Hormonal regulation of tumor suppressor proteins in breast cancer cells

This laboratory is studying hormonal regulation of tumor suppressor proteins, p53 and retinoblastoma (pRB). Estrogen receptor and progesterone receptor positive human breast cancer cell lines, T47D and MCF-7, were utilized for determining influence of hormonal and antihormonal agents on the level of expression of p53, state of phosphorylation of pRB, and rate of cell proliferation. The expression of p53 in T47D cells grown for 4–5 days in culture medium containing charcoal-treated (stripped) fetal bovine serum declined gradually to 10% of the level seen in control (whole serum, non charcoal-treated) groups. Supplementation of culture medium containing stripped serum with 0.1–1 nM estradiol (E₂) restored p53 to its level seen in the control within 6–24 h. Under above conditions, treatment of cells with R5020 or RU486 reduced (15–30%) the level of p53. Incubation of cells in E₂-containing growth medium caused cell proliferation and hyperphosphorylation of pRB; the latter effect was seen maximally between 24–72 h. The E₂-induced hyperphosphorylation of pRB and increase in the level of p53 were sensitive to the presence of ICI and 4-hydroxy tamoxifen (OHT). T47D and MCF-7 cells were also transiently transfected with a P1CAT reporter plasmid containing c-Myc responsive element and the levels of chloramphenicol acetyltransferase (CAT) activity were observed in response to various treatments. E₂ and OHT caused P1CAT induction as seen by increased CAT activity: E₂ caused an endogenous increase in the expression of an ICI-sensitive c-Myc form. These data suggest that estrogen upregulates p53 expression while progesterone downregulates this process. Further, E₂ regulates p53 level and pRB activity in a coordinated manner.     

Intranuclear Redistribution of SV40T, p53, and PML in a Conditionally SV40T-Immortalized Cell Line

We have previously reported that EBNA-5, one of the Epstein–Barr virus-encoded proteins, accumulates in the nuclear bodies containing PML, the promyelocytic leukemia associated protein. In this study, we examine the intranuclear distribution of SV40 large T-antigen (SV40T), the p53 tumor suppressor protein (p53), and PML in a conditionally immortalized cell line, IDH4. In IDH4 cells, the expression of SV40T is regulated by a dexamethasone (Dex)-driven promoter. Withdrawal of Dex results in down-regulation of SV40T and growth arrest, whereas addition of Dex to the growth-arrested cells results in up-regulation of SV40T and proliferation. In proliferating IDH4 cells, SV40T is concentrated in nuclear dots that are also positive for p53. Many of these dots are juxtaposed to PML positive structures but do not colocalize with them. After removal of Dex, SV40T–p53 dots gradually disappear, while the PML structures remain. Induction of SV40T in nonproliferating IDH4 cells causes a coordinated redistribution of SV40T and p53. The immunostaining for SV40T and p53 is first weak, then strong with a homogeneous distribution, and 3–4 days later becomes dot-like again. This reappearance of SV40T–p53 dots coincides with the recovery of proliferation in restimulated IDH4 cells. Also, the p53 pattern correlates with the SV40T pattern with regard to both morphology and intensity during both suppression and induction of SV40T. Taken together, our data suggest that (i) the level of p53 is coregulated with the level of SV40T in a dose-dependent fashion; (ii) the formation of SV40T–p53 nuclear dots correlates with the transformed phenotype; (iii) the SV40T–p53 dots localize preferentially to the neighborhood of PML bodies which are already present in normal cells.     

Inactivated tumor suppressor Rb by nitric oxide promotes mitosis in human breast cancer cells

Inactivation of tumor suppressor protein retinoblastoma (Rb) is important mechanism for the G1/S transition during cell cycle progression. Human breast cancer cells T47D release great amount of nitric oxide (NO), but its relation to tumor suppressor Rb is unknown. In this study, it is shown that NO induces phosphorylation and inactivation of Rb tumor suppressor protein, increasing G2/M phase and cell proliferation of breast cancer cells T47D. NO did not induce changes in p53 ser-15 phosphorylation, the most phosphorylated site of p53 during its activation. These data indicate that NO induces cell proliferation through the Rb pathway. NO phosphorylates and inactivates tumor suppressor protein Rb inducing mitosis by the p53 independent pathway in breast cancer cell.     

