Publisher Correction: An integrated bacterial system for the discovery of chemical rescuers of disease-associated protein misfolding.

In the version of this Article originally published, in Fig. 1c-e, on the x axes, the lines labelled 'Aß42' and 'Aß42(F19S;L34P)' grouped the data incorrectly; the line labelled Aß42 should have grouped the data for Random 1-2 and Clones 1-10, and the line labelled Aß42(F19S;L34P) should have only grouped the data for Random 1-2 on the right end of the plots and blots. These figures have now been corrected in all versions of the Article.

An integrated bacterial system for the discovery of chemical rescuers of disease-associated protein misfolding.

Protein misfolding and aggregation are common pathological features of several human diseases, including Alzheimer's disease and type 2 diabetes. Here, we report an integrated and generalizable bacterial system for the facile discovery of chemical rescuers of disease-associated protein misfolding. In this system, large combinatorial libraries of macrocyclic molecules are biosynthesized in Escherichia coli cells and simultaneously screened for their ability to rescue pathogenic protein misfolding and aggregation using a flow cytometric assay. We demonstrate the effectiveness of this approach by identifying drug-like, head-to-tail cyclic peptides that modulate the aggregation of the Alzheimer's disease-associated amyloid ß peptide. Biochemical, biophysical and biological assays using isolated amyloid ß peptide, primary neurons and various established Alzheimer's disease nematode models showed that the selected macrocycles potently inhibit the formation of neurotoxic amyloid ß peptide aggregates. We also applied the system to the identification of misfolding rescuers of mutant Cu/Zn superoxide dismutase-an enzyme linked with inherited forms of amyotrophic lateral sclerosis. Overall, the system enables the identification of molecules with therapeutic potential for rescuing the misfolding of disease-associated polypeptides.

Prevalence of BRCA1 mutations in familial and sporadic greek ovarian cancer cases.

Germline mutations in the BRCA1 and BRCA2 genes contribute to approximately 18% of hereditary ovarian cancers conferring an estimated lifetime risk from 15% to 50%. A variable incidence of mutations has been reported for these genes in ovarian cancer cases from different populations. In Greece, six mutations in BRCA1 account for 63% of all mutations detected in both BRCA1 and BRCA2 genes. This study aimed to determine the prevalence of BRCA1 mutations in a Greek cohort of 106 familial ovarian cancer patients that had strong family history or metachronous breast cancer and 592 sporadic ovarian cancer cases. All 698 patients were screened for the six recurrent Greek mutations (including founder mutations c.5266dupC, p.G1738R and the three large deletions of exon 20, exons 23-24 and exon 24). In familial cases, the BRCA1 gene was consequently screened for exons 5, 11, 12, 20, 21, 22, 23, 24. A deleterious BRCA1 mutation was found in 43/106 (40.6%) of familial cancer cases and in 27/592 (4.6%) of sporadic cases. The variant of unknown clinical significance p.V1833M was identified in 9/698 patients (1.3%). The majority of BRCA1 carriers (71.2%) presented a high-grade serous phenotype. Identifying a mutation in the BRCA1 gene among breast and/or ovarian cancer families is important, as it enables carriers to take preventive measures. All ovarian cancer patients with a serous phenotype should be considered for genetic testing. Further studies are warranted to determine the prevalence of mutations in the rest of the BRCA1 gene, in the BRCA2 gene, and other novel predisposing genes for breast and ovarian cancer.

A nonsynonymous polymorphism in IRS1 modifies risk of developing breast and ovarian cancers in BRCA1 and ovarian cancer in BRCA2 mutation carriers.

