Decreased Cyclin D1 Levels Research Articles

Specific targeting of insulin-like growth factor 1 receptor signaling in human estrogen dependent breast cancer cell by a novel tyrosine-based benzoxazepine derivative

The present study sought to investigate the in vitro and in vivo effects of a tyrosine-based benzoxazepine, 4-[4-(toluene-4-sulfonyl)-2,3,4,5-tetrahydro-benzo[f][1,4]oxazepin-3-ylmethyl]-phenol) [THBP] in human breast cancer cells, with a focus on determining its molecular target. THBP had growth inhibitory effect on MCF-7 and MDA-MD-231 cells. At IC 50 value (∼20 μM), THBP resulted in G1 arrest, decrease in cyclin D1 levels and induction of apoptosis of MCF-7 cells. Mechanistically, activation of caspase 8 contributes critically to the induction of apoptotic cell death as copresence of selective inhibition of caspase 8 effectively abrogates the cytotoxic effect of THBP in MCF-7 cells. Further, THBP increased pro-apoptotic protein, Bax; decreased anti-apoptotic protein, Bcl-2; and decreased mitochondrial membrane potential in MCF-7 cells, indicating involvement of an intrinsic pathway of apoptosis following caspase 8 activation. Out of the various growth factors/hormones, THBP selectively abrogated increased viability of MCF-7 cells by insulin-like growth factor 1 (IGF-1). Molecular docking studies revealed that THBP occupied the ATP binding pocket of IGF-1 receptor (IGF-1R). Accordingly THBP was found to inhibit IGF-1-induced phosphorylation of IGF-1R and insulin receptor substrate-1 (IRS-1) without inhibiting insulin signaling in MCF-7 cells. In athymic nude mice, compared with vehicle, THBP treatment significantly reduced the growth of MCF-7 xenograft tumors through inhibition of cancer cell proliferation as well as promotion of cell death that correlated with reduced phospho-IGF-1R levels. We suggest that interfering with the IGF-1R signaling by the benzoxazepine THBP offers a novel and selective therapeutic strategy for estrogen receptor-positive, postmenopausal breast cancer patients.

New targeted therapies in myelodysplastic syndrome: the role of farnesyltransferase and proteasome inhibitors

One of the main mechanisms responsible for MDS molecular pathogenesis involves the activation of tyrosine-kinase receptors, such as FLT3, RAS proteins, and deregulation of apoptotic pathways. Regarding this, new drugs have been developed to target pathways involved in malignancy, such as Farnesyltransferase Inhibitors (IFTs) and proteasome inhibitors (PI). This work aims to clarify the role of IFTs and PI as potential therapeutic agents in Myelodysplastic Syndrome (MDS). For this, F-36P cells, were incubated with different concentrations of α-HFPA (IFT) and MG262 (PI), as single agents and in association with the conventional therapeutic drug, Cytosine Arabinoside (Ara-C). Cell growth and viability was evaluated by Trypan Blue test. Cell death was analyzed by optic microscopy and flow cytometry (FC). Expression of proteins involved in apoptosis and cell cycle regulation was evaluated by FC. The detection of RAS and FLT3 mutations was accessed by sequentiation and PCR, respectively. Our results show that α-HFPA and MG262, in monotherapy, induce a decrease in cell growth and viability in a time and dose-dependent manner (IC50, α-HFPA 125 μM; MG262 100 nM). The antiproliferative effect of α-HFPA could be related to RAS/MAPK pathway inhibition, as we observed a decrease in cyclin D1 levels, while the cytotoxicity induced by MG262 to an increase in BAX expression. Our results show that α-HFPA is effective independently of RAS mutations, once we didn’t identify mutations in none of the isoforms studied, but we observe ITD mutations in FLT3 gene. These results suggest that IFTs and PIs may constitute a potential therapeutic approach as single agents in MDS.

Abstract 1235: The organic cation transporter SLC22A5 gene is regulated by the cooperative activity of the estrogen receptor and other transcription factors in breast cancer

