Downregulation Of E2F1 Research Articles

Sorafenib enhances the chemotherapeutic efficacy of S-1 against hepatocellular carcinoma through downregulation of transcription factor E2F-1

Sorafenib and S-1 (one mixed formulation containing 5-FU prodrug and dihydropyrimidine dehydrogenase inhibitor) were two effective agents against hepatocellular carcinoma (HCC), but whether they had synergistic effects remained unclear. The present study aimed at evaluating their synergistic effects against HCC and its mechanisms. Inhibitory effects of sorafenib, 5-FU and their combination on HCC cells PLC/PRF/5 and SK-HEP-1 were evaluated. Expressions of transcription factor E2F-1 and its downstream thymidylate synthetase (TS) in the treated cells were determined using real-time PCR and Western blot. In vivo anti-tumoral efficacy of S-1 plus sorafenib on HCC was evaluated in NOD/SCID mice. E2F-1 and TS expressions in tumors were determined by immunohistochemical staining. Sorafenib inhibited growth of HCC cells in dose-dependent manner, with IC50 of 5.4 ± 0.3 μmol/L for PLC/PRF/5 and 5.3 ± 0.5 μmol/L for SK-HEP-1. Sorafenib (1 μmol/L) enhanced inhibitory efficacy of 5-FU on HCC cells in vitro, dropping IC50 of 5-FU from 167.7 ± 12.1 to 105.4 ± 8.4 μmol/L for PLC/PRF/5 and 115 ± 10.2 to 82 ± 7.4 μmol/L for SK-HEP-1 (both p < 0.01). Sorafenib downregulated E2F-1 and TS expressions on HCC cells, and its combination with 5-FU yielded a synergistic downregulation of TS expression on HCC cells. In NOD/SCID mice with subcutaneously inoculated HCC, sorafenib combined with S-1 yielded greater inhibition on tumor growth and remarkable TS suppression when compared with sorafenib or S-1 alone (all p < 0.05). Sorafenib enhanced therapeutic efficacy of 5-FU/S-1 against HCC through downregulation of E2F-1 and TS expressions. Sorafenib combined with S-1 might represent as valuable therapeutic regimen against HCC.

Protective effect of nectandrin B, a potent AMPK activator on neointima formation: inhibition of Pin1 expression through AMPK activation

Neointima is considered a critical event in the development of vascular occlusive disease. Nectandrin B from nutmeg functions as a potent AMP-activated protein kinase (AMPK) activators. The present study addressed whether nectandrin B inhibits intimal hyperplasia in guide wire-injured arteries and examined its molecular mechanism. Neointima was induced by guide wire injury in mouse femoral arteries. Cell proliferation and mechanism studies were performed in rat vascular smooth muscle cells (VSMC) culture model. Nectandrin B increased AMPK activity in VSMC. Nectandrin B inhibited the cell proliferation induced by PDGF and DNA synthesis. Moreover, treatment of nectandrin B suppressed neointima formation in femoral artery after guide wire injury. We have recently shown that Pin1 plays a critical role in VSMC proliferation and neointima formation. Nectandrin B potently blocked PDGF-induced Pin1 and cyclin D1 expression and nectandrin B's anti-proliferation effect was diminished in Pin1 overexpressed VSMC. PDGF-induced phosphorylation of ERK and Akt was marginally affected by nectandrin B. However, nectandrin B increased the levels of p53 and its downstream target p21 and, also reversibly decreased the expression of E2F1 and phosphorylated Rb in PDGF-treated VSMC. AMPK inhibition by dominant mutant form of adenovirus rescued nectandrin B-mediated down-regulation of Pin1 and E2F1. Nectandrin B inhibited VSMC proliferation and neointima formation via inhibition of E2F1-dependent Pin1 gene transcription, which is mediated through the activation of an AMPK/p53-triggered pathway.

