Inhibition of AKT with the Orally Active Allosteric AKT Inhibitor, MK-2206, Sensitizes Endometrial Cancer Cells to Progestin

Progestin resistance is a major obstacle to treating early stage, well-differentiated endometrial cancer as well as recurrent endometrial cancer. The mechanism behind the suboptimal response to progestin is not well understood. The PTEN tumor suppressor gene is frequently mutated in type I endometrial cancers and this mutation results in hyperactivation of the PI3K/AKT pathway. We hypothesized that increased activation of AKT promotes an inadequate response to progestins in endometrial cancer cells. Ishikawa cells stably transfected with progesterone receptor B (PRB23 cells) were treated with the AKT inhibitor, MK-2206, which effectively decreased levels of p(Ser473)-AKT in a dose-dependent (10 nM to 1 uM) and time-dependent manner (0.5 h to 24 h). MK-2206 inhibited levels of p(Thr308)-AKT and a downstream target, p(Thr246)-PRAS40, but did not change levels of p(Thr202/Tyr204)ERK or p(Thr13/Tyr185)SAPK/JNK, demonstrating specificity of MK-2206 for AKT. Additionally, MK-2206 treatment of PRB23 cells resulted in a significant increase in levels of progesterone receptor B (PRB) protein. Microarray analysis of PRB23 cells identified PDK4 as the most highly upregulated gene among 70 upregulated genes in response to R5020. Inhibition of AKT further upregulated progestin-mediated expression of PDK4 but did not affect another progestin-responsive gene, SGK1. Treatment of PRB23 cells with R5020 and MK-2206 independently decreased viability of cells while the combination of R5020 and MK-2206 caused the greatest decrease in cell viability. Furthermore, mice with xenografted tumors treated with MK-2206 alone or with progesterone alone exhibited modest reductions in their tumor volume. The largest decrease in tumor size was observed in the mice treated with both MK-2206 and progesterone; these tumors exhibited the least proliferation (Ki67) and the most apoptosis (cleaved caspase-3) of all the treatment groups. In summary, inhibition of AKT stabilizes the Progesterone Receptor B and augments progesterone response in endometrial cancer cells that have hyperactivated AKT.     

Phase I clinical trial of systemically administered TUSC2(FUS1)-nanoparticles mediating functional gene transfer in humans

Background

Tumor suppressor gene TUSC2/FUS1 (TUSC2) is frequently inactivated early in lung cancer development. TUSC2 mediates apoptosis in cancer cells but not normal cells by upregulation of the intrinsic apoptotic pathway. No drug strategies currently exist targeting loss-of–function genetic abnormalities. We report the first in-human systemic gene therapy clinical trial of tumor suppressor gene TUSC2.

Methods

Patients with recurrent and/or metastatic lung cancer previously treated with platinum-based chemotherapy were treated with escalating doses of intravenous N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP):cholesterol nanoparticles encapsulating a TUSC2 expression plasmid (DOTAP:chol-TUSC2) every 3 weeks.

Results

Thirty-one patients were treated at 6 dose levels (range 0.01 to 0.09 milligrams per kilogram). The MTD was determined to be 0.06 mg/kg. Five patients achieved stable disease (2.6–10.8 months, including 2 minor responses). One patient had a metabolic response on positron emission tomography (PET) imaging. RT-PCR analysis detected TUSC2 plasmid expression in 7 of 8 post-treatment tumor specimens but not in pretreatment specimens and peripheral blood lymphocyte controls. Proximity ligation assay, performed on paired biopsies from 3 patients, demonstrated low background TUSC2 protein staining in pretreatment tissues compared with intense (10–25 fold increase) TUSC2 protein staining in post-treatment tissues. RT-PCR gene expression profiling analysis of apoptotic pathway genes in two patients with high post-treatment levels of TUSC2 mRNA and protein showed significant post-treatment changes in the intrinsic apoptotic pathway. Twenty-nine genes of the 82 tested in the apoptosis array were identified by Igenuity Pathway Analysis to be significantly altered post-treatment in both patients (Pearson correlation coefficient 0.519; p<0.01).