BACKGROUND: We previously reported significant associations between genetic variants in insulin receptor substrate 1 (IRS1) and breast cancer risk in women carrying BRCA1 mutations. The objectives of this study were to investigate whether the IRS1 variants modified ovarian cancer risk and were associated with breast cancer risk in a larger cohort of BRCA1 and BRCA2 mutation carriers. METHODS: IRS1 rs1801123, rs1330645, and rs1801278 were genotyped in samples from 36 centers in the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Data were analyzed by a retrospective cohort approach modeling the associations with breast and ovarian cancer risks simultaneously. Analyses were stratified by BRCA1 and BRCA2 status and mutation class in BRCA1 carriers. RESULTS: Rs1801278 (Gly972Arg) was associated with ovarian cancer risk for both BRCA1 (HR, 1.43; 95% confidence interval (CI), 1.06-1.92; P = 0.019) and BRCA2 mutation carriers (HR, 2.21; 95% CI, 1.39-3.52, P = 0.0008). For BRCA1 mutation carriers, the breast cancer risk was higher in carriers with class II mutations than class I mutations (class II HR, 1.86; 95% CI, 1.28-2.70; class I HR, 0.86; 95%CI, 0.69-1.09; P(difference), 0.0006). Rs13306465 was associated with ovarian cancer risk in BRCA1 class II mutation carriers (HR, 2.42; P = 0.03). CONCLUSION: The IRS1 Gly972Arg single-nucleotide polymorphism, which affects insulin-like growth factor and insulin signaling, modifies ovarian cancer risk in BRCA1 and BRCA2 mutation carriers and breast cancer risk in BRCA1 class II mutation carriers. IMPACT: These findings may prove useful for risk prediction for breast and ovarian cancers in BRCA1 and BRCA2 mutation carriers.

Prevalence of BRCA1 mutations among 403 women with triple-negative breast cancer: implications for genetic screening selection criteria: a Hellenic Cooperative Oncology Group Study.

In spite the close association of the triple-negative breast cancer immunophenotype with hereditary breast cancers and the BRCA1 pathway, there is a lack of population studies that determine the frequency of BRCA1 mutations among triple-negative breast cancer patients. To address this, we have screened a large sample of 403 women diagnosed with triple-negative invasive breast cancer, independently of their age or family history, for germline BRCA1 mutations. Median age at diagnosis was 50 years (range 20-83). The overall prevalence of triple-negative cases among the initial patient group with invasive breast cancer was 8%. BRCA1 was screened by direct DNA sequencing in all patients, including all exons where a mutation was previously found in the Greek population (exons 5, 11, 12, 16, 20, 21, 22, 23, 24-77% of the BRCA1 coding region), including diagnostic PCRs to detect the three Greek founder large genomic rearrangements. Sixty-five deleterious BRCA1 mutations were identified among the 403 triple-negative breast cancer patients (16%). Median age of onset for mutation carriers was 39 years. Among a total of 106 women with early-onset triple-negative breast cancer (<40 years), 38 (36%) had a BRCA1 mutation, while 27% of women with triple-negative breast cancer diagnosed before 50 years (56/208) had a BRCA1 mutation. A mutation was found in 48% (50/105) of the triple-negative breast cancer patients with family history of breast or ovarian cancer. It is noteworthy, however, that of the 65 carriers, 15 (23%) had no reported family history of related cancers. All but one of the carriers had grade III tumors (98%). These results indicate that women with early-onset triple-negative breast cancer, and ideally all triple-negative breast cancer patients, are candidates for BRCA1 genetic testing even in the absence of a family history of breast or ovarian cancer.

The novel molecule 2-[5-(2-chloroethyl)-2-acetoxy-benzyl]-4-(2-chloroethyl)-phenyl acetate inhibits phosphoinositide 3-kinase/Akt/mammalian target of rapamycin signalling through JNK activation in cancer cells.