Syncytin-1 modulates placental trophoblast cell proliferation by promoting G1/S transition

Placental syncytiotrophoblasts formed by the fusion of cytotrophoblasts constitute the interface between maternal and fetal circulations. The syncytium, composed of a continuous layer of syncytiotrophoblasts, assumes the fetal–maternal nutrient exchange, placental barrier, and endocrine functions important for the maintenance of normal pregnancy. Syncytin-1, an endogenous retroviral gene product, mediates the fusion of cytotrophoblasts. While the fusogenic function of syncytin-1 has been well established, little is known regarding its nonfusogenic activities. This study investigates the role of syncytin-1 in trophoblast proliferation. We found that syncytin-1 knockdown significantly inhibited BeWo cell growth and DNA synthesis. Moreover, time course studies on key cell cycle regulators demonstrated an upregulation of p15 and downregulation of CDK4, E2F1, PCNA, and c-Myc, which consequently led to a reduced level of CDK1. These results, together with those from flow cytometry analysis, indicated that syncytin-1 knockdown blocked the G1/S transition phase of the cell cycle. Moreover, syncytin-1 overexpression promoted CHO cell proliferation and led to changes opposite to those observed in syncytin-1 knockdown experiments, confirming the critical role of syncytin-1 for G1/S transition. Thus, syncytin-1, through both nonfusogenic and fusogenic, functions, may co-regulate the input (proliferation) and output (fusion) of the cytotrophoblast “pool”. Such co-regulation could be an efficient way to achieve the balance between these two opposing processes, which is required for syncytium homeostasis. Since decreased syncytin-1 expression has been shown to be associated with preeclamptic and hypoxic condition, insufficient replenishing of the cytotrophoblast “pool” may contribute to syncytium deficiency, a critical pathological change frequently found in preeclamptic placentas.

DNA Damage–Specific Control of Cell Death by Cryptochrome in p53-Mutant Ras–Transformed Cells

The main feedback loop driving circadian rhythm in mice is controlled, in part, by the genes encoding the cryptochromes Cry1 and Cry2. Targeted mutation of both Cry1 and Cry2 delay the early onset of tumor formation in p53-null mutant mice. Furthermore, Ras-transformed p53- and Cry-null mouse skin fibroblasts are more sensitive than p53 mutants to apoptotic cell death initiated by agents that activate either the intrinsic or the extrinsic apoptosis pathways. Here, we investigated the effect of Cry1 and Cry2 mutations on cell death by other genotoxic agents that generate alkylated bases, interstrand crosslinks, DNA-protein crosslinks, and double-strand breaks. Both ultraviolet (UV) and the UV mimetic compound oxaliplatin and the radiomimetic compound doxorubicin promoted apoptosis by upregulating the tumor suppressor p73. However, only the UV and oxaliplatin-induced upregulation of p73 mediated by the transcription factor Egr1, but not the doxorubicin-induced upregulation mediated by the transcription factor E2F1, was enhanced by Cry1/Cry2 double mutation. Accordingly, Egr1 downregulation reduced oxaliplatin-induced apoptosis, whereas E2F1 downregulation reduced doxorubicin-induced apoptosis. Our findings establish distinct roles for cryptochromes in intrinsic apoptosis induced by UV mimetic and radiomimetic agents.

Abstract B56: Activation of Chk1 by Piperine causes cell cycle arrest and apoptosis in melanoma cells

Abstract The National Cancer Institute estimates that about 76,250 new cases of melanomas will be diagnosed in 2012 in the United States leading to at least 9810 related deaths. Therefore, novel strategies to control and treat melanoma are of prime importance. In this study, we determined the cytotoxic effects of piperine, a major constituent of black (Piper nigrum Linn) and long pepper (Piper longum Linn), in melanoma cell lines. Piperine treatment inhibited the growth of SKMEL-28 and B16-F0 cells in a dose and time-dependent manner. The growth inhibitory effects of piperine were mediated by cell cycle arrest of both the cell lines in G1 phase. The G1 arrest by piperine correlated with the down-regulation of cyclin D1 and induction of p21. Furthermore, this growth arrest was associated with DNA damage caused by piperine treatment as indicated by phosphorylation of H2A.X at Ser139, activation of ataxia telangiectasia and Rad3-related protein (ATR) and checkpoint kinase 1 (Chk1). Pretreatment with AZD 7762, a Chk1 inhibitor not only abrogated the activation of Chk1 but also piperine mediated G1 arrest. Furthermore, transfection of cells with Chk1 siRNA completely protected the cells from G1 arrest induced by piperine treatment. The G1 arrest by piperine was also confirmed with the down-regulation of E2F1 and decreased phosphorylation of retinoblastoma protein (Rb), indicating the inhibition of the dynamic transcription complex. In addition, apoptosis induced by piperine was associated with down-regulation of XIAP and Bid (uncleaved) and cleavage of Caspase-3 and PARP. In addition, our results showed that piperine treatment generated ROS in melanoma cells. Blocking ROS generation by Tiron prevented the cells from piperine mediated cell cycle arrest. Taken together, our results demonstrate that activation of Chk1 is required for piperine induced G1 phase cell cycle arrest. [Supported in part by R01 grants CA 106953 and CA 129038 awarded to S.K.S by National Cancer Institute, NIH] Citation Format: Neel Fofaria, Sanjay K. Srivastava. Activation of Chk1 by Piperine causes cell cycle arrest and apoptosis in melanoma cells. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B56.