Abstract The solute carrier family 22 member 5 (SLC22A5) gene encodes a polyspecific organic cation transporter that is associated with many diseases. However, the regulation of SLC22A5 and its relationship with breast cancer is not clearly defined. In a previous global gene expression analysis from our group, SLC22A5 was identified as an estrogen-regulated gene. In this study, we investigated the molecular mechanism by which estrogen receptor (ER) and other transcription factors regulate SLC22A5 gene expression in breast cancer. SLC22A5 is highly expressed in ER-positive breast cancer cell lines and ER-positive mammary tumor tissues compared to ER-negative cells and tissues. SLC22A5 expression is also correlated with the levels of ER expression in breast cancer cell lines and in mammary tumor tissue specimens. SLC22A5 mRNA expression is induced in MCF-7 breast cancer cells within half an hour after estrogen treatment and remains elevated for over 24 hours. Actinomycin D, but not cycloheximide, can inhibit this estrogen induction, indicating new protein synthesis is not needed for induction. These results suggest that estrogen directly activates SLC22A5 transcription. We next cloned the 6kb SLC22A5 promoter and luciferase experiments demonstrated that this portion of the SLC22A5 promoter is not responsive to estrogen treatment. However, we discovered that an intronic enhancer located approximately 9kb from the transcriptional start site confers estrogen responsiveness when inserted downstream of the SLC22A5 gene promoter-luciferase reporter construct. Several transcription factors binding sites, such as a cyclic adenosine monophosphate (cAMP) response element (CRE), an estrogen-response element (ERE), an activator protein-1 (AP-1) site and a half ERE, are juxtaposed in the middle of this intronic enhancer. Mutation or deletion of these elements abolishes the estrogen responsiveness. Chromatin-immunoprecipitation (ChIP) assay demonstrates the recruitment of the transcription factors onto the intronic enhancer region in an estrogen-dependent manner. We next investigated the effect of SLC22A5 knockdown on breast cancer cell growth. Results from these studies show that shRNA knockdown of SLC22A5 inhibits the proliferation of MCF-7 cells. This reduced proliferation is accompanied by decreased cyclin D1 levels. These results suggest that SLC22A5 is an estrogen-induced gene that is involved in regulating breast cell growth. Our results also suggest that this estrogen induction is mediated by a complex intronic enhancer. This estrogen-induced gene may serve as a novel breast cancer biomarker that may be targeted for improved treatment and prevention of breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1235.

Control of Cyclin D1 and Breast Tumorigenesis by the EglN2 Prolyl Hydroxylase

2-Oxoglutarate-dependent dioxygenases, including the EglN prolyl hydroxylases that regulate HIF, can be inhibited with drug-like molecules. EglN2 is estrogen inducible in breast carcinoma cells and the lone Drosophila EglN interacts genetically with Cyclin D1. Although EglN2 is a nonessential gene, we found that EglN2 inactivation decreases Cyclin D1 levels and suppresses mammary gland proliferation in vivo. Regulation of Cyclin D1 is a specific attribute of EglN2 among the EglN proteins and is HIF independent. Loss of EglN2 catalytic activity inhibits estrogen-dependent breast cancer tumorigenesis and can be rescued by exogenous Cyclin D1. EglN2 depletion also impairs the fitness of lung, brain, and hematopoietic cancer lines. These findings support the exploration of EglN2 inhibitors as therapeutics for estrogen-dependent breast cancer and other malignancies.

Mammalian target of rapamycin inhibitors rapamycin and RAD001 (everolimus) induce anti-proliferative effects in GH-secreting pituitary tumor cells in vitro

The effect of mammalian target of rapamycin (mTOR) inhibitors on pituitary tumors is unknown. Akt overexpression was demonstrated in pituitary adenomas, which may render them sensitive to the anti-proliferative effects of these drugs. The objective of the study was to evaluate the anti-proliferative efficacy of the mTOR inhibitor, rapamycin, and its orally bioavailable analog RAD001 on the GH-secreting pituitary tumor GH3 and MtT/S cells and in human GH-secreting pituitary adenomas (GH-omas) in primary cell cultures. Treatment with rapamycin or RAD001 significantly decreased the number of viable cells and cell proliferation in a dose- and time-dependent manner. This was reflected by decreased phosphorylation levels of the downstream mTOR target p70S6K. Rapamycin treatment of GH3 cells induced G0/G1 cell cycle arrest. In other tumor cell types, this was attributed to a decrease in cyclin D1 levels. However, rapamycin did not affect cyclin D1 protein levels in GH3 cells. By contrast, it decreased cyclin D3 and p21/CIP, which stabilizes cyclin D/cyclin-dependent kinase 4 (cdk4) complexes. Rapamycin inhibited FCS-induced retinoblastoma phosphorylation and subsequent E2F-transcriptional activity. In response to decreased E2F activity, the expression of the E2F-regulated genes cyclin E and cdk2 was reduced. Our results showed that mTOR inhibitors potently inhibit pituitary cell proliferation, suggesting that mTOR inhibition may be a promising anti-proliferative therapy for pituitary adenomas. This therapeutic manipulation may have beneficial effects particularly for patients harboring invasive pituitary tumors resistant to current treatments.

