Signaling molecules implicated in heregulin induction of growth arrest and apoptosis.

Heregulin (HRG) is one of the groups of polypeptide growth factors that activate the erbB-2 receptor via induction of heterodimerization with erbB-3 and erbB-4 receptors. The biological effects of HRG have been extensively studied. The vast majority of the reports indicate that HRG induces cell growth in breast cancer cells expressing normal levels of erbB-2 and growth inhibition and apoptosis in cells over-expressing erbB-2. However, the mechanism by which HRG promotes cell growth inhibition and apoptosis is still unknown. Previously we reported that constitutive expression of HRG in an erbB-2-overexpressing cell line (SKBr-3) induced growth arrest and apoptosis. We also demonstrated that constitutive expression of HRG promoted a marked morphological change, G2/M delay of the cell cycle, and DNA fragmentation. In this study, we demonstrate the mechanism by which HRG induces these cellular effects. The doubling time of the SK/HRG cells increased in relation to the level of HRG expression, and the level of HRG expression dictates the morphological change of the cells as well as their ability to grow or not grow in an anchorage-independent manner. We demonstrate that these effects are accompanied by downregulation of both erbB-2 and erbB-3 receptors at the transcriptional and translational levels and that down-regulation of the erbB-receptors results in reduced receptor tyrosine phosphorylation. The decrease in erbB-receptor phosphorylation in turn results in a marked reduction of ERK activity and a significant increase in JNK activity. Consequently, overexpression of HRG promoted the expression of PEA3, an Ets nuclear transcription factor. Taken together, our data demonstrate that the cellular effects induced by constitutive expression of HRG in SKBr-3 cells are correlated with the level of HRG expression. This is a first report demonstrating that HRG induction of apoptosis is directly correlated with decreased MAPK activity, increased JNK activity resulting in upregulation of PEA3 and down-regulation of the erbB-2 receptor. Overall, these data provide important clues regarding the mechanism and downstream molecules involved in HRG induction of apoptosis that can be used as targets for therapeutic prevention.

Expression and regulation of estrogen receptor beta in human breast tumors and cell lines.

Expression of estrogen receptor beta (ER-beta) and its regulation by estradiol and anti-estrogens was analyzed in breast cancer cells. We determined that ER-beta is expressed in normal and tumor human breast tissue as well as in breast cancer cell lines. We observed moderate levels of ER-beta expression in both T47D and T47D-V22 (a T47D variant cell line) cells, in contrast with T47DCo (a T47D variant cell line) cells when compared to ER-alpha expression. While T47DCo (a T47D variant cell line), BT474, MDA-MB-231, MDA-MB-453, MDA-MB-468 and MCF-7 express low levels of ER-beta, other cell lines including the T47D-Y (a T47D variant cell line), MDA-MB-435, BT-549, and SKBr-3 cells express undetectable levels of ER-beta. Interestingly, ER-beta and ER-alpha are apparently not co-expressed in the breast tissue analyzed. Estradiol induced 30-40-fold increased ER-beta mRNA expression in T47D cells over control untreated cells. Moreover, the anti-estrogen, 4-hydroxy-tamoxifen (4OH-Tam) strongly inhibited estradiol induction of ER-beta expression, but had little or no effect on estradiol induction of ER-alpha. A pure anti-estrogen, ICI-182,780, completely abolished the ability of estradiol to up-regulate the expression of ER. In addition, both actinomycin D and cyclohexymide inhibited estradiol induction of ER-beta mRNA, indicating that de novo mRNA and protein synthesis are probably required for this induction. In summary, this study demonstrates that ER-beta is expressed in breast cancer, and it is regulated by estradiol. Moreover, the studies demonstrate that estradiol up-regulation of ER-beta mRNA in T47D cells can be abolished by anti-estrogens. Thus, ER-beta expression may serve as a prognostic, diagnostic and/or therapeutic marker for breast cancer. To the best of our knowledge, this is the first report regarding hormonal regulation of ER-beta in human mammary cells.