Role of transcriptional and posttranscriptional regulation of methionine adenosyltransferases in liver cancer progression

Down-regulation of the liver-specific MAT1A gene, encoding S-adenosylmethionine (SAM) synthesizing isozymes MATI/III, and up-regulation of widely expressed MAT2A, encoding MATII isozyme, known as MAT1A:MAT2A switch, occurs in hepatocellular carcinoma (HCC). Here we found Mat1A:Mat2A switch and low SAM levels, associated with CpG hypermethylation and histone H4 deacetylation of Mat1A promoter, and prevalent CpG hypomethylation and histone H4 acetylation in Mat2A promoter of fast-growing HCC of F344 rats, genetically susceptible to hepatocarcinogenesis. In HCC of genetically resistant BN rats, very low changes in the Mat1A:Mat2A ratio, CpG methylation, and histone H4 acetylation occurred. The highest MAT1A promoter hypermethylation and MAT2A promoter hypomethylation occurred in human HCC with poorer prognosis. Furthermore, levels of AUF1 protein, which destabilizes MAT1A messenger RNA (mRNA), Mat1A-AUF1 ribonucleoprotein, HuR protein, which stabilizes MAT2A mRNA, and Mat2A-HuR ribonucleoprotein sharply increased in F344 and human HCC, and underwent low/no increase in BN HCC. In human HCC, Mat1A:MAT2A expression and MATI/III:MATII activity ratios correlated negatively with cell proliferation and genomic instability, and positively with apoptosis and DNA methylation. Noticeably, the MATI/III:MATII ratio strongly predicted patient survival length. Forced MAT1A overexpression in HepG2 and HuH7 cells led to a rise in the SAM level, decreased cell proliferation, increased apoptosis, down-regulation of Cyclin D1, E2F1, IKK, NF-κB, and antiapoptotic BCL2 and XIAP genes, and up-regulation of BAX and BAK proapoptotic genes. In conclusion, we found for the first time a post-transcriptional regulation of MAT1A and MAT2A by AUF1 and HuR in HCC. Low MATI/III:MATII ratio is a prognostic marker that contributes to determine a phenotype susceptible to HCC and patients' survival. Interference with cell cycle progression and I-kappa B kinase (IKK)/nuclear factor kappa B (NF-κB) signaling contributes to the antiproliferative and proapoptotic effect of high SAM levels in HCC.

FTY720 induces cell cycle arrest and apoptosis of rat glomerular mesangial cells

The present study aimed to examine the effect of FTY720, a new immunosuppressive agent, on the proliferation and apoptosis of glomerular mesangial cells (GMC), and investigate the underlying mechanisms. Cultured rat GMC were treated by FTY720, and the cell viability, apoptosis and cell cycle progression were examined. Furthermore, cell cycle related gene expression profile was analyzed by cDNA microarray, and the protein expression of cell cycle related genes as well as Bax and Bcl-2 were examined by Western blot. The results showed that FTY720 inhibited GMC proliferation and induced apoptosis of GMC in a dose- and time-dependent manner, and induced G(1) phase cell cycle arrest in GMC in a dose-dependent manner as well. cDNA microarray analysis revealed that FTY720 regulated the expression of cell cycle-related gene. Western blot analysis showed that FTY720 induced the downregulation of cyclin D1, cyclin E, CDK2, CDK4, Bcl-2 and E2F1 and the upregulation of Kip1/p27, Cip1/p21, Bax and Rb in GMC in a dose-dependent manner. These results demonstrated that FTY720 could inhibit the proliferation of GMC through inducing cell cycle arrest and apoptosis, probably via the regulation of the expression of cell cycle-related genes and Bax/Bcl-2.