Docosahexaenoic acid induces proteasome-dependent degradation of estrogen receptor α and inhibits the downstream signaling target in MCF-7 breast cancer cells

About two thirds of breast cancers in women are hormone-dependent and require estrogen for growth, its effects being mainly mediated through estrogen receptor α (ERα). Docosahexaenoic acid (DHA, 22:6n-3) and arachidonic acid (AA, 20:4n-6) have opposite effects on carcinogenesis, with DHA suppressing and AA promoting tumor growth both in vitro and in vivo. However, the mechanism is not clear. Here, we examined whether the effect is mediated through changes in ERα distribution. MCF-7 cells, an ERα-positive human breast cancer cell line, was cultured in estrogen-free medium containing 0, 10 or 60 μM DHA or AA, then were stimulated with estradiol. DHA supplementation resulted in down-regulation of ERα expression (particularly in the extranuclear fraction), a reduction in phosphorylated MAPK, a decrease in cyclin D1 levels and an inhibition in cell viability. In contrast, AA had no such effects. The DHA-induced decrease in ERα expression resulted from proteasome-dependent degradation and not from decreased ERα mRNA expression. We propose that breast cancer cell proliferation is inhibited by DHA through proteasome-dependent degradation of ERα, reduced cyclin D1 expression and inhibition of MAPK signaling.

TIS21/BTG2/PC3 and cyclin D1 are key determinants of nuclear diacylglycerol kinase-ζ-dependent cell cycle arrest

In addition to lipid second messengers derived from the plasma membrane, increasing evidence supports the existence of nuclear lipid-dependent signaling networks. Diacylglycerol is a key second messenger, generated at the nuclear level, which is metabolized by diacylglycerol kinases (DGKs). It has been demonstrated that nuclear DGK-ζ negatively regulates cell cycle progression. The aim of this study was to identify key determinants of nuclear DGK-ζ-dependent cell cycle arrest in C2C12 mouse myoblasts. Using DNA microarrays, Real-Time RT-PCR and western blot, we demonstrated that nuclear DGK-ζ downregulated the expression of cyclin D1 and increased the expression of TIS21/BTG2/PC3, a transcriptional regulator of cyclin D1 with a strong anti-proliferative function. Overexpression of TIS21/BTG2/PC3 blocked the cells in G1 phase of the cell cycle and decreased the levels of Ser807/811 phosphorylated retinoblastoma protein, similarly to overexpression of DGK-ζ. Moreover, during myogenic differentiation of C2C12 cells, we showed an increase of TIS21/BTG2/PC3 expression and a decrease in cyclin D1 levels. siRNA downregulation of TIS21/BTG2/PC3 impaired myogenic differentiation by opposing cell cycle arrest. In summary, these data identify TIS21/BTG2/PC3 and cyclin D1 as downstream effectors of nuclear DGK-ζ and highlight the importance of this DGK isoform in the regulation of myoblast proliferation and differentiation.

Thiazolidinediones regulate expression of cell cycle proteins in human prostate cancer cells via PPARγ-dependent and PPARγ-independent pathways

Thiazolidinediones (TZDs) are peroxisome proliferator activated receptor gamma (PPARγ) ligands that have been reported to reduce proliferation of human prostate cancer cells. However, the mechanisms by which TZDs inhibit prostate cancer cell proliferation are not fully understood. In addition, it is not known if the anti-proliferative effects of TZDs require activation of PPARγ or are mediated by PPARγ –independent pathways. The goals of this study were to assess whether TZDs regulate expression of proteins that control the transition from G1 to S phase of the cell cycle and define the role of PPARγ in these TZD-induced responses in androgen-independent human prostate cancer cell lines. Western blot analysis revealed that growth inhibitory concentrations of the TZDs rosiglitazone and ciglitazone induced expression of the cyclin dependent kinase inhibitor p21 and decreased cyclin D1 levels in the androgen independent PC-3 cell line. Phosphorylation of retinoblastoma protein at Serine 780 was also reduced in PC-3 cells exposed to ciglitazone. Furthermore, growth inhibitory concentrations of ciglitazone increased p21 and lowered cyclin D1 expression within C4-2 cells. PPARγ-directed siRNAs inhibited the ability of rosiglitazone to regulate expression of cyclin D1 and p21. However, knockdown of PPARγ did not significantly reduce ciglitazone-induced alterations in cyclin D1 and p21. Furthermore PPARγ siRNA did not prevent inhibition of PC-3 cell proliferation by either TZD. Thus, activation of PPARγ is involved in rosiglitazone-induced alterations in cell cycle protein expression. However, the alterations in protein expression and proliferation induced by ciglitazone occur primarily via PPARγ-independent signaling pathways.