Screening a compound library of compound 48/80 analogues, we identified 2-[5-(2-chloroethyl)-2-acetoxy-benzyl]-4-(2-chloroethyl)-phenyl acetate (E1) as a novel inhibitor of the phosphoinositide 3-kinase/Akt pathway. In order to determine the mechanism of action of E1, we analysed the effect of E1 on components of the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway. E1 demonstrated dose-dependent and time-dependent repression of Akt and mTOR activity in prostate and breast cancer cell lines, PC-3 and MCF-7, respectively. Inhibition of Akt and mTOR activity by E1 also coincided with increased c-Jun NH2-terminal kinase (JNK) phosphorylation. However, the mode of action of E1 is different from that of the mTOR inhibitor rapamycin. Proliferation and cell cycle analysis revealed that E1 induced cell cycle arrest and cell death in PC-3 and MCF-7 cells. Moreover, pretreatment of cancer cells with the JNK inhibitor SP600125 abolished the repression of Akt and mTOR activity by E1, indicating that the inhibition of Akt and mTOR by E1 is mediated through JNK activation. Consistently, E1 repressed Akt and mTOR activity in wild-type and p38-null mouse embryonic fibroblasts (MEFs), but not in MEFs lacking JNK1/2, and JNK-null MEFs were less sensitive to the antiproliferative effects of E1. We further showed that E1 can function cooperatively with suboptimal concentrations of paclitaxel to induce cell death in PC-3 and MCF-7 cells. Taken together, these data suggest that E1 induces cancer cell death through the JNK-dependent repression of Akt and mTOR activity and may provide a valuable compound for further development and research.

Transcriptional cross talk between the forkhead transcription factor forkhead box O1A and the progesterone receptor coordinates cell cycle regulation and differentiation in human endometrial stromal cells.

Differentiation of human endometrial stromal cells (HESCs) into decidual cells is associated with induction of the forkhead transcription factor forkhead box O1A (FOXO1). We performed a genomic screen to identify decidua-specific genes under FOXO1 control. Primary HESCs were transfected with small interfering RNA targeting FOXO1 or with nontargeting control small interfering RNA before treatment with a cAMP analogue and the progestin, medroxyprogesterone acetate for 72 h. Total RNA was processed for whole genome analysis using high-density oligonucleotide arrays. We identified 3405 significantly regulated genes upon decidualization of HESCs, 507 (15.3%) of which were aberrantly expressed upon FOXO1 knockdown. Among the most up-regulated FOXO1-dependent transcriptional targets were WNT signaling-related genes (WNT4, WNT16 ), the insulin receptor (INSR), differentiation markers (PRL, IGFBP1, and LEFTY2), and the cyclin-dependent kinase inhibitor p57(Kip2) (CDKN1C). Analysis of FOXO1-dependent down-regulated genes uncovered several factors involved in cell cycle regulation, including CCNB1, CCNB2, MCM5, CDC2 and NEK2. Cell viability assay and cell cycle analysis demonstrated that FOXO1 silencing promotes proliferation of differentiating HESCs. Using a glutathione-S-transferase pull-down assay, we confirmed that FOXO1 interacts with progesterone receptor, irrespectively of the presence of ligand. In agreement, knockdown of PR disrupted the regulation of FOXO1 target genes involved in differentiation (IGFBP1, PRL, and WNT4) and cell cycle regulation (CDKN1, CCNB2 and CDC2) in HESCs treated with either cAMP plus medroxyprogesterone acetate or with cAMP alone. Together, the data demonstrate that FOXO1 engages in transcriptional cross talk with progesterone receptor to coordinate cell cycle regulation and differentiation of HESCs.

Role of glycogen synthase kinase 3 beta (GSK3beta) in mediating the cytotoxic effects of the histone deacetylase inhibitor trichostatin A (TSA) in MCF-7 breast cancer cells.