Altered MicroRNA Expression Profile in Human Pituitary GH Adenomas: Down-Regulation of miRNA Targeting HMGA1, HMGA2, and E2F1

MicroRNA (miRNA) are an important class of regulators of gene expression. Altered miRNA expression has been constantly found in human neoplasias and plays an important role in the process of carcinogenesis. The aim of this study was to identify specific miRNA whose expression is altered in GH-secreting pituitary adenomas. Using a miRNACHIP microarray, we have analyzed the miRNA expression profile of human GH adenomas vs. normal pituitary gland. We report the identification of a set of miRNA, including miR-34b, miR-326, miR-432, miR-548c-3p, miR-570, and miR-603, drastically and constantly down-regulated in GH adenomas. We demonstrate that these miRNA target genes such as high-mobility group A1 (HMGA1), HMGA2, and E2F1, whose overexpression and/or activation plays a critical role in pituitary tumorigenesis. We also show that the enforced expression of the down-regulated miRNA has a negative role on the growth regulation of pituitary adenoma cells. Finally, an inverse correlation is found between the expression of these miRNA and HMGA1 and HMGA2 protein levels in GH adenomas. Our study identifies a specific subset of miRNA, whose down-regulation might contribute to pituitary tumorigenesis.

Abstract 4208: Post-translational modification of E2F1 in malignant melanoma

Abstract Malignant melanoma has the highest fatality among all skin cancers. No current treatment significantly enhances survival once metastasis is established. Previous studies have shown that E2F1, a transcription factor, is over-expressed in melanoma cells. However, the mechanism through which E2F1 is elevated remains unknown. E2F1 is known to be involved in many important biological processes, including cell cycle progression, DNA damage, DNA repair, differentiation, development, autophagy and apoptosis. This underscores the importance of E2F1 in carcinogenesis. Normally in late S phase, E2F1 is recognized by the E3 ligase p45 SKP2 and undergoes ubiquitination and finally degradation in 26S proteasome. Our overall hypothesis is that E2F1 escapes proteasomal degradation in melanoma cells due to changes in post-translational modification. In this study we have used melanoma cells of increasing disease progression. Our results show that although the primary melanoma cells have high level of E2F1 message and protein, it is unstable. On the other hand the more metastatic melanoma cells have low levels of E2F1 which is however very stable. Consistent with this result, post-translational modification of E2F1 including the phosphorylation at Serine 337 and the acetylation at Lysine 117 increase with melanoma progression, which suggests that these post-translational modification sites might play an important role in stabilization of E2F1 and keep it functionally active in the more metastatic cell lines. E2F1 knockdown data also shows that E2F1 KD can inhibit cell proliferation in early stage melanoma cells but not in the more metastatic cells, which also suggest that inhibition of post-translational modifications and stability of E2F1 may be a more effective way to inhibit melanoma progression to more advanced stages. Abrogation of acetylation is expected to cause E2F1 protein instability. On the other hand, if acetylation is potentiated, the stabilized E2F1 will continue to promote progression through the cell cycle. We are currently testing whether the ectopic expression of the continuously acetylated E2F1 protein can rescue proliferation inhibition caused by E2F1 down-regulation, and drive metastasis and a mutant E2F1 with abrogated acetylation can block rescue of the proliferation inhibition in the E2F1 down-regulated melanoma cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4208. doi:1538-7445.AM2012-4208

Selective inhibition of rRNA transcription downregulates E2F-1: a new p53-independent mechanism linking cell growth to cell proliferation

The tumour suppressor p53 negatively controls cell cycle progression in response to perturbed ribosome biogenesis in mammalian cells, thus coordinating growth with proliferation. Unlike mammalian cells, p53 is not involved in the growth control of proliferation in yeasts and flies. We investigated whether a p53-independent mechanism of response to inadequate ribosome biogenesis rate is also present in mammalian cells. We studied the effect of specific inhibition of rRNA synthesis on cell cycle progression in human cancer cell lines using the small-interfering RNA procedure to silence the POLR1A gene, which encodes the catalytic subunit of RNA polymerase I. We found that interference of POLR1A inhibited the synthesis of rRNA and hindered cell cycle progression in cells with inactivated p53, as a consequence of downregulation of the transcription factor E2F-1. Downregulation of E2F-1 was due to release of the ribosomal protein L11, which inactivated the E2F-1-stabilising function of the E3 ubiquitin protein ligase MDM2. These results demonstrated the existence of a p53-independent mechanism that links cell growth to cell proliferation in mammalian cells, and suggested that selective targeting of the RNA polymerase I transcription machinery might be advisable to hinder proliferation of p53-deficient cancer cells.