The Antidiabetic Biguanide Metformin Induces Growth Arrest in Multiple Myeloma Cells in Vitro, overcoming the Effect of Stromal Cells

Obesity and insulin resistance are associated with increased risk for several malignancies, including multiple myeloma (MM), possibly due to hyperinsulinemia and the resulting increased circulating free IGF-I levels that can have a growth-promoting and anti-apoptotic effect on MM cells. It has been proposed that improving insulin sensitivity, with a low-fat diet or with the use of the biguanide metformin, could suppress circulating insulin and free IGF-I levels and lower cancer risk. Metformin has been widely used as an antidiabetic agent since the 1950s and has an excellent safety profile. Interestingly, epidemiologic studies have suggested that patients with type 2 diabetes mellitus who are treated with metformin have lower cancer incidence and lower cancer-related mortality than diabetics who receive sulfonylurea monotherapy or insulin. Even more intriguing is the recent finding that metformin can have direct anticancer activity in vitro against prostate and breast carcinoma cell lines. In breast carcinoma cells, metformin inhibits mammalian target of rapamycin (mTOR) activity and suppresses translation initiation and global protein synthesis, via an AMP-activated protein kinase (AMPK)-dependent pathway. We investigated the direct effects of metformin in vitro in a panel of 17 MM cell lines and sublines using MTT, flow cytometry, annexin V-PI, as well as compartment-specific bioluminescence (CS-BLI) assays in the absence or presence of bone marrow stromal cells (BMSCs). We found that metformin suppressed the growth of all MM cell lines and sublines, including those that are resistant to conventional and several investigational anti-MM agents. The anti-MM activity of metformin was achieved with clinically relevant sub-mM or low mM levels, and was in the same order of magnitude or stronger than its activity reported in solid tumor cell line models. The anti-MM activity of metformin did not involve apoptotic/necrotic cell death, but inhibition of proliferation (G0/G1 arrest). Western blot analyses showed that metformin treatment of RPMI-8226/S MM cells led to increased phosphorylation of AMPK; suppression of p70S6K phosphorylation; and decreased cyclin D1 levels. These results are consistent with the known effect of metformin in triggering AMPK activity and thereby suppressing the function of its downstream targets mTOR and p70S6K. Co-culture with BMSCs did not abrogate the anti-MM effect of metformin, but further sensitized MM cell to this agent, at concentrations which did not affect BMSC viability. This sensitization was more prominent in MM cell lines that respond to BMSCs with increased proliferation/survival (e.g. MM-1S, MM-1R, KMS-11, L363) than in cell lines independent/unresponsive to stromal stimulation (e.g. OPM-2). To further probe the mechanistic basis for this stroma-induced sensitization to metformin, we studied the transcriptional signature of genes suppressed by mTOR inhibition, a downstream effect of metformin, in MM cells cultured alone vs. co-cultured with BMSCs. We observed that stroma triggers a significant increase in the average expression of mTOR-responsive genes in stroma responsive MM cell lines, but not in stroma unresponsive lines, in which this transcriptional signature is constitutively upregulated. This observation suggests that the increased metformin sensitivity of MM cells co-cultured with BMSCs may reflect the ability of metformin to counteract the upregulation of p70S6K/mTOR pathway activity triggered by stroma. Our studies demonstrate a direct, growth-suppressive effect of metformin in MM cells in vitro, which can be potentiated by interactions with the bone marrow microenvironment. Taken together with the beneficial effect of this antidiabetic agent on circulating insulin and free IGF-I levels and its favorable safety profile, these data support the clinical evaluation of metformin in patients with MM.

A Novel mTOR Kinase Inhibitor Causes Growth Inhibition, Cell Cycle Arrest, Apoptosis and Autophagic Cell Death in Mantle Cell Lymphoma Cell Lines: A Distinct Profile from Rapamycin.

