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Human Growth Hormone-regulated HOXA1 Is a Human Mammary Epithelial Oncogene
Increased mammary epithelial expression of the human growth hormone hGH gene is associated with the acquisition of pathological proliferation. We report here that autocrine hGH production by human mammary carcinoma cells increased the expression and transcriptional activity of the homeobox domain containing protein HOXA1. Forced expression of HOXA1 in human mammary carcinoma cells resulted in increased total cell number primarily by the promotion of cell survival mediated by the transcriptional up-regulation of Bcl HOXA1 also abrogated the apoptotic response of mammary carcinoma cells to doxorubicin.
Forced expression of HOXA1 in mammary carcinoma cells, in a Bcldependent manner, resulted in dramatic enhancement of anchorage-independent proliferation and colony formation in soft agar.
Finally, forced expression of HOXA1 was sufficient to result in the oncogenic transformation of immortalized human mammary epithelial cells with aggressive in vivo tumor formation. Herein, we have therefore provided a molecular mechanism by which autocrine hGH stimulation of human mammary epithelial cells may result in oncogenic transformation.
Signal transduction pathways that regulate mammary epithelial cell proliferation, differentiation, apoptosis, and transformation have been delineated and utilized as therapeutic targets for the treatment of mammary gland neoplasia 1 , 2. Examples of such include selective estrogen modulators 3 and herceptin 4 targeting estrogen receptor and HER-2 pathways, respectively.
However, many carcinomas of the mammary gland neither express the estrogen receptor 5 nor overexpress HER-2 6 and are, therefore, not responsive to these specific therapeutic strategies. Clinical data for the treatment of cancer has clearly demonstrated the superiority of combinatorial therapy over a single-agent approach 7. Growth hormone GH 1 is obligatory for normal pubertal mammary gland development 8.
Specifically, GH acts on both the mammary stromal and epithelial components to result in ductal elongation and the differentiation of ductal epithelia into terminal end buds 9 , Expression of the hGH transgene in mice results in precocious development of the mammary gland 11 , 12 and the development of neoplasia Conversely, spontaneous or experimentally engineered functional deficiency of GH results in severely impaired mammary gland development and virtual resistance to the spontaneous development of hyperplastic alveolar nodules 13 and chemically induced mammary carcinogenesis 14 , Similarly, in a primate model, hGH administration results in marked hyperplasia of the mammary gland with an increased epithelial proliferation index Thus, the somatotropic axis represents one potential and unutilized approach for novel therapeutic approaches to the treatment of mammary epithelial neoplasia.
The hGH gene is also expressed in epithelial cells of the normal human mammary gland Increased epithelial expression of the hGH gene is associated with the acquisition of pathological proliferation, and the highest level of hGH gene expression is observed in metastatic mammary carcinoma cells We have recently generated a model system to study the role of autocrine-produced hGH in mammary carcinoma by stable transfection of either the hGH gene or a translation-deficient hGH gene into mammary carcinoma cells The autocrine production of hGH by mammary carcinoma cells results in a hyperproliferative state with marked synergism between trophic agents such as insulin-like growth factor 1 The increase in mammary carcinoma cell number as a consequence of autocrine production of hGH is a result of both increased mitogenesis and decreased apoptosis Thus, autocrine production of hGH by mammary carcinoma cells may direct mammary carcinoma cell behavior to impact on the final clinical prognosis, and therefore, systematic analysis of the relevant mechanistic features by which it exerts its cellular effects is required.
One major mechanism by which GH affects cellular and somatic function is by regulating the level of specific mRNA species We have previously utilized cDNA microarray analyses to identify genes regulated by autocrine production of hGH in human mammary carcinoma cells MCF-7 Homeobox-containing genes are a family of genes encoding transcription factors that possess pivotal roles in development For example, HOXA1 has been demonstrated to be required for vertebrate hindbrain segmentation Several reports also suggest the involvement of homeobox-containing genes in the control of cell proliferation and, when dysregulated, in oncogenesis 26 , Moreover, alterations of HOX gene expression have been detected in a variety of human tumors , including those of the mammary gland 29 , Accordingly, HOXA1 has been detected in carcinoma of the mammary gland in the mouse but not in normal mammary tissue 31 , and HOXA1 expression has also been detected in neoplastic lesions of the human mammary gland Herein we provide a molecular mechanism by which hGH stimulation of human mammary epithelial cells results in oncogenic transformation.