Histone deacetylase inhibitors (HDACIs) have been shown to induce apoptotic and autophagic cell death in vitro and in vivo. The molecular mechanisms that underlie these cytotoxic effects are not yet clearly understood. Recently, HDACIs were shown to induce Akt dephosphorylation by disrupting HDAC-protein phosphatase 1 (PP1) complexes. This disruption results in the increased association of PP1 with Akt, resulting in the dephosphorylation and consequent inactivation of the kinase. Akt enhances cellular survival through the phosphorylation-dependent inhibition of several pro-apoptotic proteins. Akt is an important negative regulator of GSK3beta, a kinase that has been shown to regulate apoptosis in response to various stimuli. In the present study, we investigated the role of GSK3beta in mediating the cytotoxic effects in MCF-7 breast cancer cells treated with trichostatin A (TSA), a prototype HDACI. We show that TSA induces Akt dephosphorylation in a PP1-dependent manner, resulting in activation of GSK3beta in MCF-7 cells. Similarly, knockdown of HDAC1 and-2 by small interfering RNA (siRNA) resulted in the dephosphorylation of Akt and GSK3beta. Selective inhibition of GSK3beta attenuated TSA induced cytotoxicity and resulted in enhanced proliferation following drug removal. Our findings identify GSK3beta as an important mediator of TSA-induced cytotoxicity in MCF-7 breast cancer cells.

In vivo biological activity of the histone deacetylase inhibitor LAQ824 is detectable with 3'-deoxy-3'-[18F]fluorothymidine positron emission tomography.

Histone deacetylase inhibitors (HDACI) are emerging as growth inhibitory compounds that modulate gene expression and inhibit tumor cell proliferation. We assessed whether 3'-deoxy-3'-[(18)F]fluorothymidine-positron emission tomography ([18F]FLT-PET) could be used to noninvasively measure the biological activity of a novel HDACI LAQ824 in vivo. We initially showed that thymidine kinase 1 (TK1; EC2.7.1.21), the enzyme responsible for [18F]FLT retention in cells, was regulated by LAQ824 in a drug concentration-dependent manner in vitro. In HCT116 colon carcinoma xenograft-bearing mice, LAQ824 significantly decreased tumor [18F]FLT uptake in a dose-dependent manner. At day 4 of treatment, [18F]FLT tumor-to-heart ratios at 60 minutes (NUV60) were 2.16 +/- 0.15, 1.86 +/- 0.13, and 1.45 +/- 0.20 in vehicle, and 5 and 25 mg/kg LAQ824 treatment groups, respectively (P < or = 0.05). LAQ825 at 5 mg/kg also significantly reduced both TK1 levels and [18F]FLT uptake at day 10 but not at day 2 (P < or = 0.05). [18F]FLT NUV60 correlated significantly with cellular proliferation (r = 0.68; P = 0.0019) and was associated with drug-induced histone H4 hyperacetylation. Of interest to [18F]FLT-PET imaging, both TK1 mRNA copy numbers and protein levels decreased in the order vehicle >5 mg/kg LAQ824 > 25 mg/kg LAQ824, providing a rationale for the use of [18F]FLT-PET in this setting. We also observed increases in Rb hypophosphorylation and p21 levels, factors that could have contributed to the alteration in TK1 transcription in vivo. In conclusion, we have shown the utility of [18F]FLT-PET for monitoring the biological activity of the HDACI, LAQ824. Drug-induced changes in tumor [18F]FLT uptake were due, at least in part, to reductions in TK1 transcription and translation.

The cyclin D1 proto-oncogene is sequestered in the cytoplasm of mammalian cancer cell lines.

BACKGROUND: The cyclin D1 proto-oncogene is an important regulator of G1 to S-phase transition and an important cofactor for several transcription factors in numerous cell types. Studies on neonatal cardiomyocytes and postmitotic neurons indicate that the activity of cyclin D1 may be regulated through its cytoplasmic sequestration. We have demonstrated previously, that TSA induces the ubiquitin-dependent degradation of cyclin D1 in MCF-7 breast cancer cells. Additional studies were initiated in order to further investigate the effect of TSA on cyclin D1 regulation using sub-cellular fractionation techniques. RESULTS: Our studies revealed cyclin D1 to be localized predominantly within the cytoplasmic fraction of all cell lines tested. These observations were confirmed by confocal microscopy. GSK3beta was found to be localized within both the nucleus and cytoplasm throughout the cell cycle. Inhibition of GSK3beta or CRM1-dependent nuclear export resulted in only modest nuclear accumulation, suggesting that the cytoplasmic localization of cyclin D1 results from the inhibition of its nuclear import. CONCLUSION: We have shown by several different experimental approaches, that cyclin D1 is in fact a predominantly cytoplasmic protein in mammalian cancer cell lines. Recent studies have shown that the cytoplasmic sequestration of cyclin D1 prevents apoptosis in neuronal cells. Our results suggest that cytoplasmic sequestration may additionally serve to regulate cyclin D1 activity in mammalian cancer cells.