Selective inhibition of rRNA transcription downregulates E2F-1: a new p53-independent mechanism linking cell growth to cell proliferation

The tumour suppressor p53 negatively controls cell cycle progression in response to perturbed ribosome biogenesis in mammalian cells, thus coordinating growth with proliferation. Unlike mammalian cells, p53 is not involved in the growth control of proliferation in yeasts and flies. We investigated whether a p53-independent mechanism of response to inadequate ribosome biogenesis rate is also present in mammalian cells. We studied the effect of specific inhibition of rRNA synthesis on cell cycle progression in human cancer cell lines using the small-interfering RNA procedure to silence the POLR1A gene, which encodes the catalytic subunit of RNA polymerase I. We found that interference of POLR1A inhibited the synthesis of rRNA and hindered cell cycle progression in cells with inactivated p53, as a consequence of downregulation of the transcription factor E2F-1. Downregulation of E2F-1 was due to release of the ribosomal protein L11, which inactivated the E2F-1-stabilising function of the E3 ubiquitin protein ligase MDM2. These results demonstrated the existence of a p53-independent mechanism that links cell growth to cell proliferation in mammalian cells, and suggested that selective targeting of the RNA polymerase I transcription machinery might be advisable to hinder proliferation of p53-deficient cancer cells.

ATM and p53 Regulate FOXM1 Expression via E2F in Breast Cancer Epirubicin Treatment and Resistance

In this report, we investigated the role and regulation of forkhead box M1 (FOXM1) in breast cancer and epirubicin resistance. We generated epirubicin-resistant MCF-7 breast carcinoma (MCF-7-EPI(R)) cells and found FOXM1 protein levels to be higher in MCF-7-EPI(R) than in MCF-7 cells and that FOXM1 expression is downregulated by epirubicin in MCF-7 but not in MCF-7-EPI(R) cells. We also established that there is a loss of p53 function in MCF-7-EPI(R) cells and that epirubicin represses FOXM1 expression at transcription and gene promoter levels through activation of p53 and repression of E2F activity in MCF-7 cells. Using p53(-/-) mouse embryo fibroblasts, we showed that p53 is important for epirubicin sensitivity. Moreover, transient promoter transfection assays showed that epirubicin and its cellular effectors p53 and E2F1 modulate FOXM1 transcription through an E2F-binding site located within the proximal promoter region. Chromatin immunoprecipitation analysis also revealed that epirubicin treatment increases pRB (retinoblastoma protein) and decreases E2F1 recruitment to the FOXM1 promoter region containing the E2F site. We also found ataxia-telangiectasia mutated (ATM) protein and mRNA to be overexpressed in the resistant MCF-7-EPI(R) cells compared with MCF-7 cells and that epirubicin could activate ATM to promote E2F activity and FOXM1 expression. Furthermore, inhibition of ATM in U2OS cells with caffeine or depletion of ATM in MCF-7-EPI(R) with short interfering RNAs can resensitize these resistant cells to epirubicin, resulting in downregulation of E2F1 and FOXM1 expression and cell death. In summary, our data show that ATM and p53 coordinately regulate FOXM1 via E2F to modulate epirubicin response and resistance in breast cancer.

Analysis of Let-7a and Mir-17-5p Micro-RNAs Expression In Patients with Adult De Novo Myelodysplastic Syndromes