Functional antagonism of hGH, and the molecular pathways it utilizes, will therefore constitute novel adjunct therapeutic approaches to the treatment of mammary gland neoplasia. A detailed description of the characterization of these cell lines has been published previously Vincenzo Zappavigna Milano, Italy.
Individual colonies were selected to determine the HOXA1 expression level. Transient transfection was performed by use of Effectene as described After a further 24 h, cells were washed with PBS, and luciferase assays were performed as described previously The Bcl-2 P1 promoter reporter plasmid was a kind gift of Dr.
John Kurland Houston, TX. Cells were treated and harvested as described. Whole cell lysates and crude nuclear extracts were prepared according to the protocol described Mitogenesis was directly assayed by measuring the incorporation of BrdUrd as described San Francisco, CA according to the manufacturer's instructions.
A total population of over three times cells was analyzed in several arbitrarily chosen microscopic fields to determine the BrdUrd labeling index percentage of cells synthesizing DNA.
After 48 h, cells were trypsinized with 0. On the days indicated, cells were harvested and counted as for monolayers. For soft agar colony formation assay, MCF-7 cell and its derived cell lines were cultured in six-well plates first covered with an agar layer RPMI with 0. Medium was added as the top layer to prevent drying of the agarose gels. The plates were incubated for 9 days for MCF-7 cells or 14 days for MCFA cells , after which the cultures were inspected and photographed.
Apoptotic cell death was measured by fluorescent microscopic analysis of cell DNA staining patterns with Hoechst from Sigma Chemical Co. St Louis, MO Cells were trypsinized with 0. Apoptotic cells were distinguished from viable cells by their nuclear morphology characterized by nuclear condensation and fragmentation as well as the higher intensity of the blue fluorescence of the nuclei.
Severe combined immunodeficient SCID 4-week-old mice were purchased from the animal holding facility of the National University of Singapore and acclimated for 7—10 days. At necropsy, primary tumors and all organs were evaluated macroscopically for the presence of tumors.
All experiments were repeated at least three to five times. HOXA1 transcriptional activity in the presence and absence of its binding partner PBX1 was determined by reporter assay C as indicated. Thus autocrine hGH production by mammary carcinoma cells results in increased HOXA1-mediated transcriptional activity. We therefore examined the effect of forced expression of HOXA1 in mammary carcinoma cells on total cell number.
In any case, forced expression of HOXA1 in mammary carcinoma cells resulted in a significant increase in cell number. Increased cell number is achieved by either increased proliferation or decreased apoptotic cell death, and we therefore proceeded to determine the relative contribution of these processes to the observed increase in cell number as a consequence of forced expression of HOXA1.
Effect of forced expression of HOXA1 in mammary carcinoma cells on cell number, cell cycle progression, and apoptosis. The D family of cyclins is pivotal to initiate progression through the G 1 phase of the cell cycle CyclinD1 is the predominant member of this family expressed in mammary gland 40 , and we have previously demonstrated its requirement for autocrine hGH-stimulated mammary carcinoma cell cycle progression We next examined the protein level of the cyclin-dependent kinase inhibitor p27 Kip1.
We therefore examined the effect of forced expression of HOXA1 on a variety of proteins involved in the apoptotic process. Exogenous hGH slightly reduced apoptotic cell death in both cell lines. Forced expression of HOXA1 in mammary carcinoma cells resulted in decreased apoptotic cell death associated with a specific increase in Bcl-2 protein.
