Gerald E. Stoica

Effect of estradiol on estrogen receptor- α gene expression and activity can be modulated by the ErbB2/PI 3-K/Akt pathway

Epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I), and heregulin-β1 (HRG-β1), can modulate the expression and activity of the estrogen receptor-α (ER-α) via the phosphatidylinositol 3-kinase (PI 3-K)/Akt pathway in the ER-α-positive breast cancer cell line, MCF-7. Estradiol can also rapidly activate PI 3-K/Akt in these cells (nongenomic effect). The recent study examines whether Akt is involved in the ER-α regulation by estradiol (genomic effect). Stable transfection of parental MCF-7 cells with a dominant-negative Akt mutant, as well as the PI 3-K inhibitors wortmannin and LY 294,002, blocked the effect of estradiol on ER-α expression and activity by 70–80 and 55–63%, respectively. Stable transfection of MCF-7 cells with a constitutively active Akt mimicked the effect of estradiol. The changes in ER-α expression and activity were abrogated in response to estradiol by an arginine to cysteine mutation in the pleckstrin homology (PH) domain of Akt (R25C), suggesting the involvement of this amino acid in the interaction between Akt and ER-α. Experiments employing selective ErbB inhibitors demonstrate that the effect of estradiol on ER-α expression and activity is mediated by ErbB2 and not by EGFR. Moreover, anchorage-dependent and -independent growth assays, cell cycle and membrane ruffling analyses showed that Akt exerts estrogen-like activity on cell growth and membrane ruffling and that a selective ErbB2 inhibitor, but not anti-ErbB2 antibodies directed to the extracellular domain, can block these effects. In the presence of constitutively active Akt, tamoxifen only partially inhibits cell growth. In contrast, in cells stably transfected with either a dominant-negative Akt or with R25C-Akt, as well as in parental cells in the presence of a selective ErbB2 inhibitor, the effect of estradiol on anchorage-dependent and -independent cell growth was inhibited by 50–75 and 100%, respectively. Dominant-negative Akt inhibited membrane ruffling by 54%; however, R25C-Akt did not have any effect, suggesting that kinase activity plays an important role in this process. Scatchard analysis demonstrated a 67% reduction in estrogen-binding capacity in cells transfected with constitutively active Akt. No change in binding affinity of estradiol to the receptor was observed upon transfection with either Akt mutant. Taken together, our results suggest that estradiol treatment results in binding to membrane ER-α and interaction with a heterodimer containing ErbB2, leading to tyrosine phosphorylation. This results in the activation of PI 3-K and Akt. Akt, in turn, may interact with nuclear ER-α, altering its expression and activity.

Progressive Loss of Syk and Abnormal Proliferation in Breast Cancer Cells

The tumor suppressor gene Syk tyrosine kinase is absent or reduced in invasive breast cancer tissues and cell lines; its loss in breast tissues is linked to poor prognosis and metastasis. Also, evidence shows that in vitro Syk is involved in regulating proliferation. Here, we show by in situ hybridization on breast tissue sections that the loss of Syk expression is progressive during tumor development. Strikingly, Syk is already partially lost in normal epithelial tissue adjacent to the cancer lesion. In vivo, cell proliferation (as measured by the proliferative index Ki67) increased from normal to ductal carcinoma in situ to invasive, whereas Syk in situ staining in the same tissues decreased. In vitro, the presence of Syk was associated with reduced cell proliferation in an epidermal growth factor receptor-overexpressing breast cancer cell line, BT549, whereas changes in apoptosis were undetected. Concomitantly, the kinase activity of the proto-oncogene Src was reduced by ∼30%. A 5-fold increase in abnormal mitoses was observed in the Syk-transfected cells compared with vector control. We propose that Syk is involved in the regulation of cell proliferation, possibly by controlling mechanisms of mitosis and cytokinesis via Src signal transduction pathway(s). Because of its progressive and early loss during tumor onset and development, monitoring of Syk loss in breast epithelial cells by noninvasive techniques such as ductal lavage may be a powerful tool for screening purposes.

Heregulin-β1 regulates the estrogen receptor-α gene expression and activity via the ErbB2/PI 3-K/akt pathway

This study examines whether the serine/threonine protein kinase, Akt, is involved in the crosstalk between the ErbB2 and estrogen receptor-α (ER-α) pathways. Treatment of MCF-7 cells with 10−9 M heregulin-β1 (HRG-β1) resulted in a rapid phosphorylation of Akt and a 15-fold increase in Akt activity. Akt phosphorylation was blocked by inhibitors of phosphatidylinositol 3-kinase (PI 3-K), by antiestrogens, the protein tyrosine kinase inhibitor, genistein, and by AG825, a selective ErbB2 inhibitor; but not by AG30, a selective EGFR inhibitor. Akt phosphorylation by HRG-β1 was abrogated by an arginine to cysteine mutation (R25C) in the pleckstrin homology (PH) domain of Akt, and HRG-β1 did not induce Akt phosphorylation in the ER-negative variant of MCF-7, MCF-7/ADR. Transient transfection of ER-α into these cells restored Akt phosphorylation by HRG-β1, suggesting the requirement of ER-α. HRG-β1 did not activate Akt in MCF-7 cells stably transfected with an anti-ErbB2-targeted ribozyme, further confirming a role for ErbB2. Stable transfection of the cells with a dominant negative Akt or with the R25C-Akt mutant, as well as PI 3-K inhibitors, blocked the effect of HRG-β1 on ER-α expression and activity and on the growth of MCF-7 cells. Stable transfection of MCF-7 cells with a constitutively active Akt mimicked the effect of HRG-β1. Experiments employing selective ErbB inhibitors demonstrate that the effect of HRG-β1 on ER-α expression and activity is also mediated by ErbB2 and not by EGFR, demonstrating that ErbB2 is the primary mediator of the effects of HRG-β1 on ER-α regulation. Taken together, our data suggest that HRG-β1, bound to the ErbB2 ErbB3 heterodimer, in the presence of membrane ER-α, interacts with and activates PI 3-K/Akt. Akt leads to nuclear ER-α phosphorylation, thereby altering its expression and transcriptional activity.

Recruitment of insulin receptor substrate-1 and activation of NF-κB essential for midkine growth signaling through anaplastic lymphoma kinase

Anaplastic lymphoma kinase (ALK) is a transmembrane receptor tyrosine kinase in the insulin receptor superfamily. We recently demonstrated that the growth factors pleiotrophin (PTN) and midkine (MK) are ligands for ALK and that upon ALK activation, insulin receptor substrate-1 (IRS-1) and other substrates are phosphorylated. Here, the role of IRS-1 in ligand-mediated ALK signaling is investigated in interleukin-3 (IL-3)-dependent 32D murine myeloid cells. These cells do not express ALK and IRS family members, and do not respond to exogenously added PTN or MK. We show that expression of ALK plus IRS-1 renders these cells independent of IL-3 owing to the activation of ALK by endogenous MK. Mutational analysis reveals that this transformed phenotype of 32D cells requires kinase-active ALK as well as the interaction of ALK with IRS-1. Furthermore, 32D/IRS-1/ALK cells display an enhanced activation of mitogen-activated protein kinase and PI3-kinase pathways, and a selective transcriptional activation of nuclear factor (NF)-κB. Small interfering RNA-mediated knockdown of the endogenous MK or p65/NF-κB revealed that both these are rate limiting for the transformed phenotype induced by ALK plus IRS-1. We conclude that the recruitment of IRS-1 to activated ALK and the activation of NF-κB are essential for the autocrine growth and survival signaling of MK.