Hormone-independent Mammary Tumors Research Articles

Abstract 1316: Overactivation of AKT promotes hormone-independent mammary tumors

Abstract Using a mouse mammary tumor model induced by medroxyprogesterone acetate (MPA) that transits through different stages of hormone dependence, we have previously reported that the activation of the PI3K/AKT pathway is critical for the growth of hormone-independent (HI) mammary tumors, but not for the growth of a hormone-dependent (HD) tumor variant. The main aim of this work was to explore whether the activation of the PI3K/AKT pathway per se is responsible for the transition from a HD to a HI tumor phenotype. To answer this question we infected primary cultures of epithelial cells obtained from HD tumors with a retrovirus that directs the expression of a constitutively active form of AKT1 (myristoylated AKT1, myrAKT1), or with the empty vector as control. We inoculated 103 cells in nude mice (n = 6). MyrAKT1 expressing HD-derived cells were able to form mammary tumors even in the absence of MPA, that is, they behaved as HI cells. These tumors were highly differentiated and displayed a ductal phenotype with a pattern of expression of cytokeratin 8 and laminin-1 typical of HI tumors. Ligand-independent phosphorylation of progesterone receptor (PR) in Ser 190 was also increased in myrAKT1 tumors supporting our hypothesis that overactivation of AKT is directly involved in the acquisition of hormone independence. Furthermore, comparing myrAKT tumors with uninfected HD tumors revealed that overactivation of AKT did not increase the incidence of lung metastasis, indicating that AKT1 is involved specifically with tumor differentiation and growth rather than with tumor cell invasiveness. In vitro, primary cultures of mammary tumor cells modified to express myrAKT1 showed similar levels of proliferation determined by Ki67 and H3-tymidine incorporation as compared to control (cells infected with the empty vector), suggesting that the in vivo tumorigenic effect of myrAKT1 cells described above might require cell-cell or cell-matrix interactions absent in vitro. Interestingly, we found that conditioned medium from myrAKT1 tumor cells increase endogenous AKT1 activity when tested in untransfected cells. Moreover, conditioned medium from HI carcinoma associated fibroblasts (CAF-HI) increased the level of activation of the PI3K/AKT pathway in epithelial cells. Altogether, these results indicate that paracrine signals derived from the tumor cells and/or its microenvironment participate in the activation of the PI3K/AKT pathway, ultimately leading to tissue differentiation and ligand-independent mammary tumor growth. Thus, the PI3K/AKT pathway might be a key therapeutic target to prevent progression to autonomous and endocrine resistant breast tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1316. doi:10.1158/1538-7445.AM2011-1316

Phosphorylation and estradiol binding of estrogen receptor in hormone-dependent and hormone-independent GR mouse mammary tumors.

The effect of phosphorylation on the hormone-binding capacity of the estrogen receptor (ER) was investigated in hormone-dependent (HD) and hormone-independent (HI) mammary carcinomas of GR mice. Tumor cytosols were incubated with ATP under conditions previously used to study the tyrosine kinase which confers hormone binding to phosphatase-treated or in vitro-synthesized ER. The ATP-dependent increases in hormone-binding capacity of 8 out of 20 HI tumors ranged from values of 23 to 124 fmol/mg cytosol protein. The enhancement by ATP of hormone binding to ER was significantly less marked in HD and HR tumors than in HI tumors. In only 3 out of 13 HD and HR tumors was an increase ranging from 15 to 20 fmol/mg protein detected. Analysis by Scatchard plot of estradiol binding to ER showed that cytosol incubation of HI tumors with ATP markedly increased the hormone binding without any change in affinity. The data suggest that ER of HI tumors is less phosphorylated in vivo than the ER of HD/HR tumors, so that the receptor of HI tumors is more susceptible to gamma-32P-ATP phosphorylation and ATP-induced hormone binding in vitro. Western blot of ER with antiphosphotyrosine antibody showed that, in HI tumors, the large ATP-induced increase in hormone binding to ER was associated with phosphorylation on tyrosine of the receptor itself. Our findings indicate that the process of activation-inactivation of binding through tyrosine-phosphorylation/phosphotyrosine-dephosphorylation of ER observed in estrogen target tissues is altered in some HI mammary tumors.