AbstractAbstract 4965Introduction. MicroRNAs are small non-coding RNAs that act at the post-transcriptional level, regulating protein expression by repressing translation or destabilizing mRNA target. Since their discovery, microRNAs have been associated with almost every normal cell function, including proliferation, differentiation and apoptosis. Several studies suggest that they have an important role in normal hematopoiesis and hematological malignancies. Let-7a negatively regulates the expression of Ras proteins, which are overexpressed in patients with Myelodysplastic syndromes (MDS). In addition mir-17-5p contributes to the down-regulation of E2F1, which is overexpressed in MDS. Purpose. The purpose of the current study was to evaluate the expression of let-7a and mir-17-5p in CD34 cells from the bone marrow of patients with MDS. Material and methods. We evaluated 29 patients with MDS (25 men, 4 women) with median age 73 years. FAB classification was as follows: 9 RA, 15 RAEB, 2 RAEB-t and 3 AML. According to IPSS our study included 9 patients with low, 8 with Int-1, 7 with Int-2 and 5 with high risk disease. We isolated CD34+ cells from bone marrow of patients using magnetic beads. Extraction of microRNA and total RNA was performed and cDNA of let-7a and mir-17-5p was amplified using real time PCR, to study the relative expression of these microRNAS according to the expression of RNU48 (a snoRNA used as control). The control group included donors of CD34+ cells for stem cell transplantation. Results. The median miR17-5p expression level in patients was lower compared to controls [23vs 47.4]. The median let-7a expression levels were higher in MDS patients than in healthy donors [32.85 vs 8]. However these differences were not significant as shown by the Mann-Whitney U test. Moreover, ROC analysis demonstrated that neither miR17-5p nor let-7a expression had significant discriminatory value to efficiently distinguish MDS patients from non-MDS. In addition Spearman analysis did not reveal any correlations between these two microRNA expression levels and other continuous variables examined in the current study (age, hemoglobin concentration, numbers of neutrophils, platelets, and BM blasts). Finally, we found no associations between either miR17-5p or let-7a expression and clinicopathological parameters of MDS patients using chi-square (χ2) or Fisher's exact test where appropriate.Conclusion. The micro RNA let-7 was overexressed although not significantly in CD34 cells from MDS patients demonstrating that this is not a mechanism contributing to the increased expression RAS proteins in MDS. The micro RNA-mir17-5 is underexpressed in CD34 cells and needs further investigation for the establishment of a role in the overepxression of E2F in MDS. The lack of significant differences in the expression levels of both micro RNAs could be related to the admixture of normal and dysplastic CD34 cells in the bone marrow of MDS or to the small sample size. Disclosures:No relevant conflicts of interest to declare.

Knockdown of Inwardly Rectifying Potassium Channel Kir2.2 Suppresses Tumorigenesis by Inducing Reactive Oxygen Species–Mediated Cellular Senescence

Senescence is an important determinant of treatment outcome in cancer therapy. In the present study, we show that knockdown of the inwardly rectifying K(+) channel Kir2.2 induced growth arrest without additional cellular stress in cancer cells lacking functional p53, p16, and/or Rb. Kir2.2 knockdown also induced senescence-associated β-galactosidase activity and upregulated senescence marker proteins in multiple cancer cell lines derived from different tissues, including prostate, stomach, and breast. Interestingly, knockdown of Kir2.2 induced a significant increase in reactive oxygen species (ROS) that was accompanied by cell cycle arrest, characterized by significant upregulation of p27, with concomitant downregulation of cyclinA, cdc2, and E2F1. Kir2.2 knockdown cells displayed increased levels of PML bodies, DNA damage (γH2AX) foci, senescence-associated heterochromatin foci, mitochondrial dysfunction, secretory phenotype, and phosphatase inactivation. Conversely, overexpression of Kir2.2 decreased doxorubicin-induced ROS accumulation and cell growth inhibition. Kir2.2 knockdown-induced cellular senescence was blocked by N-acetylcysteine, indicating that ROS is a critical mediator of this pathway. In vivo tumorigenesis analyses revealed that tumors derived from Kir2.2 knockdown cells were significantly smaller than those derived from control cells (P < 0.0001) and showed a remarkable increase in senescence-associated proteins, including senescence-associated β-galactosidase, p27, and plasminogen activator inhibitor-1. Moreover, the preestablished tumors are reduced in size after the injection of siKir2.2 (P = 0.0095). Therefore, we propose for the first time that Kir2.2 knockdown induces senescence of cancer cells by a mechanism involving ROS accumulation that requires p27, but not Rb, p53, or p16.

The RB-E2F1 Pathway Regulates Autophagy

Autophagy is a protective mechanism that renders cells viable in stressful conditions. Emerging evidence suggests that this cellular process is also a tumor suppressor pathway. Previous studies showed that cyclin-dependent kinase inhibitors (CDKI) induce autophagy. Whether retinoblastoma protein (RB), a key tumor suppressor and downstream target of CDKIs, induces autophagy is not clear. Here, we show that RB triggers autophagy and that the RB activators p16INK4a and p27/kip1 induce autophagy in an RB-dependent manner. RB binding to E2 transcription factor (E2F) is required for autophagy induction and E2F1 antagonizes RB-induced autophagy, leading to apoptosis. Downregulation of E2F1 in cells results in high levels of autophagy. Our findings indicate that RB induces autophagy by repressing E2F1 activity. We speculate that this newly discovered aspect of RB function is relevant to cancer development and therapy.