HMG-I(Y) recognizes base-unpairing regions of matrix attachment sequences and its increased expression is directly linked to metastatic breast cancer phenotype.

Base-unpairing regions (BURs) contain a specialized DNA context with an exceptionally high unwinding propensity, and are typically identified within various matrix attachment regions. A BUR affinity column was used to purify a doublet of Mr 20,000 proteins from human breast carcinoma cells. These proteins were identified as the high-mobility group (HMG) protein, HMG-I, and its splicing variant, HMG-Y. We show that HMG-I(Y) specifically binds BURs. Mutating BURs so as to abrogate their unwinding property greatly reduced their binding affinity to HMG-I(Y). Numerous studies have indicated that elevated HMG-I(Y) expression is correlated with more advanced cancers and with increased metastatic potential. We studied whether the expression of HMG-I(Y) responds to signaling through the heregulin (HRG)-erbB pathway and the extracellular matrix. HMG-I(Y) expression was increased in MCF-7 cells after stable transfection with an HRG expression construct that led cells to acquire estrogen independence and metastasizing ability. A high level of HMG-I(Y) expression was detected in metastatic MDA-MB-231 cells, but the expression was virtually diminished, and the metastasizing ability was lost after cells were stably transfected with an antisense HRG cDNA construct. HMG-I(Y) was also decreased in MDA-MB-231 cells when treated with a chemical inhibitor for matrix metalloproteinase-9 that led to a reduction of invasive capability in vitro. The level of HMG-I(Y) expression, therefore, is dynamically regulated in human breast cancer cells in response to varying types of signaling that affect metastatic ability, including the HRG-erbB pathway and those from the extracellular matrix.

Constitutive expression of Heregulin induces apoptosis in an erbB-2 overexpressing breast cancer cell line SKBr-3.

We have previously reported that Heregulin (HRG)/neu differentiation factor (NDF) induced growth arrest and cellular differentiation in breast cancer cells overexpressing erbB-2 receptor. To elucidate the cellular mechanisms underlying the growth inhibition by HRG, we developed an in vitro model by transfection of HRG cDNA into the erbB-2 overexpressing breast cancer cell line, SKBr-3. We showed that the enforced expression of HRG in SKBr-3 cells induces dramatic morphological changes and pronounced inhibition of anchorage-dependent and -independent growth. Most SKBr-3/HRG-transfected (SK/HRG) cells exhibited about 15-fold increase in size and persisted as multinucleated cells with extended flattened vacuole-filled cytoplasm with reduced cell attachment. The growth suppression of SK/HRG cells was accompanied by a reduction in S phase, the presence of a G2-M cell cycle delay, and an increase in DNA aneuploid components. In addition, DNA fragmentation assays showed that HRG induced apoptosis of SKBr-3 cells. In contrast, while HRG treatment of other erbB-2 overexpressing breast cancer cell lines led to growth arrest and cell detachment, it did not induce apoptotic features. Thus, this study demonstrates that while growth arrest and cell detachment are general effects of HRG towards erbB-2 overexpressing cells, the ability of HRG to induce apoptosis is a phenomenon confined to selective cells including SKBr-3 cells.

Expression of estrogen receptor beta messenger RNA variant in breast cancer.

Estrogen receptor (ER) beta is expressed in a number of tissues, including the breast. We have recently shown that ER-beta mRNA is regulated by estradiol (E2) and that antiestrogens antagonize E2 induction of ER-beta mRNA. Here, we identify by reverse transcription-PCR and by the RNase protection assay a mRNA coding for a variant of ER-beta that is coexpressed with wild-type ER-beta in the ER-alpha-negative, estrogen-independent breast cancer cell line MDA-MB-231 and in malignant breast tumor specimens. In contrast, this variant was not seen in the tested normal breast tissue. Sequence analysis of the ER-beta variant PCR product revealed the absence of 139 bp within the hormone-binding domain. This ER-beta deletion corresponds precisely to the entire exon 5 of ER-alpha. The ER-beta variant protein is predicted to lack part of the hormone-binding domain and may bind E2 with lower affinity than the wild-type ER-beta protein.