alveologenesis) as a master regulator of differentiation of cells to the milk-producing lineage. We generated a Roma null/reporter mouse to allow us to investigate the function of Roma in mammary gland development. Surprisingly, Gata-3 (which is a good prognostic factor for breast cancer) becomes dispensable in the absence of Roma. This results in rescue of lactation failure which occurs in Gata3 deficient mice. We noticed that the ‘rescued’ Gata3 null alveolar cells were morphologically abnormal with an elongated shape and luminally displaced nuclei suggestive of mild epithelial dysplasia which could be predisposing for tumourigenesis. Double knockout cells have two nuclei, suggesting that they have failed to undergo cell division, and have markers of the DNA damage response such as the appearance of foci of H2AX followed by recruitment of 53BP1 to gammaH2AX foci. These exciting results suggest that Roma regulates the DNA damage response and is a novel tumour suppressor.We have also shown that the transcription factor Stat3, which if often aberrantly active in cancer cells, is required for mammary stem cells to maintain the full differentiation potential of these stem cells. This is an important discovery as it is likely that Stat3 is required for stem cell function in breast cancer stem cells. The main aim of this project was to address the cellular origin of breast cancer using cutting-edge genetic approaches to delete genes in specific cell populations. There are two outstanding questions in relation to breast cancer: 1) Do specific cancer-causing genes (oncogenes) target preferentially different types of cells within the breast and 2) is it the initiating genetic mutation or the type of cell which becomes transformed that is the main factor in determining the tumour type generated? In order to address these questions, one of the approaches we are utilising is a transgenic mouse model expressing an inducible Diphtheria Toxin Receptor (iDTR). Specific populations of cells will be killed upon administration of Diphtheria Toxin (DT) since mice do not normally express the receptor for this toxin. Therefore, only DTR-expressing cells will be ablated by DT treatment. We are selectively depleting the basal sub-population of cells in vivo. This allows a more relevant recapitulation of human breast tumourigenesis. Another aim was to generate mammary tumours from single-cell oncogenic events. This is how we believe breast cancer arises and using such a model much more accurately recapitulates what occurs in humans. We have investigated the use of slippage cassette consisting of a dinucleotide repeat tract (up to 30 repeats of the DNA sequence CA) which is highly susceptible to mutations as a result of slippage of DNA polymerase on this tract. We have shown that this works in mammary gland in vivo. However, the efficiency was not optimal for our cancer studies and this is being further developed. We have identified genes that perturb mammary tissue architecture and cellular composition. Unexpectedly, we have also genetically ablated lineages and have focussed some time in characterising these genetic changes further as this could impact on tumourigenesis if the cell of origin has been ablated. A previously uncharacterized gene Roma (Regulator of Mammary Alveologenesis) as a master regulator of differentiation of cells to the milk-producing lineage. We generated a Roma null/reporter mouse to allow us to investigate the function of Roma in mammary gland development. Surprisingly, Gata-3 (which is a good prognostic factor for breast cancer) becomes dispensable in the absence of Roma. This results in rescue of lactation failure which occurs in Gata3 deficient mice. We noticed that the ‘rescued’ Gata3 null alveolar cells were morphologically abnormal with an elongated shape and luminally displaced nuclei suggestive of mild epithelial dysplasia which could be predisposing for tumourigenesis. Double knockout cells have two nuclei, suggesting that they have failed to undergo cell division, and have markers of the DNA damage response such as the appearance of foci of gammaH2AX followed by recruitment of 53BP1 to gammaH2AX foci. These exciting results suggest that Roma regulates the DNA damage response and is a novel tumour suppressor.We have also shown that the transcription factor Stat3, which if often aberrantly active in cancer cells, is required for mammary stem cells to maintain their full differentiation potential. This is an important discovery as it is likely that Stat3 is required for stem cell function in breast cancer stem cells. This work will be important in furthering the understanding of breast tumourigenesis and the development of specific therapies that target sub-populations of cells.