Suppression of A549 lung cancer cell migration by precursor let-7g microRNA

Let-7g miRNAs, short non-coding RNAs approximately 21 nucleotides long, repress protein translation by binding to the 3'UTR of target mRNAs. Aberrant expression of let-7g is associated with the poor prognosis of lung cancer patients. Compared to normal lung cells, let-7g expression is absent in non-small cell lung cancer (NSCLC) cells. Furthermore, K-Ras and HMGA2 are well known as targets of let-7g. In this study, we evaluated the potential role of precursor (pre)-let-7g in lung cancer cell metastasis, focusing on the two targets of let-7g, HMGA2 and K-Ras. We found that pre-let-7g inhibited the migration of A549 lung cancer cells through HMGA2-mediated E2F1 down-regulation. Thus, our results suggest that pre-let-7g could be used as a suitable target for the suppression of lung cancer cell migration.

Mouse Prkar1a haploinsufficiency leads to an increase in tumors in the Trp53+/− or Rb1+/− backgrounds and chemically induced skin papillomas by dysregulation of the cell cycle and Wnt signaling

PRKAR1A inactivation leads to dysregulated cAMP signaling and Carney complex (CNC) in humans, a syndrome associated with skin, endocrine and other tumors. The CNC phenotype is not easily explained by the ubiquitous cAMP signaling defect; furthermore, Prkar1a(+/-) mice did not develop skin and other CNC tumors. To identify whether a Prkar1a defect is truly a generic but weak tumorigenic signal that depends on tissue-specific or other factors, we investigated Prkar1a(+/-) mice when bred within the Rb1(+/-) or Trp53(+/-) backgrounds, or treated with a two-step skin carcinogenesis protocol. Prkar1a(+/-) Trp53(+/-) mice developed more sarcomas than Trp53(+/-) mice (P < 0.05) and Prkar1a(+/-) Rb1(+/-) mice grew more (and larger) pituitary and thyroid tumors than Rb1(+/-) mice. All mice with double heterozygosity had significantly reduced life-spans compared with their single-heterozygous counterparts. Prkar1a(+/-) mice also developed more papillomas than wild-type animals. A whole-genome transcriptome profiling of tumors produced by all three models identified Wnt signaling as the main pathway activated by abnormal cAMP signaling, along with cell cycle abnormalities; all changes were confirmed by qRT-PCR array and immunohistochemistry. siRNA down-regulation of Ctnnb1, E2f1 or Cdk4 inhibited proliferation of human adrenal cells bearing a PRKAR1A-inactivating mutation and Prkar1a(+/-) mouse embryonic fibroblasts and arrested both cell lines at the G0/G1 phase of the cell cycle. In conclusion, Prkar1a haploinsufficiency is a relatively weak tumorigenic signal that can act synergistically with other tumor suppressor gene defects or chemicals to induce tumors, mostly through Wnt-signaling activation and cell cycle dysregulation, consistent with studies in human neoplasms carrying PRKAR1A defects.

BMS-214662 induces mitochondrial apoptosis in chronic myeloid leukemia (CML) stem/progenitor cells, including CD34+38− cells, through activation of protein kinase Cβ

AbstractChronic myeloid leukemia (CML) is a hematopoietic stem cell disorder maintained by cancer stem cells. To target this population, we investigated the mechanism of action of BMS-214662, developed as a farnesyl transferase inhibitor (FTI) and unique in inducing apoptosis in these cells. By contrast, a related congener and equally effective FTI, BMS-225975 does not induce apoptosis, indicating a novel mechanism of action. BMS-214662 significantly and selectively induced apoptosis in primitive CD34+38− CML compared with normal cells. Apoptosis proceeded via the intrinsic pathway: Bax conformational changes, loss of mitochondrial membrane potential, generation of reactive oxygen species, release of cytochrome c, and caspase-9/3 activation were noted. Up-regulation of protein kinase Cβ (PKCβ), down-regulation of E2F1, and phosphorylation of cyclin A–associated cyclin-dependent kinase 2 preceded these changes. Cotreatment of CML CD34+ and CD34+38− cells with PKC modulators, bryostatin-1, or hispidin markedly decreased these early events and the subsequent apoptosis. None of these events was elicited by BMS-214662 in normal CD34+ cells or by BMS-225975 in CML CD34+ cells. These data suggest that BMS-214662 selectively elicits a latent apoptotic pathway in CML stem cells that is initiated by up-regulation of PKCβ and mediated by Bax activation, providing a molecular framework for development of novel therapeutics.