Isolation of autonomously growing dog mammary tumor cell lines cultured in medium supplemented with serum treated to inactivate growth factors.

Conventional methods for isolation of cell lines from carcinomas suffer inherently from a lack of advantage for proliferation of transformed cells as opposed to contaminating fibroblasts and normal epithelial cells. To isolate cell lines from metastases of estrogen receptor-negative mammary carcinomas in dogs, we applied a novel method using medium supplemented with serum treated to inactivate growth factors. Under these conditions, autonomously growing tumor cells are selectively allowed to proliferate. In this way, four autonomously growing tumor cell lines were obtained from metastases of two dogs. Tumors formed from cells implanted in C3H nude mice closely resembled the original dog tumors, indicating that the main result of this selective procedure was suppression of normal cell proliferation. Serum treated to inactivate growth factors seems to be an important medium supplement for isolation of autonomously growing tumor cell lines, which may be valuable tools for future studies on regulation of cell proliferation in advanced hormone-independent mammary tumors.

Estrogen influence on maturational pathway of murine mammary tumor virus: An immunoelectron microscopy study

Modifications induced by estrogens on hormone-independent murine mammary tumor (MMT) and its main etiological agent, the MMT virus (MMTV), are reported. High doses of estrogens released continuously from silastic capsules delay significantly the development of transplanted tumors into syngeneic hosts. Neoplastic cells present a striking cytoplasmic vacuolization and changes in the MMTV differentiation pattern. Mature virions are detected budding into cytoplasmic vacuoles instead of the extracellular space as in spontaneous and untreated transplanted tumors. This phenomenon is reversed after estrogen withdrawal at the first sign of tumor development. Application of electron microscope immunocytochemistry with colloidal gold-protein A complex and multiple monospecific antibodies reveals several interesting features. In spontaneous and untreated tumor grafts, structural viral proteins p14 and p25 appear in both intracytoplasmic capsids and mature extracellular viruses. By contrast glycoprotein gp55 labels only the envelope of mature virus. In estrogentreated tumors this antigenic pattern is modified and the gp55 is detected in those atypical virions maturing into the intracytoplasmic vacuoles. These observations led to the conclusions that the delay in the development of hormone-independent mammary tumors caused by estrogen is due to an abnormal maturational viral process and that estrogens induce alterations of polarity in the translocation process of viral envelope glycoproteins.

Autoradiographic evidence of a loss of iodination within hormone-dependent GR mouse mammary tumors as they progress to independence.

The purpose of this study was to determine by use of light- and electron-microscope autoradiography whether or not iodination occurred in mammary tumors of female GR mice. Of the sixty tumors studied it was found that pregnancy-dependent and hormone-induced tumors possessed iodinating ability. Although mammary glands from nonpregnant GR mice lacked the ability to iodinate, by the 16th day of pregnancy in response to hormonal stimulation the glands readily iodinated casein, and some epithelial cells contained ultrastructural cytochemical evidence of mammary peroxidase. Preneoplastic mammary gland lesions known as hyperplastic alveolar nodules were also able to iodinate, as were plaques, the disc-shaped lesions which give rise to the hormone-responsive mammary tumors in this strain. Plaques also contained epithelial cells with mammary peroxidase activity. When hormone-induced mammary tumors were transplanted into syngeneic mice they retained the ability to iodinate for several generations. However, as the tumors progressed to hormone independence, the ability to iodinate was gradually lost. Hormone-independent mammary tumors from GR mice lacked both iodinating ability and cytochemical evidence of mammary peroxidase. These findings suggest that iodination depends upon hormone-responsive cells within the mammary tumors and that as these cells become hormone unresponsive, the ability to iodinate is lost.

CAMP-dissociation kinetics in hormone-dependent and -independent rat mammary tumor cytosols.