Conclusions

DOTAP:chol-TUSC2 can be safely administered intravenously in lung cancer patients and results in uptake of the gene by human primary and metastatic tumors, transgene and gene product expression, specific alterations in TUSC2-regulated pathways, and anti-tumor effects (to our knowledge for the first time for systemic DOTAP:cholesterol nanoparticle gene therapy).

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00059605","term_id":"NCT00059605"}}NCT00059605     

Integrin-binding Protein Nischarin Interacts with Tumor Suppressor Liver Kinase B1 (LKB1) to Regulate Cell Migration of Breast Epithelial Cells

Biallelic inactivation of LKB1, a serine/threonine kinase, has been detected in 30% of lung adenocarcinomas, and inhibition of breast tumor growth has been demonstrated. We have identified the tumor suppressor, Nischarin, as a novel binding partner of LKB1. Our mapping analysis shows that the N terminus of Nischarin interacts with amino acids 44–436 of LKB1. Time lapse microscopy and Transwell migration data show that the absence of both Nischarin and LKB1 from an invasive breast cancer cell line (MDA-MB-231) enhances migration as measured by increased distance and speed of migrating cells. Our data suggest that this is a result of elevated PAK1 and LIMK1 phosphorylation. Moreover, the absence of Nischarin and LKB1 increased tumor growth in vivo. Consistent with this, the percentage of S phase cells was increased, as demonstrated by flow cytometry and enhanced cyclin D1. The absence of Nischarin and LKB1 also led to a dramatic increase in the formation of lung metastases. Our studies, for the first time, demonstrate functional interaction between LKB1 and Nischarin to inhibit cell migration and breast tumor progression. Mechanistically, we show that these two proteins together regulate PAK-LIMK-Cofilin and cyclin D1/CDK4 pathways.     

A Novel Aurora-A Inhibitor,BPR1K0609S1, Sensitizes Colorectal Tumor cells to 5-Fluorofracil (5-FU) Treatment

Small synthetic compounds have been implicated in treatment of human cancers. We have synthesized a small compound, BPR1K0609S1 (hereafter, BP), which inhibits Aurora-A kinase. In the present study, we studied the mechanism of BP suppression of tumorigenesis induced by Aurora-A. Given our previous results that inactivation of p53 accelerates MMTV-Aurora-A-mediated tumorigenesis in vivo, we studied the roles of p53 pathway using the isogenic human colon carcinoma cell lines of HCT116, in which p53, Puma, Bax, p21 or Chk2 is deleted. When these isogenic cell lines are treated with BP for 48 h, accumulation of G2M phase and aneuploidy are commonly observed, and HCT116 p21(-) cells show increase in apoptosis. In xenograft assay, s.c. injection of BP efficiently inhibits tumorigenesis of HCT116 deficient for Chk2 or p21. Re-transplantation of BP-resistant tumors indicates that these resistant cells do not acquire advanced tumor growth. Significantly, 5-FU (5-fluorouracil) treatment further induces apoptosis of BP-resistant HCT116 deficient for Chk2 or Puma. These results demonstrate that p21 deficiency enhances BP-mediated suppression of tumor growth, and that BP and 5-FU can collaborate for tumor regression.     