Prolonged progestin treatment induces the promoter of CDK inhibitor p21 Cip1,Waf1 through activation of p53 in human breast and endometrial tumor cells

Progestins are frequently used in the treatment of advanced breast and endometrial cancer. The human breast carcinoma cell line T47D shows a biphasic response to progestins. Short-term progestin treatment leads to enhanced DNA synthesis, while this line is growth inhibited upon prolonged exposure. An important protein involved in growth regulation by progestins in this cell is the CDK inhibitor p21(Cip1,Waf1). We show that after 1 day of progestin treatment in T47D cells, the p21 promoter-proximal region containing Sp1 binding sites is crucial in the induction by progestins. However, after 3 days the activity of the promoter-distal region becomes predominant in T47D cells or the endometrial carcinoma cell line ECC1. This is dependent upon two domains within this region that contain p53 response elements. In ECC1 and T47D cells 3-day progestin treatment induces a reporter containing a p53 response element, but not a mutated version. This induction is due to activation of p53 by progestin, which may be caused by nuclear translocation of p53. These data indicate that upon prolonged exposure, progestins activate p53, in human breast and endometrial tumor cells, which up-regulates the p21(Cip1,Waf1) promoter. This may be an important mechanism involved in progestin-inhibited cellular proliferation in these cells.     

Characterization of ligand binding,DNA binding and phosphorylation of progesterone receptor by two novel progesterone receptor antagonist ligands

In order to gain a better understanding of the distinctive mechanisms of the various types of antiprogestins, we have characterized in vitro ligand binding, specific DNA binding and phosphorylation of progesterone receptor (PR) from T47D cells after treatment of cells with progestins (progesterone, R5020) and antiprogestins (RU486, ZK98299, Org 31806 and Org 31710). Treatment of the cells with R5020 or PR antagonists, with the exception of ZK98299, resulted in a quantitative upshift of PR-A and PR-B indicative of ligand/DNA-induced phosphorylation of PR. Treatment of cells with RU486, Org 31710 or Org 31806, but not R5020 or ZK98299 resulted in detectable PR-progesterone response element complexes (PR-PREc) as assessed by gel mobility shift assay. Although treatment of cells with ZK98299, a type I PR antagonist, did not induce phosphorylation, the antiprogestins, Org 31806 and Org 31710, in a manner identical to RU486, did. Our data suggest that Org 31806 and Org 31710 affect propertie s of PR from T47D cells that are similar to RU486. (Mol Cell Biochem 175: 205–212, 1997)     

Progestin inhibition of cell death in human breast cancer cell lines

Previously, we have shown that progestins both stimulate proliferation of the progesterone receptor (PR)-rich human breast cancer cell line T47D and protect from cell death, in charcoal-stripped serum-containing medium. To lessen the variability inherent in different preparations of serum, we decided to further characterize progestin inhibition of cell death using serum starvation to kill the cells, and find that progestins protect from serum-starvation-induced apoptosis in T47D cells. This effect exhibits specificity for progestins and is inhibited by the antiprogestin RU486. While progestin inhibits cell death in a dose–responsive manner at physiological concentrations, estradiol-17β surprisingly does not inhibit cell death at any concentration from 0.001 nM to 1 μM. Progestin inhibition of cell death also occurs in at least two other human breast cancer cell lines, one with an intermediate level of PR, MCF-7 cells, and, surprisingly, one with no detectable level of PR, MDA-MB-231 cells. Further, we have found progestin inhibition of cell death caused by the breast cancer chemotherapeutic agents doxorubicin and 5-fluorouracil. These data are consistent with the building body of evidence that progestins are not the benign hormones for breast cancer they have been so long thought to be, but may be harmful both for undiagnosed cases and those undergoing treatment.     

Peptide immunisation of HLA-DR–transgenic mice permits the identification of a novel HLA-DRβ1*0101– and HLA-DRβ1*0401–restricted epitope from p53

Because of the central role of CD4⁺ T cells in antitumour immunity, the identification of the MHC class II–restricted peptides to which CD4⁺ T cells respond has become a priority of tumour immunologists. Here, we describe a strategy permitting us to rapidly determine the immunogenicity of candidate HLA-DR–restricted peptides using peptide immunisation of HLA-DR–transgenic mice, followed by assessment of the response in vitro. This strategy was successfully applied to the reported haemaglutinin influenza peptide HA(307–319), and then extended to three candidate HLA-DR–restricted p53 peptides predicted by the evidence-based algorithm SYFPEITHI to bind to HLA-DR1*0101 (HLA-DR1) and HLA-DR1*0401 (HLA-DR4) molecules. One of these peptides, p53(108–122), consistently induced responses in HLA-DR1– and in HLA-DR4–transgenic mice. Moreover, this peptide was naturally processed by dendritic cells (DCs), and induced specific proliferation in the splenocytes of mice immunised with p53 cDNA, demonstrating that immune responses could be naturally mounted to the peptide. Furthermore, p53(108–122) peptide was also immunogenic in HLA-DR1 and HLA-DR4 healthy donors. Thus, the use of this transgenic model permitted the identification of a novel HLA-DR–restricted epitope from p53 and constitutes an attractive approach for the rapid identification of novel immunogenic MHC class II–restricted peptides from tumour antigens, which can ultimately be incorporated in immunotherapeutic protocols.     