Histone deacetylase inhibitor, trichostatin A induces ubiquitin-dependent cyclin D1 degradation in MCF-7 breast cancer cells.

BACKGROUND: Cyclin D1 is an important regulator of G1-S phase cell cycle transition and has been shown to be important for breast cancer development. GSK3beta phosphorylates cyclin D1 on Thr-286, resulting in enhanced ubiquitylation, nuclear export and degradation of the cyclin in the cytoplasm. Recent findings suggest that the development of small-molecule cyclin D1 ablative agents is of clinical relevance. We have previously shown that the histone deacetylase inhibitor trichostatin A (TSA) induces the rapid ubiquitin-dependent degradation of cyclin D1 in MCF-7 breast cancer cells prior to repression of cyclin D1 gene (CCND1) transcription. TSA treatment also resulted in accumulation of polyubiquitylated GFP-cyclin D1 species and reduced levels of the recombinant protein within the nucleus. RESULTS: Here we provide further evidence for TSA-induced ubiquitin-dependent degradation of cyclin D1 and demonstrate that GSK3beta-mediated nuclear export facilitates this activity. Our observations suggest that TSA treatment results in enhanced cyclin D1 degradation via the GSK3beta/CRM1-dependent nuclear export/26S proteasomal degradation pathway in MCF-7 cells. CONCLUSION: We have demonstrated that rapid TSA-induced cyclin D1 degradation in MCF-7 cells requires GSK3beta-mediated Thr-286 phosphorylation and the ubiquitin-dependent 26S proteasome pathway. Drug induced cyclin D1 repression contributes to the inhibition of breast cancer cell proliferation and can sensitize cells to CDK and Akt inhibitors. In addition, anti-cyclin D1 therapy may be highly specific for treating human breast cancer. The development of potent and effective cyclin D1 ablative agents is therefore of clinical relevance. Our findings suggest that HDAC inhibitors may have therapeutic potential as small-molecule cyclin D1 ablative agents.

Histone deacetylase inhibitor trichostatin A represses estrogen receptor alpha-dependent transcription and promotes proteasomal degradation of cyclin D1 in human breast carcinoma cell lines.

PURPOSE: Estrogen receptor alpha (ERalpha)-positive breast cancer cell lines are up to 10 times more sensitive than ERalpha-negative cell lines to the antiproliferative activity of the histone deacetylase inhibitor trichostatin A (TSA). The purpose of the study was to investigate the mechanisms underlying this differential response. EXPERIMENTAL DESIGN AND RESULTS: In the ERalpha-positive MCF-7 cell line, TSA repressed ERalpha and cyclin D1 transcription and induced ubiquitin dependent proteasomal degradation of cyclin D1, leading primarily to G(1)-S-phase cell cycle arrest. By contrast, cyclin D1 degradation was enhanced but its transcription unaffected by TSA in the ERalpha-negative MDA-MB-231 cell line, which arrested in G(2)-M phase. Cyclin D1 degradation involved Skp2/p45, a regulatory component of the Skp1/Cullin/F-box complex; silencing SKP2 gene expression by RNA interference stabilized cyclin D1 and abrogated the cyclin D1 down-regulation response to TSA. CONCLUSIONS: Tamoxifen has been shown to inhibit ERalpha-mediated cyclin D1 transcription, and acquired resistance to tamoxifen is associated with a shift to ERalpha-independent cyclin D1 up-regulation. Taken together, our data show that TSA effectively induces cyclin D1 down-regulation through both ERalpha-dependent and ERalpha-independent mechanisms, providing an important new strategy for combating resistance to antiestrogens.