Mantle cell lymphoma (MCL) is a distinct sub-type non-Hodgkin lymphoma characterized by overexpression of cyclin D1 (CCND1) in 95% of patients due to the cytogenetic change of chromosome translocation t(11;14) (q13;q32). It remains one of the most challenging lymphomas associated with shorter response duration to conventional chemotherapy as well as continuous relapses and refractory to current drugs. However, dysregulation of cyclin D1 biology alone is insufficient to develop MCL. The emerging data suggest that the mammalian target of rapamycin (mTOR) plays a crucial role in the proper transmission of proliferative and anti-apoptotic signals through the PI3K/AKT pathway that makes it an attractive therapeutic target for hematological malignances including mantle cell lymphoma. As a single agent, rapamycin analogs such as temsirolimus (CCI-779) achieved 38% overall response rate in heavily pretreated MCL and prolonged progression free survival (PFS) in relapsed and refractory mantle cell lymphoma (4.8 months in temsirolimus vs. 1.9 months in investigator's choice, ASCO 2008). mTOR regulates two distinct complexes TORC1 and TORC2. TORC1 complex is involved in cell cycle regulation by phosphorylating p70S6K and 4E-BP1, two molecules that are important for translational control of cyclin D1 and c-myc as well as ribosomal biogenesis whereas TORC2 complex mainly regulates phospho- AKT serine 473 leading to cell survival and proliferation. mTOR kinase also negatively regulates autophagy, a process of cellular bulk protein degradation by fusion to lysosomes upon the nutrient deprivation. We have developed mTOR kinase selective inhibitors which exhibit distinct biological profile from rapamycin in many cancer cell lines. Here we demonstrate that a selective mTOR kinase inhibitor displays potent anti-proliferative activity in JeKo-1 and Mino cells associated with decreased phosphorylation of S6, p70S6K, AKT S473, 4E-BP1 as well as decreased cyclin D1 levels leading to G1 arrest. The inhibitor also promotes autophagic cell death at 72h and 96h post-treatment. Furthermore a selective mTOR kinase inhibitor but not rapamycin induces a significant apoptosis in JeKo-1 and Mino cells. The observed apoptosis is correlated with caspases mediated PARP cleavage as well as inhibition of anti-apoptotic protein Mcl-1, suggesting TORC2/AKT S473 complex may provide survival signaling for mantle cell lymphoma. A timecourse study demonstrated that JeKo-1 and Mino cells undergo apoptosis at 24h and 48h followed by significant autophagic cell death at 72h and 96h in a dose dependent manner when exposed to our mTOR kinase inhibitor. In conclusion, mTOR kinase inhibitors are able to induce G1 cell cycle arrest, caspase-dependent apoptosis and autophagic cell death that contribute to the anti-tumor activity. Therefore it may provide a powerful alternative targeted therapy for mantle cell lymphoma.

Cytotoxic effects of a novel pyrazolopyrimidine derivative entrapped in liposomes in anaplastic thyroid cancer cells in vitro and in xenograft tumors in vivo

In this study, we evaluated the activity of two novel pyrazolopyrimidine derivatives (Si 34 and Si 35) against ARO cells, a human anaplastic thyroid cancer cell line. ARO cells exposed to different concentrations of the drugs showed a reduced growth rate and an increase of mortality. After 72 h incubation, doses of 5 and 10 microM Si 34 determined a decrease of cell counts by approximately 25% and approximately 75% compared with those of control cells respectively. Similar findings were observed using Si 35. Treatment with both Si 34 and Si 35 at 10 microM increased cell mortality also ( approximately 29% and approximately 18% respectively). At these concentrations, a decrease in cyclin D1 levels was observed. To improve the biopharmaceutical properties, a liposome formulation was prepared. When entrapped in unilamellar liposomes, Si 34 exerted its cytotoxic effects even at lower doses (maximal inhibition at 5 microM) and after shorter incubation time (48 h) either in ARO or other thyroid cancer cell lines. The effects were associated with weak apoptotic death. Inhibition of epidermal growth factor-stimulated src and ERK phosphorylation, as well as reduction of migration properties of ARO cells was also observed. Moreover, the growth of tumor xenografts induced in severe combined immunodeficiency (SCID) mice was inhibited by i.v. administration of 25-50 mg/kg of the drug liposomal formulation. In conclusion, the liposomal preparation of this novel pyrazolopyrimidine derivative appears to be a promising tool for the treatment of anaplasic thyroid cancer.

Lentiviral overexpression of Peroxisome Proliferator‐activated receptor gamma (PPARγ) induces apoptosis in human multiple myeloma cells

Peroxisome proliferator‐activated receptor γ (PPAR‐γ) is a transcription factor that has key role in adipocyte differentiation. PPARγ also regulates immune and inflammatory responses. Our laboratory has demonstrated that normal and malignant B cells express PPARγ and that exposure to certain PPARγ ligands inhibits their proliferation and induces apoptosis. These studies support a key role for PPARγ in B lymphocyte activation and death. We first examined the functional consequences of PPARγ overexpression in human multiple myeloma cells using a lentiviral vector approach. Human multiple myeloma 8226 cells were infected with a lentivirus expressing PPARγ. Here, PPARγ overexpression decreased multiple myeloma cell proliferation and induced spontaneous apoptosis, even in the absence of exogenous ligand, as determined by 3H thymidine and BrdU incorporation, annexin‐V‐7AAD staining, DAPI staining and caspase activation. The decrease in proliferation was associated with increased levels in p21 and decreased cyclin D1 levels. These cells were dramatically more sensitive to PPARγ‐ligand exposure, as treatment with ciglitazone enhanced apoptosis. Collectively, these findings support a regulatory role for PPARγ in the proliferation and apoptosis of malignant B cells and suggest the potential of PPARγ as a therapeutic target for multiple myeloma. Supported by DE011390 and the Leukemia and Lymphoma Society