HOXA1 expression in human mammary carcinoma cells prevents apoptotic cell death in a Bcldependent manner. Both Bcl-2 antisense oligonucleotide D and Bcl-2 inhibitor F abrogated protection from apoptotic cell death as a consequence of forced expression of HOXA1 as indicated. To further verify the Bcl-2 dependence of the survival effect of forced expression of HOXA1 in mammary carcinoma cells, we utilized a novel Bcl-2 inhibitor that antagonizes Bcl-2 function by blocking the binding of the Bak BH3 peptide to Bcl-2 in vitro Bcl-2 expression was not altered by cellular treatment with the Bcl-2 inhibitor Fig.
Use of the Bcl-2 inhibitor abrogated the protection from apoptotic cell death as a consequence of forced expression of HOXA1 Fig. The anti-apoptotic effects of forced expression of HOXA1 in mammary carcinoma cells are therefore mediated by increased Bcl-2 gene transcription.
As forced expression of HOXA1 in mammary carcinoma cells offered dramatic protection from apoptosis, we reasoned that increased HOXA1 expression would also result in decreased sensitivity to cell death as a result of exposure to anti-neoplastic agents. We therefore examined the effect of forced expression of HOXA1 on the apoptotic response of mammary carcinoma cells to doxorubicin.
Bcl-xL protein was also slightly decreased after doxorubicin treatment but to equivalent levels in both cell lines. The level of Bak and Bax did not differ between the two cell lines, and no effect of doxorubicin was observed.
Therefore, increased expression of HOXA1 in human mammary carcinoma cells produces a chemoresistant phenotype. Forced expression of HOXA1 in mammary carcinoma cells protects against doxorubicin-induced apoptosis.
Doxorubicin-induced apoptotic cell death D was abrogated by forced expression of HOXA1 in mammary carcinoma cells as indicated. Anchorage-independent growth is one pivotal characteristic of malignant transformation 2. As is observed in Fig. Thus, forced expression of HOXA1 enhances oncogenicity of human mammary carcinoma cells. Forced expression of HOXA1 enhances anchorage-independent growth of mammary carcinoma cells in a Bcldependent manner.
To determine if forced expression of HOXA1 would result in oncogenic transformation of human mammary epithelial cells, we utilized the immortalized human mammary epithelial cell line MCFA When grown attached to a plastic substrate these cells display a typical epithelial morphology, do not form colonies in soft agar, and are not capable of growth in immunocompromised mice Comparison of nuclear BrdUrd incorporation between the two cell lines under serum-free conditions, 50 n m exogenous hGH, or FBS demonstrated that forced expression of HOXA1 significantly increased cell cycle progression of mammary carcinoma cells Fig.
Thus forced expression of HOXA1 conferred tumorigenic potential upon human mammary epithelial cells. We also observed that forced expression of HOXA1 in mouse NIH-3T3 cells resulted in their oncogenic transformation as indicated by dramatic colony formation in soft agar in comparison to an effective lack of colony formation by vector transfected NIH-3T3 cells data not shown.
Forced expression of HOXA1 in immortalized mammary epithelial cells results in oncogenic transformation and tumor formation in vivo. In vitro analyses of oncogenic transformation are not always concordant with tumorigenic potential in vivo. Necropsy revealed that the tumors were attached to the underlying axillary muscle and surrounded by a vascular fibrous capsule.
Histologically the neoplastic cells were locally invasive and associated with fibrous connective tissue Fig. The cells exhibited moderate cytoplasmic and nuclear pleomorphism and formed a solid mass often with areas of central necrosis. We have demonstrated here that forced expression of HOXA1 in immortalized human mammary epithelial cells MCFA results in oncogenic transformation and the development of a rapidly growing carcinoma in vivo.
This is remarkable, given that forced expression of other oncogenes e. The aberrant expression of homeobox-containing genes has been reported in a variety of neoplasias for review see Ref. Herein, we provide the first direct evidence that a homeobox-containing protein is a bona fide mammary gland oncogene, the enhanced expression of which is sufficient for tumorigenesis of human mammary epithelial cells.