The pea3 subfamily ets genes are required for HER2/Neu-mediated mammary oncogenesis

AbstractBackground: The PEA3 Ets transcription factor is overexpressed in the vast majority of human breast tumors and in nearly all of those of the HER2/Neu-positive subclass. PEA3 is also overexpressed in various transgenic mouse models of this disease. Whether PEA3 plays an essential role in HER2/Neu-mediated oncogenesis has heretofore not been addressed.Results: Here, we report that each of the three highly related ets genes of the pea3 subfamily (pea3, er81, and erm) were coordinately overexpressed in mammary tumors of MMTV-neu transgenic mice. Other ets genes normally expressed in the mammary gland were not upregulated in these tumors. Expression of a dominant-negative pea3 transgene under the control of the MMTV promoter in mammary epithelial cells of MMTV-neu transgenic mice dramatically delayed the onset of mammary tumors and reduced the number and size of such tumors in individual mice. Those tumors that arose in bitransgenic mice expressed the MMTV-neu transgene, but not the MMTV-dominant-negative pea3 transgene.Conclusions: These findings imply that one or more of the PEA3 subfamily Ets proteins or other Ets proteins with related DNA binding specificity play an essential role in Neu-mediated mammary oncogenesis. Hence, agents that inhibit the expression or activity of the PEA3 subfamily proteins may prove efficacious in the treatment of breast cancer

Single Unpurified Breast Tumor-Initiating Cells from Multiple Mouse Models Efficiently Elicit Tumors in Immune-Competent Hosts

<div><p>The tumor-initiating cell (TIC) frequency of bulk tumor cell populations is one of the criteria used to distinguish malignancies that follow the cancer stem cell model from those that do not. However, tumor-initiating cell frequencies may be influenced by experimental conditions and the extent to which tumors have progressed, parameters that are not always addressed in studies of these cells. We employed limiting dilution cell transplantation of minimally manipulated tumor cells from mammary tumors of several transgenic mouse models to determine their tumor-initiating cell frequency. We determined whether the tumors that formed following tumor cell transplantation phenocopied the primary tumors from which they were isolated and whether they could be serially transplanted. Finally we investigated whether propagating primary tumor cells in different tissue culture conditions affected their resident tumor-initiating cell frequency. We found that tumor-initiating cells comprised between 15% and 50% of the bulk tumor cell population in multiple independent mammary tumors from three different transgenic mouse models of breast cancer. Culture of primary mammary tumor cells in chemically-defined, serum-free medium as non-adherent tumorspheres preserved TIC frequency to levels similar to that of the primary tumors from which they were established. By contrast, propagating the primary tumor cells in serum-containing medium as adherent populations resulted in a several thousand-fold reduction in their tumor-initiating cell fraction. Our findings suggest that experimental conditions, including the sensitivity of the transplantation assay, can dramatically affect estimates of tumor initiating cell frequency. Moreover, conditional on cell culture conditions, the tumor-initiating cell fraction of bulk mouse mammary tumor cell preparations can either be maintained at high or low frequency <i>in vitro</i> thus permitting comparative studies of tumorigenic and non-tumorigenic cancer cells.</p> </div

Tumors arising from transplant of single tumor cells recapitulate the histology and cellular composition characteristic of the parental tumors.

<p>(<b>A</b>), Histopathology of a primary mammary tumor from an MMTV-Neu transgenic mouse (subpanel 1), that of a tumor seeded by transplant of a single cell (subpanel 2) from the same primary tumor, and that of a tumor derived from transplant of a single cell dissociated from tumorspheres of the same transgenic strain (subpanel 3). H&E staining of tumor sections illustrates the solid and nodular cytoarchitecture characteristic of Neu-induced tumors. (<b>B</b>), Immunofluorescence analyses of an MMTV-Neu tumor (subpanel 1), that of a tumor seeded by transplant of a single cell (subpanel 2) from the same primary tumor, and that of a tumor (subpanel 3) seeded by transplant of a single tumorsphere-derived cell stained with antibodies to CK8 and CK14. (<b>C</b>), Immunofluorescence analyses of an MMTV-Neu tumor (subpanel 1), that of a tumor seeded by transplant of a single cell (subpanel 2) from the same primary tumor, and that of a tumor (subpanel 3) seeded by transplant of a single tumorsphere-derived cell stained with antibodies to CK8 and α-SMA. Note that distinct sections of the same tumor were used for the analyses shown in panels a–c. Scale bar (inset) represents 40 µm in all panels.</p

Tumor-initiating cell frequencies in primary tumors.

<p>Tumor-initiating cell frequencies in primary tumors.</p

Limiting dilution transplants of dispersed primary MMTV-Neu transgenic mammary tumor cells.