Colored Potato Bioactive Compounds Suppress Proliferation and Induce Apoptosis in Both Androgen‐Dependent and –Independent Prostate Cancer Cell Lines

We recently reported that colored potato extracts induce apoptosis in LNCaP (androgen‐dependent) and PC‐3 (androgen‐independent) prostate cancer cell lines via caspase‐dependent and ‐independent pathways. In order to identify the most active growth inhibiting and pro‐apoptotic compounds in the potato extract, we have further investigated the effects of different phenolic compounds (chlorogenic acid, caffeic acid, gallic acid, catechin and malvidin) and glycoalkaloids (α‐ chaconine and solanine) found in these extracts on PC‐3 and LNCaP cells. α‐ chaconine (5 μg/ml) and gallic acid (10 μg/ml) showed potent antiproliferative properties and increased levels of the cyclin‐dependent kinase inhibitor p27 in both cell lines. Gallic acid decreased cyclin D1 levels in LNCaP cells. The growth inhibitory effects of α‐ chaconine were potentiated by SP600125, a c‐jun N‐terminal kinase (JNK) inhibitor, and atractyloside, an ADP phosphorylation inhibitor in both cell lines. Both α‐ chaconine and gallic acid induced caspase‐dependent apoptosis through PARP cleavage and activation of caspase‐3 in LNCaP cells. However, induction of apoptosis in PC‐3 cells was caspase‐independent. These results suggest that apoptosis induced by whole potato extracts in prostate cancer cell lines may be in part due to α‐ chaconine and gallic acid. CSREES special research grant.

PKR and PKR-like Endoplasmic Reticulum Kinase Induce the Proteasome-dependent Degradation of Cyclin D1 via a Mechanism Requiring Eukaryotic Initiation Factor 2α Phosphorylation

PKR and PKR-like Endoplasmic Reticulum Kinase Induce the Proteasome-dependent Degradation of Cyclin D1 via a Mechanism Requiring Eukaryotic Initiation Factor 2α Phosphorylation

ICA512 signaling enhances pancreatic β-cell proliferation by regulating cyclins D through STATs

Changes in metabolic demands dynamically regulate the total mass of adult pancreatic beta-cells to adjust insulin secretion and preserve glucose homeostasis. Glucose itself is a major regulator of beta-cell proliferation by inducing insulin secretion and activating beta-cell insulin receptors. Here, we show that islet cell autoantigen 512 (ICA512)/IA-2, an intrinsic tyrosine phosphatase-like protein of the secretory granules, activates a complementary pathway for beta-cell proliferation. On granule exocytosis, the ICA512 cytoplasmic domain is cleaved and the resulting cytosolic fragment (ICA512-CCF) moves into the nucleus where it enhances the levels of phosphorylated STAT5 and STAT3, thereby inducing insulin gene transcription and granule biogenesis. We now show that knockdown of ICA512 decreases cyclin D1 levels and proliferation of insulinoma INS-1 cells, whereas beta-cell regeneration is reduced in partially pancreatectomized ICA512-/- mice. Conversely, overexpression of ICA512-CCF increases both cyclin D1 and D2 levels and INS-1 cell proliferation. Up-regulation of cyclin D1 and D2 by ICA512-CCF is affected by knockdown of STAT3 and STAT5, respectively, whereas it does not require insulin signaling. These results identify ICA512 as a regulator of cyclins D and beta-cell proliferation through STATs and may have implication for diabetes therapy.

Induction of the SUMO-specific Protease 1 Transcription by the Androgen Receptor in Prostate Cancer Cells

