The estrogen receptor alpha:insulin receptor substrate 1 complex in breast cancer: structure-function relationships

Background: Insulin receptor substrate 1 (IRS-1) is a signaling molecule that exerts a key role in mediating cross talk between estrogen receptor a (ERa) and insulin-like growth factor 1 (IGF-1) in breast cancer cells. Previously, we demonstrated that a fraction of IRS-1 binds ERa, translocates to the nucleus, and modulates ERa-dependent transcription at estrogen response elements (ERE). Here, we studied structure–function relationships of the ERa:IRS-1 complex under IGF-1 and/or estradiol (E2) stimulation. Materials and methods: ERa and IRS-1 deletion mutants were used to analyze structural and functional ERa/IRS-1 interactions. IRS-1 binding to ERE and IRS-1 role in ERa-dependent ERE transcription was examined by chromatin immunoprecipitation and gene reporter analysis, respectively. The requirement for IRS-1 in ERa function was tested with RNAi technology. Results: Nuclear translocation of IRS-1 was induced by E2, IGF-1, and a combination of both stimuli. ERa/IRS-1 binding was direct and involved the activation function-1 (AF-1)/DNA binding domain (DBD) region of ERa and two discrete regions of IRS-1 (the N-terminal pleckstrin homology domain and a region within the C-terminus). IRS-1 knock down abrogated IGF-1-dependent transcriptional activity of unliganded ERa, but induced the activity of liganded ERa. Conclusions: ERa/IRS-1 interactions are direct and involve the ERa AF-1/DBD domain and IRS-1 domains mapping within N- and C-terminus. IRS-1 may act as a repressor of liganded ERa and coactivator of unliganded ERa

FoxO3a as a positive prognostic marker and a therapeutic target in Tamoxifen-resistant breast cancer

Background: Resistance to endocrine treatments is a major clinical challenge in the management of estrogen receptor positive breast cancers. Although multiple mechanisms leading to endocrine resistance have been proposed, the poor outcome of this subgroup of patients demands additional studies. Methods: FoxO3a involvement in the acquisition and reversion of tamoxifen resistance was assessed in vitro in three parental ER+ breast cancer cells, MCF-7, T47D and ZR-75-1, in the deriving Tamoxifen resistant models (TamR) and in Tet-inducible TamR/FoxO3a stable cell lines, by growth curves, PLA, siRNA, RT-PCR, Western blot, Immunofluorescence, Transmission Electron Microscopy, TUNEL, cell cycle, proteomics analyses and animal models. FoxO3a clinical relevance was validated in silico by Kaplan−Meier survival curves. Results: Here, we show that tamoxifen resistant breast cancer cells (TamR) express low FoxO3a levels. The hyperactive growth factors signaling, characterizing these cells, leads to FoxO3a hyper-phosphorylation and subsequent proteasomal degradation. FoxO3a re-expression by using TamR tetracycline inducible cells or by treating TamR with the anticonvulsant lamotrigine (LTG), restored the sensitivity to the antiestrogen and strongly reduced tumor mass in TamR-derived mouse xenografts. Proteomics data unveiled novel potential mediators of FoxO3a anti-proliferative and pro-apoptotic activity, while the Kaplan−Meier analysis showed that FoxO3a is predictive of a positive response to tamoxifen therapy in Luminal A breast cancer patients. Conclusions: Altogether, our data indicate that FoxO3a is a key target to be exploited in endocrine-resistant tumors. In this context, LTG, being able to induce FoxO3a, might represent a valid candidate in combination therapy to prevent resistance to tamoxifen in patients at risk

po 058 unravelling the protective role of androgens androgen receptorin breast cancer when bad goes good

Introduction Androgen receptor (AR) expression in breast cancer growth and progression appears to be clinically relevant and disease context specific. In oestrogen receptor (ER)α-positive primary breast cancers, AR positivity correlates with lower tumour grade and a better clinical outcome. These clinical-pathological findings mirror the capability of androgens to counteract ERα-dependent proliferation in both normal mammary epithelium and ERα-positive breast cancer preclinical models in which androgen/AR-dependent pro-apoptotic effects have been also evidenced. Here we report a novel additional mechanism underlining the protective, anti-proliferative role exerted by AR signalling. This mechanism involves modulation of the expression, cellular distribution and function of BAD, a pro-apoptotic member of the Bcl-2 family proteins, whose expression is related to a significantly better disease free survival in (ER)α-positive human breast cancers. Material and methods MCF-7, TD47D, ZR-75 breast cancer cells; qReal Time PCR; western blotting (WB); immunofluorescence analysis (IF); immunoprecipitation assay (IP); DNA affinity precipitation assay; Chromatin Immunoprecipitation Assay. Results and discussions The expression of a panel of pro/anti-apoptotic proteins was investigated in cellular protein lysates from ERα/AR-positive MCF-7 cells cultured for 1, 3 and 6 days under androgen treatment. The expression of the anti-apoptotic Bcl-2 protein, or the pro-apoptotic BID and BAX remained unchanged, while a sustained increase in the expression of the pro-apoptotic BAD could be observed, reducing the Bcl-2/BAD ratio and, thus, shifting the delicate balance between inhibitors and inducers of cell death. Interestingly, androgens induced a marked BAD levels increase into the nuclear compartment in ERα/AR-positive MCF-7, T47D and ZR-75 as well as in ERα negative/AR-positive SKBR3 cells. The androgen-regulated intracellular localization of BAD involved an AR/BAD physical interaction, suggesting a nuclear role for BAD upon androgen stimulation. Indeed, androgens induced both AR and BAD recruitment at a AP-1 and at a ARE site within the cyclin D1 promoter region, contributing to explain the anti-proliferative effect of androgens in breast cancer cells. Conclusion We defined a novel mechanism by which androgens modulate BAD expression and force its ability to act as a cell cycle inhibitor through modulation of cyclin D1 gene transcriptional activity, strengthening the protective role of androgen signalling in estrogen-responsive breast cancer