The Deubiquitinase Inhibitor PR-619 Sensitizes Normal Human Fibroblasts to Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-mediated Cell Death

TNF-related apoptosis-inducing ligand (TRAIL) is a potential cancer therapy that selectively targets cancer cell death while non-malignant cells remain viable. Using a panel of normal human fibroblasts, we characterized molecular differences in human foreskin fibroblasts and WI-38 TRAIL-resistant cells and marginally sensitive MRC-5 cells compared with TRAIL-sensitive human lung and colon cancer cells. We identified decreased caspase-8 protein expression and protein stability in normal fibroblasts compared with cancer cells. Additionally, normal fibroblasts had incomplete TRAIL-induced caspase-8 activation compared with cancer cells. We found that normal fibroblasts lack the ubiquitin modification of caspase-8 required for complete caspase-8 activation. Treatment with the deubiquitinase inhibitor PR-619 increased caspase-8 ubiquitination and caspase-8 enzymatic activity and sensitized normal fibroblasts to TRAIL-mediated apoptosis. Therefore, posttranslational regulation of caspase-8 confers resistance to TRAIL-induced cell death in normal cells through blockade of initiation of the extrinsic cell death pathway.     

Methylation of the Putative Tumor Suppressor Gene RASSF1A in Primary Cervical Tumors

Methylation-sensitive restriction endonuclease analysis (MSRA) followed by polymerase chain reaction (PCR) have been used to estimate the methylation level of 13 CpG dinucleotides in the promoter region of the putative suppressor gene RASSF1A (3p21.31) in squamous cell carcinomas of the uterine cervix (SCCs) carrying human papillomavirus (HPV) types 16, 18, and related types. Methylation of 3 to 13 CpG pairs has been found in 64% (25 out of 39) tumor DNA samples, 22% (2 out of 9) DNA samples from morphologically normal tissues adjacent to the tumor (P = 0.0306), and two out of three DNA samples from peripheral blood leukocytes of carcinoma patients. These CpG pairs are not methylated in the DNA of leukocytes of healthy donors (0 out of 10). The methylation level of the RASSF1A promoter region studied in tumors of the patients with regional lymph node metastases is significantly higher than in tumors of the patient that have no metastases (P = 8.5 × 10^–12). The methylation frequency of gene RASSF1A is two times higher than the frequency of hemi- and homozygous deletions in the chromosome 3 region where the gene is located. The data obtained indicate that methylation is one of the main mechanisms of the RASSF1A gene inactivation in HPV-positive human cervical tumors. The methylation of this gene may be an early event in the genesis of cervical tumors, the methylation level increasing with tumor progression.     

Evidence for a Tumor Suppressor Role for the Large Tumor Suppressor Genes LATS1 and LATS2 in Human Cancer

The role of Large tumor suppressor LATS/Warts in human cancer is not clearly understood. Here we show that hLATS1/2 cancer mutations affect their expression and kinase activity. hLATS1/2 mutants exhibit a decreased activity in inhibiting YAP and tissue growth. Therefore, hLATS1/2 alleles from human cancer can be loss-of-function mutations.     

人类抑癌基因beclin 1在胃癌和直结肠癌中表达下调的研究

人类抑癌基因beclin 1通过自噬作用调节细胞生长,但在胃癌和直结肠癌中其表达水平和调控机制仍不清楚．通过检测胃癌和直结肠肿瘤组织中beclin 1基因的表达水平,及DNA异常甲基化和杂合子缺失对其表达的影响,发现与癌旁组织相比,35％的胃癌标本和30％的直结肠癌标本中beclin 1基因表达显著下调．同时发现,beclin 1基因5’端存在一高密度CpG岛,在胃癌和直结肠癌中beclin 1的启动子区域和第二个内含子区域存在甲基化,而杂合子缺失仅在胃癌中发生．这些发现表明beclin 1基因的异常甲基化和杂合子缺失对其在胃癌和直结肠癌中的表达起调控作用．     

The Tumor Suppressor Gene Retinoblastoma-1 Is Required for Retinotectal Development and Visual Function in Zebrafish