Prostate cancer, the most frequently diagnosed carcinoma in males, is readily modulated via the transcriptional activity of androgen receptors. Our recent publication reported that androgen receptor-dependent transcription is significantly elevated with expression of the human sentrin/SUMO-specific protease (SENP1) in the androgen-sensitive human prostate cancer cell line (LNCaP). In situ hybridization studies indicated an elevation of SENP1 message in prostatic intraepithelial neoplasia and prostate cancer lesions as compared with normal prostate epithelia. This study aimed to delineate the mechanism for the regulation of SENP1 message and to determine the pathophysiological consequence of SENP1 induction with respect to prostate cancer. Real-time PCR confirmed the elevation of SENP1 mRNA in prostate cancer cells as compared with normal prostate epithelial cells. Chronic androgen exposure of LNCaP cells prompted an enhancement in the SENP1 transcript selectively. This androgen-mediated augmentation of SENP1 was absent with co-administration of the androgen receptor antagonist bicalutamide and in androgen receptor-negative prostate cancer PC-3 cells, indicating an androgen receptor-dependent event. Activation of the androgen receptor was required for binding an identified androgen response element and positively regulating SENP1 promoter activity. Abrogation of elevated SENP1 mRNA in prostate cancer cells significantly decreased androgen-mediated cell growth. Because increased SENP1 expression directly modulated androgen receptor-dependent cell proliferation and transcription, SENP1 could play an important role in prostate carcinogenesis.

Iron Chelation in Mantle Cell Lymphoma.

Cell proliferation is dependent upon iron, and numerous studies have shown that iron limitation arrests cells in the G1 phase of the cell cycle. A recent study of the molecular basis of these observations (Richardson, et al. Blood 2007; 109:4045) examined the ability of iron chelators to inhibit cell proliferation and to induce apoptosis, focusing on the role of iron chelation on cyclin D1. Cyclin D1 assembles with cdk-4 or cdk-6, generating an active holo-enzyme that catalyzes a rate limiting step in G1/S progression. This complex phosphorylates substrates, including the retinoblastoma protein, which regulate S phase entrance. Richardson's group demonstrated that the G1/S arrest after Fe depletion is mediated, in part, by a decrease in cyclin D1 via ubiquitin-independent proteasomal degradation. Studies looking specifically at mantle cell lymphoma cell lines, however, have not yet been reported. Mantle Cell lymphoma is an interesting target for potential iron chelation as it is associated with a balanced translocation (t11;14) which leads to upregulation of BCL1 and to the constitutive overproduction of cyclin D1. We studied five different cell lines - JeKo (Mantle Cell Lymphoma), BL-41 (Burkitt Cell Lymphoma), DG-75 (Burkitt Cell Lymphoma), SUDHL-6 (Diffuse Large B cell Lymphoma) and EBV-immortalized lymphocytes from normal controls - and incubated them with four different iron chelators - deferoxamine (DFO), 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311), Pyridoxal Isonicotinoyl Hydrazone (PIH), and Salicylaldehyde Isocotinoyl Hydrazone (SIH). We then measured and compared cell cycle proliferation (using the Cellometer Auto T4, an instrument that measures cell count, cell viability, and cell size) and the rate of apoptosis (via propidium iodide FACS analysis). At 24 hours incubation, the mantle cell lymphoma lines showed significantly increased rates of apoptosis compared with non-chelated mantle cell controls (5% vs. 48%, p=0.04). The diffuse large B cell lymphoma line showed a lesser increase in apoptosis that did not reach statistical significant (6.5% vs. 14%, p=0.07), while the Burkitt's lymphoma lines and the EBV immortalized lymphocytes showed no significant difference (BL-41, 3.4% vs. 4.1%, p=0.50; DG-75, 6% vs. 5.9%, p=0.99; EBV lymphocytes, 12.5% vs. 12.7%, p=0.96). At 72 hours of incubation with chelators, the EBV lymphocytes showed increased apoptosis compared to untreated controls (2.5% vs. 44.5%, p=0.002), while the apoptotic rate increased in the diffuse large B cell lymphoma line (3.8% vs. 48%, p=0.001) and even more dramatically in the mantle cell lymphoma line (1.5% vs. 64%, p=0.0006). The two Burkitt's lymphoma lines were affected to a lesser degree at 72 hours by the presence of iron chelators (BL-41, 0.9% vs, 3.9%, p=0.02; DG-75, 5.5% vs. 8.9%, p=0.11). Although iron chelation, especially at longer incubation times, did affect all cell lines to various degrees, the chelator-mediated effects do appear to be specific for cell type, with mantle cell lymphoma cells displaying higher rates of apoptosis compared with other lymphomas and normal lymphocytes. These initial results will now be followed by examination of cyclin D1 expression after iron chelation. If overexpression of cyclin D1 in mantle cell lymphoma releases cells from their normal controls and acts as an oncogene, then a decrease in cyclin D1 levels via iron chelation could be added to the therapeutic armamentarium of mantle cell lymphoma.