Inhibition of ERβ Induces Resistance to Cisplatin by Enhancing Rad51–Mediated DNA Repair in Human Medulloblastoma Cell Lines

Cisplatin is one of the most widely used and effective anticancer drugs against solid tumors including cerebellar tumor of the childhood, Medulloblastoma. However, cancer cells often develop resistance to cisplatin, which limits therapeutic effectiveness of this otherwise effective genotoxic drug. In this study, we demonstrate that human medulloblastoma cell lines develop acute resistance to cisplatin in the presence of estrogen receptor (ER) antagonist, ICI182,780. This unexpected finding involves a switch from the G2/M to G1 checkpoint accompanied by decrease in ATM/Chk2 and increase in ATR/Chk1 phosphorylation. We have previously reported that ERβ, which is highly expressed in medulloblastomas, translocates insulin receptor substrate 1 (IRS-1) to the nucleus, and that nuclear IRS-1 binds to Rad51 and attenuates homologous recombination directed DNA repair (HRR). Here, we demonstrate that in the presence of ICI182,780, cisplatin-treated medulloblastoma cells show recruitment of Rad51 to the sites of damaged DNA and increase in HRR activity. This enhanced DNA repair during the S phase preserved also clonogenic potential of medulloblastoma cells treated with cisplatin. In conclusion, inhibition of ERβ considered as a supplemental anticancer therapy, has been found to interfere with cisplatin–induced cytotoxicity in human medulloblastoma cell lines

Role of cyclic AMP response element-binding protein in insulin-like growth factor-I receptor up-regulation by sex steroids in prostate cancer cells

Insulin-like growth factor-I receptor (IGF-IR) overexpression may play a role in prostate cancer progression. We found previously that, in prostate cancer cells, IGF-IR is up-regulated by both androgens and estrogens via a nongenotropic pathway. We now show that, in prostate cancer cells, stimulation with either androgens or estrogens up-regulates IGF-IR by inducing cyclic AMP response element-binding protein (CREB) activation. Both sex steroids phosphorylated CREB at Ser(133) in a dose-dependent manner in androgen receptor (AR)-positive LNCaP cells, whereas only estrogens phosphorylated CREB in AR-negative PC3 cells. CREB phosphorylation involved c-Src-dependent extracellular signal-regulated kinase 1/2 activation, but not protein kinase A, protein kinase C, or calmodulin-dependent kinase II, and occurred also in cells transfected with AR or estrogen receptor mutants that do not localize into the nucleus. CREB silencing abrogated IGF-IR up-regulation and promoter activation. We also showed that CREB binds to IGF-IR promoter region and identified the relevant CREB-binding site at the 5'-untranslated region fragment of IGF-IR promoter. In conclusion, we describe a novel mechanism of IGF-IR up-regulation and promoter activity by CREB activation, induced by sex steroids, through a nongenotropic signaling

Plasma sex hormone concentrations during the reproductive cycle in the male lizard, Podarcis s. sicula.

Progesterone, 17-hydroxyprogesterone, androstenedione, 5 alpha-dihydrotestosterone, dehydroepiandrosterone, testosterone and oestradiol concentrations in the plasma were measured by simultaneous radioimmunoassay in males of the lizard Podarcis s. sicula. Hormonal determinations were performed at monthly intervals from January to December (except for August). Testosterone and androstenedione reached peak values of 174.8 ng/ml and 21.4 ng/ml in the mating season (spring) and then testosterone fell abruptly to 5.9 ng/ml in June remaining at this level during hibernation when dehydroepiandrosterone (DHA) reached a maximal level of 28.5 +/- 9.3 ng/ml. Castration resulted in a marked decrease of testosterone, androstenedione, dihydrotestosterone and DHA values, with DHA being significantly lowered only during the winter season. In castrated animals, however, testosterone and androstenedione persisted conspicuously in the plasma during the breeding period, suggesting that adrenal sex steroid output may change during the annual reproductive cycle. In intact animals, progesterone and oestradiol exhibited peak values during the refractory period after the mating season. We suggest a probable role of oestradiol in the induction of the refractory period in this lizard