<p>(<b>A</b>), Haematoxylin-stained mammary whole mounts isolated 4 weeks post-transplant reveal tumor-like masses in the fat pads of recipient mice injected with limiting dilutions of primary tumor cells. The mass of the tumor nodules correlates with the number of tumor cells injected (black arrows). Satellite tumor nodules are apparent along the needle track (white arrows). The images were photographed at a magnification of 6.4. (<b>B</b>), Tumor nodule appearing in mice transplanted with a single cell at 4 weeks post-transplantation. The image was photographed at a magnification of 6.4. (<b>C</b>), Macroscopic, palpable tumors are visible 16 weeks after transplanting single cells into the #4 mammary fat pads of a recipient mouse. (<b>D</b>), Single-cell induced tumors can develop into macroscopic tumors constituting over 10 million cells.</p

Single transplanted tumor cells isolated from the mammary tumors of MMTV-β-catenin transgenic mice recapitulate the histology and cellular composition characteristic of their parental tumors.

<p>(<b>A</b>), H&E staining of an MMTV-β-catenin primary tumor (subpanel 1) and a tumor obtained after transplanting a single tumor cell originating from the same tumor (subpanel 2). (<b>B</b>), Immunofluorescence analyses of an MMTV-β-catenin tumor (subpanel 1) and that of a tumor seeded by transplant of a single cell from the same primary tumor stained with antibodies to CK8 and CK14 (subpanel 2). (<b>C</b>), Immunofluorescence analyses of an MMTV-β-catenin tumor (subpanel 1) and that of a tumor seeded by transplant of a single cell from the same primary tumor stained with antibodies to CK8 and α-SMA (subpanel 2). Note that distinct sections of the same tumor were used for the analyses shown in panels a–c. Scale bar (inset) represents 40 µm in all panels.</p

Single transplanted tumor cells isolated from the mammary tumors of MMTV-mT transgenic mice recapitulate the histology and cellular composition characteristic of their parental tumors.

<p>(<b>A</b>), Histology of a primary MMTV-mT tumor (subpanel 1), that of a tumor resulting from transplanting a single tumor cell from the same tumor (subpanel 2), and that of a tumor that arose from transplantating a single tumorsphere-derived cell from a tumor of the same transgenic strain (subpanel 3). (<b>B</b>), Immunofluorescence analyses of an MMTV-mT tumor (subpanel 1), that of a tumor seeded by transplant of a single cell from the same primary tumor (subpanel 2), and that from a tumor resulting from transplant of a single tumorsphere-derived cell from a tumor of the same transgenic strain stained with antibodies to CK8 and CK14 (subpanel 3). (<b>C</b>), Immunofluorescence analyses of an MMTV-mT tumor (subpanel 1), that of a tumor seeded by transplant of a single cell from the same primary tumor (subpanel 2), and that of a tumor resulting from transplant of a single tumorsphere-derived cell from a tumor of the same transgenic strain stained with antibodies to CK8 and α-SMA (subpanel 3). Note that distinct sections of the same tumor were used for the analyses shown in panels a–c. Scale bar (inset) represents 40 µm in all panels.</p

Single tumorsphere-derived cells from tumors of the MMTV-Neu strain seed the growth of tumors after transplantation into syngeneic FVB/N mice.

<p>Haematoxylin-stained mammary whole mounts isolated 4 weeks post-transplant of varying numbers of tumorsphere-derived cells reveal tumor-like masses in the fat pads of recipient mice. The mass of the tumor nodules directly correlated with the number of injected tumor cells. The images were photographed at a magnification of 6.4.</p

Lef1 Haploinsufficient Mice Display a Low Turnover and Low Bone Mass Phenotype in a Gender- and Age-Specific Manner

We investigated the role of Lef1, one of the four transcription factors that transmit Wnt signaling to the genome, in the regulation of bone mass. Microcomputed tomographic analysis of 13- and 17-week-old mice revealed significantly reduced trabecular bone mass in Lef1+/− females compared to littermate wild-type females. This was attributable to decreased osteoblast activity and bone formation as indicated by histomorphometric analysis of bone remodeling. In contrast to females, bone mass was unaffected by Lef1 haploinsufficiency in males. Similarly, females were substantially more responsive than males to haploinsufficiency in Gsk3β, a negative regulator of the Wnt pathway, displaying in this case a high bone mass phenotype. Lef1 haploinsufficiency also led to low bone mass in males lacking functional androgen receptor (AR) (tfm mutants). The protective skeletal effect of AR against Wnt-related low bone mass is not necessarily a result of direct interaction between the AR and Wnt signaling pathways, because Lef1+/− female mice had normal bone mass at the age of 34 weeks. Thus, our results indicate an age- and gender-dependent role for Lef1 in regulating bone formation and bone mass in vivo. The resistance to Lef1 haploinsufficiency in males with active AR and in old females could be due to the reduced bone turnover in these mice.ISSN:1932-620

Tumor-initiating cell frequencies in adherent tumor cell preparations.

<p>Tumor-initiating cell frequencies in adherent tumor cell preparations.</p