Interaction of EGFR and IGF-1R signals to modulate the anti-proliferative and biochemical responses of head and neck squamous cancer (HNSCC) cells to EGFR inhibitors

6045 Background: Epidermal growth factor receptor (EGFR) inhibitors improve the efficacy of radiotherapy for HNSCC and as single-agents induce tumor responses or stabilization in a minority of patients. The molecular mechanisms that dictate the spectrum of observed responses are not well understood. Methods/Results: Exposure of the HNSCC lines SCC-9 and SCC-25 in vitro to the EGFR inhibitors PD158780, lapitinib, or cetuximab suppressed cell proliferation and cell cycle progression into S-phase. Immunoblots showed that PD158780 inhibited autocrine activation of the EGFR, reduced phosphorylation of the downstream signaling proteins AKT and Erk, and affected cell cycle regulatory molecules (reduced Rb phosphorylation, decreased cyclin D1 levels, and induced p27kip1). However, stimulation of the cells with an insulin-like growth factor-1 (IGF-1) analog produced a dose-dependent resistance to the anti-proliferative effects of the EGFR inhibitors. The resistance of SCC-25 cells correlated with IGF-1-induced maintenance of AKT and Rb phosphorylation and cyclin D1 expression. Conversely, the EGFRs also acted downstream of the activated IGF-1 receptors as concomitant EGFR inhibition dampened the proliferative response of the cells, reduced Erk phosphorylation and increased p27kip1 expression. Experiments with the metalloproteinase inhibitor TAPI-2 suggested that the IGF-1 activates the EGFRs through proteolytic cleavage of precursor forms of EGFR ligands. Conclusions: The proliferative and biochemical responses of HNSCC cells to EGFR inhibitors are influenced by cross-talk between the EGFR and IGF-1 receptors. One implication of this work is that the clinical response of HNSCCs to EGFR inhibitors may depend on the relative contribution of autocrine EGFR signaling to activation of specific downstream growth factor signaling pathways, whether or not these receptors are the primary or secondary source of these signals. No significant financial relationships to disclose.

Non-genomic action of resveratrol on androgen and oestrogen receptors in prostate cancer: modulation of the phosphoinositide 3-kinase pathway

Prostate cancer represents a major concern in human oncology and the phytoalexin resveratrol (RES) inhibits growth and proliferation of prostate cancer cells through the induction of apoptosis. In addition, previous data indicate that in oestrogen-responsive human breast cancer cells, RES induces apoptosis by inhibition of the phosphoinositide-3-kinase (PI3K) pathway. Here, using androgen receptor (AR)-positive LNCaP and oestrogen receptor alpha (ERα)-expressing PC-3 prostate tumour cells, we have analysed whether the antiproliferative activity of RES takes place by inhibition of the AR- or ERα-dependent PI3K pathway. Although RES treatment (up to 150 μM) decreased AR and ERα protein levels, it did not affect AR and ERα interaction with p85-PI3K. Immunoprecipitation and kinase assays showed that RES inhibited AR- and ERα-dependent PI3K activities in LNCaP and PC-3, respectively. Consistently, lower PI3K activities correlated with decreased phosphorylation of downstream targets protein kinase B/AKT (PKB/AKT) and glycogen synthase kinase-3 (GSK-3). GSK-3 dephosphorylation could be responsible for the decreased cyclin D1 levels observed in both cell lines. Importantly, RES markedly decreased PKB/AKT phosphorylation in primary cultures from human prostate tumours, suggesting that the mechanism proposed here could take place in vivo. Thus, RES could have antitumoral activity in androgen-sensitive and androgen-non-sensitive human prostate tumours by inhibiting survival pathways such as that mediated by PI3K.

Redox control of G1/S cell cycle regulators during nitric oxide‐mediated cell cycle arrest

Redox regulation of cell cycle progression during nitric oxide (NO) mediated cytostasis is not well-understood. In this study, we investigated the role of the intracellular antioxidant glutathione (GSH) in regulating specific signaling events that are associated with NO-mediated cell cycle arrest. Manipulation of intracellular GSH content through pharmacological inhibition of glutamate-cysteine ligase (GCL) indicated that GSH depletion potentiated nitrosative stress, DNA damage, phosphorylation of the tumor suppressor p53 (Ser-18) and upregulation of p21(cip1/waf1) upon NO stimulation. However, we found that neither overexpression of a dominant negative p53 nor pharmacological inhibition of p53 with cyclic pifithrin-alpha (cPFT-alpha) was sufficient to reverse NO-mediated cell cycle arrest or hypophosphorylation of retinoblastoma protein (Rb). We found that the decrease in cyclin D1 levels induced by NO was GSH-sensitive implying that the redox regulation of NO-mediated cytostasis was a multifaceted process and that both p53/p21(cip1/waf1) and p53 independent cyclin D1 pathways were involved. Together, our results demonstrate that GSH serves as an important component of cellular protective mechanisms against NO-derived nitrosative stress to regulate DNA damage checkpoint.