Mutations in the retinoblastoma tumor suppressor gene (rb1) cause both sporadic and familial forms of childhood retinoblastoma. Despite its clinical relevance, the roles of rb1 during normal retinotectal development and function are not well understood. We have identified mutations in the zebrafish space cadet locus that lead to a premature truncation of the rb1 gene, identical to known mutations in sporadic and familial forms of retinoblastoma. In wild-type embryos, axons of early born retinal ganglion cells (RGC) pioneer the retinotectal tract to guide later born RGC axons. In rb1 deficient embryos, these early born RGCs show a delay in cell cycle exit, causing a transient deficit of differentiated RGCs. As a result, later born mutant RGC axons initially fail to exit the retina, resulting in optic nerve hypoplasia. A significant fraction of mutant RGC axons eventually exit the retina, but then frequently project to the incorrect optic tectum. Although rb1 mutants eventually establish basic retinotectal connectivity, behavioral analysis reveals that mutants exhibit deficits in distinct, visually guided behaviors. Thus, our analysis of zebrafish rb1 mutants reveals a previously unknown yet critical role for rb1 during retinotectal tract development and visual function.     

Proteomic Analysis of GLUT4 Storage Vesicles Reveals Tumor Suppressor Candidate 5 (TUSC5) as a Novel Regulator of Insulin Action in Adipocytes

Insulin signaling augments glucose transport by regulating glucose transporter 4 (GLUT4) trafficking from specialized intracellular compartments, termed GLUT4 storage vesicles (GSVs), to the plasma membrane. Proteomic analysis of GSVs by mass spectrometry revealed enrichment of 59 proteins in these vesicles. We measured reduced abundance of 23 of these proteins following insulin stimulation and assigned these as high confidence GSV proteins. These included established GSV proteins such as GLUT4 and insulin-responsive aminopeptidase, as well as six proteins not previously reported to be localized to GSVs. Tumor suppressor candidate 5 (TUSC5) was shown to be a novel GSV protein that underwent a 3.7-fold increase in abundance at the plasma membrane in response to insulin. siRNA-mediated knockdown of TUSC5 decreased insulin-stimulated glucose uptake, although overexpression of TUSC5 had the opposite effect, implicating TUSC5 as a positive regulator of insulin-stimulated glucose transport in adipocytes. Incubation of adipocytes with TNFα caused insulin resistance and a concomitant reduction in TUSC5. Consistent with previous studies, peroxisome proliferator-activated receptor (PPAR) γ agonism reversed TNFα-induced insulin resistance. TUSC5 expression was necessary but insufficient for PPARγ-mediated reversal of insulin resistance. These findings functionally link TUSC5 to GLUT4 trafficking, insulin action, insulin resistance, and PPARγ action in the adipocyte. Further studies are required to establish the exact role of TUSC5 in adipocytes.     

Modulator of Apoptosis 1 (MOAP-1) Is a Tumor Suppressor Protein Linked to the RASSF1A Protein

Modulator of apoptosis 1 (MOAP-1) is a BH3-like protein that plays key roles in cell death or apoptosis. It is an integral partner to the tumor suppressor protein, Ras association domain family 1A (RASSF1A), and functions to activate the Bcl-2 family pro-apoptotic protein Bax. Although RASSF1A is now considered a bona fide tumor suppressor protein, the role of MOAP-1 as a tumor suppressor protein has yet to be determined. In this study, we present several lines of evidence from cancer databases, immunoblotting of cancer cells, proliferation, and xenograft assays as well as DNA microarray analysis to demonstrate the role of MOAP-1 as a tumor suppressor protein. Frequent loss of MOAP-1 expression, in at least some cancers, appears to be attributed to mRNA down-regulation and the rapid proteasomal degradation of MOAP-1 that could be reversed utilizing the proteasome inhibitor MG132. Overexpression of MOAP-1 in several cancer cell lines resulted in reduced tumorigenesis and up-regulation of genes involved in cancer regulatory pathways that include apoptosis (p53, Fas, and MST1), DNA damage control (poly(ADP)-ribose polymerase and ataxia telangiectasia mutated), those within the cell metabolism (IR-α, IR-β, and AMP-activated protein kinase), and a stabilizing effect on microtubules. The loss of RASSF1A (an upstream regulator of MOAP-1) is one of the earliest detectable epigenetically silenced tumor suppressor proteins in cancer, and we speculate that the additional loss of function of MOAP-1 may be a second hit to functionally compromise the RASSF1A/MOAP-1 death receptor-dependent pathway and drive tumorigenesis.     