Noncatalytic Function of ERK1/2 Can Promote Raf/MEK/ERK-mediated Growth Arrest Signaling

Kinase activity is known as the key biochemical property of MAPKs. Here, we report that ERK1/2 also utilizes its noncatalytic function to mediate certain signal transductions. Sustained activation of the Raf/MEK/ERK pathway induces growth arrest, accompanied by changes in cell cycle regulators (decreased retinoblastoma phosphorylation, E2F1 down-regulation, and/or p21(CIP1) up-regulation) and cell type-specific changes in morphology and expression of c-Myc or RET in the human tumor lines LNCaP, U251, and TT. Ablation of ERK1/2 by RNA interference abrogated all these effects. However, active site-disabled ERK mutants (ERK1-K71R, ERK2-K52R, and ERK2-D147A), which competitively inhibit activation of endogenous ERK1/2, could not block Raf/MEK-induced growth arrest as well as changes in the cell cycle regulators, although they effectively blocked phosphorylation of the ERK1/2 catalytic activity readouts, p90(RSK) and ELK1, as well as the cell type-specific changes. Because this indicated a potential noncatalytic ERK1/2 function, we generated stable lines of the tumor cells in which both ERK1 and ERK2 were significantly knocked down, and we further investigated the possibility using rat-derived kinase-deficient ERK mutants (ERK2-K52R and ERK2-T183A/Y185F) that were not targeted by human small hairpin RNA. Indeed, ERK2-K52R selectively restored Raf-induced growth inhibitory signaling in ERK1/2-depleted cells, as manifested by regained cellular ability to undergo growth arrest and to control the cell cycle regulators without affecting c-Myc and morphology. However, ERK2-T183A/Y185F was less effective, indicating the requirement of TEY site phosphorylation. Our study suggests that functions of ERK1/2 other than its "canonical" kinase activity are also involved in the pathway-mediated growth arrest signaling.

Blockade of Wnt Signaling Inhibits Angiogenesis and Tumor Growth in Hepatocellular Carcinoma

Aberrant activation of Wnt signaling plays an important role in hepatocarcinogenesis. In addition to direct effects on tumor cells, Wnt signaling might be involved in the organization of tumor microenvironment. In this study, we have explored whether Wnt signaling blockade by exogenous expression of Wnt antagonists could inhibit tumor angiogenesis and control tumor growth. Human Wnt inhibitory factor 1 (WIF1) and secreted frizzled-related protein 1 (sFRP1) were each fused with Fc fragment of human IgG1 to construct fusion proteins WIF1-Fc and sFRP1-Fc. The recombinant adenoviral vectors carrying WIF1-Fc and sFRP1-Fc driven by cytomegalovirus promoter were constructed. Ad-WIF1-Fc or Ad-sFRP1-Fc induced the expression and correct conformation of WIF1-Fc and sFRP1-Fc fusion proteins. These molecules caused down-regulation of E2F1, cyclin D1, and c-myc and promoted cell apoptosis in hepatocellular carcinoma cells. Treatment of established hepatocellular carcinoma tumors with Ad-WIF1-Fc and/or Ad-sFRP1-Fc resulted in significant inhibition of tumor growth and prolonged animal survival. The antineoplastic effect was associated with increased apoptosis of tumor cells, reduced microvessel density, and decreased expression of vascular endothelial growth factor and stromal cell-derived factor-1. Tube formation and migration of human microvascular endothelial cells and mouse endothelial progenitor cells (EPC) were significantly inhibited by both WIF1-Fc and sFRP1-Fc. In addition, these molecules blocked EPC differentiation and caused EPC apoptosis. Our data indicate that Wnt antagonists WIF1-Fc and sFRP1-Fc inhibit Wnt signaling and exert potent antitumor activity by increasing the apoptosis rate in tumor cells and by impairing tumor vascularization.