Terpenoids from Zingiber officinale (Ginger) Induce Apoptosis in Endometrial Cancer Cells through the Activation of p53

Novel strategies are necessary to improve chemotherapy response in advanced and recurrent endometrial cancer. Here, we demonstrate that terpenoids present in the Steam Distilled Extract of Ginger (SDGE) are potent inhibitors of proliferation of endometrial cancer cells. SDGE, isolated from six different batches of ginger rhizomes, consistently inhibited proliferation of the endometrial cancer cell lines Ishikawa and ECC-1 at IC₅₀ of 1.25 µg/ml. SDGE also enhanced the anti-proliferative effect of radiation and cisplatin. Decreased proliferation of Ishikawa and ECC-1 cells was a direct result of SDGE-induced apoptosis as demonstrated by FITC-Annexin V staining and expression of cleaved caspase 3. GC/MS analysis identified a total of 22 different terpenoid compounds in SDGE, with the isomers neral and geranial constituting 30–40%. Citral, a mixture of neral and geranial inhibited the proliferation of Ishikawa and ECC-1 cells at an IC₅₀ 10 µM (2.3 µg/ml). Phenolic compounds such as gingerol and shogaol were not detected in SDGE and 6-gingerol was a weaker inhibitor of the proliferation of the endometrial cancer cells. SDGE was more effective in inducing cancer cell death than citral, suggesting that other terpenes present in SDGE were also contributing to endometrial cancer cell death. SDGE treatment resulted in a rapid and strong increase in intracellular calcium and a 20–40% decrease in the mitochondrial membrane potential. Ser-15 of p53 was phosphorylated after 15 min treatment of the cancer cells with SDGE. This increase in p53 was associated with 90% decrease in Bcl2 whereas no effect was observed on Bax. Inhibitor of p53, pifithrin-α, attenuated the anti-cancer effects of SDGE and apoptosis was also not observed in the p53^neg SKOV-3 cells. Our studies demonstrate that terpenoids from SDGE mediate apoptosis by activating p53 and should be therefore be investigated as agents for the treatment of endometrial cancer.     

CG0009, a Novel Glycogen Synthase Kinase 3 Inhibitor,Induces Cell Death through Cyclin D1 Depletion in Breast Cancer Cells

Glycogen synthase kinase 3α/β (GSK3α/β) is a constitutively active serine/threonine kinase involved in multiple physiological processes, such as protein synthesis, stem cell maintenance and apoptosis, and acts as a key suppressor of the Wnt-β-catenin pathway. In the present study, we examined the therapeutic potential of a novel GSK3 inhibitor, CG0009, in the breast cancer cell lines, BT549, HS578T, MDA-MB-231, NCI/ADR-RES, T47D, MCF7 and MDA-MB-435, from the NCI-60 cancer cell line panel. Assessment of cytotoxicity, apoptosis and changes in estrogen-signaling proteins was performed using cell viability assays, Western blotting and quantitative real-time PCR. CG0009 enhanced the inactivating phosphorylation of GSK3α at Ser21 and GSK3β at Ser9 and simultaneously decreased activating phosphorylation of GSK3β at Tyr216, and induced caspase-dependent apoptosis independently of estrogen receptor α (ERα) expression status, which was not observed with the other GSK3 inhibitors examined, including SB216763, kenpaullone and LiCl. CG0009 treatment (1 µmol/L) completely ablated cyclin D1 expression in a time-dependent manner in all the cell lines examined, except T47D. CG0009 alone significantly activated p53, leading to relocation of p53 and Bax to the mitochondria. GSK3 inhibition by CG0009 led to slight upregulation of the β-catenin target genes, c-Jun and c-Myc, but not cyclin D1, indicating that CG0009-mediated cyclin D1 depletion overwhelms the pro-survival signal of β-catenin, resulting in cell death. Our findings suggest that the novel GSK3 inhibitor, CG0009, inhibits breast cancer cell growth through cyclin D1 depletion and p53 activation, and may thus offer an innovative therapeutic approach for breast cancers resistant to hormone-based therapy.     