Cytosols from 7, 12-dimethylbenz (alpha) anthracene-induced rat mammary tumors which exhibit different hormone-responsiveness were compared with respect to their cAMP-dissociation kinetics. At 22 degree C, pH 4.5, 1 micrometer cAMP, hormone-dependent mammary tumors exhibited monophasic dissociation rates with a rate constant of k-1 = 0.06 min-1. In contrast, hormone-independent mammary tumors exhibited biphasic dissociation curves with rate constants of k-1 = 0.47 and k-2 = 0.06 min-1. The binding of cAMP was completely reversible; radio-labeled ligand was completely dissociated by 1mM nonradioactive cAMP; the binding protein could be reassociated to its original binding level after dextran-coated charcoal adsorption. The mammary cytosols exhibited specific binding for cAMP which could be displaced partially by cGMP but not by ATP, ADP, AMP, or adenosine. Receptor inactivation during the course of incubation was negligible. Both mammary tissue cytosols exhibited similar association rates at 22 degree C, pH 4.5, 1 micrometer cAMP (k+1 = 5-7 x 10(5)M-1 min-1). These data indicate that mammary tissues exhibit 2 cAMP dissociation rates. Hormone-dependent mammary tumors exhibit a dissociation constant of a high affinity binding site (k-1/k+1 = 0.07 micrometer) whereas hormone-independent mammary tumors exhibit dissociation constants of one high affinity (k-1/k+1 = 0.07 micrometer) and a second low affinity site (k-1/k+1 = 0.05 micrometer).

Antagonistic action between cyclic AMP and estrogen in phosphorylation of mammary tumor nuclear proteins

A new protein kinase-dependent phosphorylation occurs in the nuclei of hormone-dependent, 7,12-dimethylbenz[alpha]anthracene (DMBA)-induced mammary carcinoma following preincubation of tumor slices with cyclic adenosine 3',5'-monophosphate (cAMP). The presence of 17beta-estradiol in the medium inhibits this effect. Both events have been observed in vivo in the nuclei of DMBA-induced tumors. The phosphorylation pattern of nuclei in hormone-independent mammary tumor, DMBA No. 1, however, is not affected by preincubation with either cAMP or estrogen. These findings suggest that the antagonistic effect of cAMP and estrogen in the growth control of mammary tumors is exerted through a specific action on nuclear protein phosphorylation and that these events correlate with the hormone-dependency of the tumors.

Prolactin receptors: a comparison between chloramine-T and lactoperoxidase iodination

The binding properties of chloramine-T iodinated oPRL (using a modified iodination procedure) to prolactin receptors of female rat liver membrane particles were compared with those of lactoperoxidase iodinated oPRL. The results indicated that both iodination methods provided 125I-oPRLs which were suitable for receptor binding studies. Our results suggested that chloramine-T 125I-oPRL was even better than lactoperoxidase 125I-oPRL in terms of lower nonspecific binding. The chloramine-T iodinated oPRL was used to study the prolactin receptors in rat ovaries and DMBA induced rat mammary tumors. The results showed that the amount of prolactin receptors in rat ovaries was related to the plasma level of prolactin as it had been reported in liver by other investigators. The study of prolactin receptors in rat mammary tumors indicated that the prolactin receptor content of hormone dependent mammary tumors was much higher than that of hormone independent mammary tumors.

Increased uptake of iodide by hormone‐responsive compared to hormone‐independent mammary tumors in GR mice

The uptake of 125-iodide by transplanted hormone-responsive (HR) or hormone-independent (HI) mammary tumors, normal mammary tissue and skeletal muscle was compared in GR mice. The uptake of 125-iodide by HR mammary tumors in mice treated with progesterone and oestrone (P+O) was about 20 times greater than the uptake of 125-iodide by HI mammary tumors in mice not treated with P+O. This difference in uptake of 125-iodide by HR and HI mammary tumors could not be attributed to the difference in the hormonal status of the mice since uptake of 125-iodide was also low in HI tumors in mice receiving P+O treatment. The uptake of 125-iodide by HR mammary tumors was greatly reduced by the simultaneous injection of either an excess of non-radioactive iodide or of perchlorate. Uptake of 125-iodide by normal mammary tissue and skeletal muscle was similar was similar in all groups of tumor bearing mice, and was not influenced by the presence of an excess of non-radioactive iodide or of perchlorate. The possible clinical significance of these findings is discussed.