cMyc-p53 feedback mechanism regulates the dynamics of T lymphocytes in the immune response

Activation and proliferation of T cells are tightly regulated during the immune response. We show here that kinetics of proliferation of PHA activated T cells follows the expression of cMyc. Expression of p53 is also elevated and remains high several days after activation. To investigate the role of p53 in activated T cells, its expression was further elevated with nultin-3 treatment, a small molecule that dissociates the E3 ubiquitin protein ligase MDM2 from p53. Concomitantly, cMyc expression and proliferation decreased. At the other end of the cMyc-p53 axis, inhibition of cMyc with 10058-F4 led to down regulation of p53, likely through the lower level of cMyc induced p14ARF, which is also known to dissociate the p53-MDM2 complex. Both compounds induced cell cycle arrest and apoptosis. We conclude that the feedback regulation between cMyc and p53 is important for the T cell homeostasis. We also show that the two compounds modulating p53 and cMyc levels inhibited proliferation without abolishing the cytotoxic function, thus demonstrating the dichotomy between proliferation and cytotoxicity in activated T cells.     

Insulin-like growth factor-binding protein-3 modulates expression of Bax and Bcl-2 and potentiates p53-independent radiation-induced apoptosis in human breast cancer cells

We report that transfection of insulin-like growth factor-binding protein-3 (IGFBP-3) cDNA in human breast cancer cell lines expressing either mutant p53 (T47D) or wild-type p53 (MCF-7) induces apoptosis. IGFBP-3 also increases the ratio of pro-apoptotic to anti-apoptotic members of the Bcl-2 family. In MCF-7, an increase in Bad and Bax protein expression and a decrease in Bcl-x(L) protein and Bcl-2 protein and mRNA were observed. In T47D, Bax and Bad proteins were up-regulated; Bcl-2 protein is undetectable in these cells. As T47D expresses mutant p53 protein, these modulations of pro-apoptotic proteins and induction of apoptosis are independent of p53. The effect of IGFBP-3 on the response of T47D to ionizing radiation (IR) was examined. These cells do not G(1) arrest in response to IR and are relatively radioresistant. Transfection of IGFBP-3 increased the radiosensitivity of T47D and increased IR-induced apoptosis but did not effect a rapid G(1) arrest. IR also caused a much greater increase in Bax protein in IGFBP-3 transfectants compared with vector controls. Thus, IGFBP-3 increases the expression of pro-apoptotic proteins and apoptosis both basally and in response to IR, suggesting it may be a p53-independent effector of apoptosis in breast cancer cells via its modulation of the Bax:Bcl-2 protein ratio.     

Fulvestrant-Induced Cell Death and Proteasomal Degradation of Estrogen Receptor α Protein in MCF-7 Cells Require the CSK c-Src Tyrosine Kinase

Fulvestrant is a representative pure antiestrogen and a Selective Estrogen Receptor Down-regulator (SERD). In contrast to the Selective Estrogen Receptor Modulators (SERMs) such as 4-hydroxytamoxifen that bind to estrogen receptor α (ERα) as antagonists or partial agonists, fulvestrant causes proteasomal degradation of ERα protein, shutting down the estrogen signaling to induce proliferation arrest and apoptosis of estrogen-dependent breast cancer cells. We performed genome-wide RNAi knockdown screenings for protein kinases required for fulvestrant-induced apoptosis of the MCF-7 estrogen-dependent human breast caner cells and identified the c-Src tyrosine kinase (CSK), a negative regulator of the oncoprotein c-Src and related protein tyrosine kinases, as one of the necessary molecules. Whereas RNAi knockdown of CSK in MCF-7 cells by shRNA-expressing lentiviruses strongly suppressed fulvestrant-induced cell death, CSK knockdown did not affect cytocidal actions of 4-hydroxytamoxifen or paclitaxel, a chemotherapeutic agent. In the absence of CSK, fulvestrant-induced proteasomal degradation of ERα protein was suppressed in both MCF-7 and T47D estrogen-dependent breast cancer cells whereas the TP53-mutated T47D cells were resistant to the cytocidal action of fulvestrant in the presence or absence of CSK. MCF-7 cell sensitivities to fulvestrant-induced cell death or ERα protein degradation was not affected by small-molecular-weight inhibitors of the tyrosine kinase activity of c-Src, suggesting possible involvement of other signaling molecules in CSK-dependent MCF-7 cell death induced by fulvestrant. Our observations suggest the importance of CSK in the determination of cellular sensitivity to the cytocidal action of fulvestrant.