Tumor Suppressor Density-enhanced Phosphatase-1 (DEP-1) Inhibits the RAS Pathway by Direct Dephosphorylation of ERK1/2 Kinases

Density-enhanced phosphatase-1 (DEP-1) is a trans-membrane receptor protein-tyrosine phosphatase that plays a recognized prominent role as a tumor suppressor. However, the mechanistic details underlying its function are poorly understood because its primary physiological substrate(s) have not been firmly established. To shed light on the mechanisms underlying the anti-proliferative role of this phosphatase, we set out to identify new DEP-1 substrates by a novel approach based on screening of high density peptide arrays. The results of the array experiment were combined with a bioinformatics filter to identify eight potential DEP-1 targets among the proteins annotated in the MAPK pathway. In this study we show that one of these potential targets, the ERK1/2, is indeed a direct DEP-1 substrate in vivo. Pulldown and in vitro dephosphorylation assays confirmed our prediction and demonstrated an overall specificity of DEP-1 in targeting the phosphorylated tyrosine 204 of ERK1/2. After epidermal growth factor stimulation, the phosphorylation of the activation loop of ERK1/2 can be modulated by changing the concentration of DEP-1, without affecting the activity of the upstream kinase MEK. In addition, we show that DEP-1 contains a KIM-like motif to recruit ERK1/2 proteins by a docking mechanism mediated by the common docking domain in ERK1/2. ERK proteins that are mutated in the conserved docking domain become insensitive to DEP-1 de-phosphorylation. Overall this study provides novel insights into the anti-proliferative role of this phosphatase and proposes a new mechanism that may also be relevant for the regulation of density-dependent growth inhibition.DEP-1⁴ (also known as CD148, HPTPη, and PTPRJ) is a class III receptor protein-tyrosine phosphatase, characterized by eight fibronectin type III repeats within the extracellular domain, a trans-membrane region, and a single cytosolic catalytic domain (1, 2). DEP-1 is expressed in all human hematopoietic cell lineages and was shown to negatively regulate T cell activation. In addition, several epithelial cell types display DEP-1 on their cell membranes (3). Homozygous DEP-1 mutant mice die before embryonic day 11.5, displaying severe defects in vascular organization (4). Interestingly, DEP-1 expression levels were found to augment with increased cell density (2), suggesting a role for this tyrosine phosphatase in sensing cell-cell contacts and in density-dependent growth inhibition (5). Moreover, accumulating evidence supports a prominent role for DEP-1 as a tumor suppressor as it negatively regulates cell proliferation and is poorly expressed in many cancer cell lines (6–10). The observed anti-proliferative effect may be accounted for by the ability of DEP-1 to down-regulate growth factor signaling through the dephosphorylation of various receptor tyrosine kinases, such as PDGFR, VEGFR2, and MET (11–13), resulting in quenching of the downstream RAS-MAPK pathway. However, given the complex pleiotropic functions of DEP-1, it is also possible that additional regulatory circuits mediated by yet unknown DEP-1 substrates may play a functional role in contact inhibition and control of cell proliferation.A variety of in vivo and in vitro approaches has led us to propose a number of DEP-1 substrates as mediators of its function. These include PDGFR, p120 catenin (CTND1), hepatocyte growth factor receptor, SRC kinase, VEGFR2, phosphatidylinositol 3-kinase regulatory subunit α (P85A), and RET receptor kinase (5, 11–16).Here we report a novel, unbiased strategy based on the screening of high density phosphopeptide arrays for their ability to bind phosphatase trapping mutants. A large portion of the phosphoproteome could be explored by this approach, thus unveiling a long list of potential substrates. A selected list of potentially relevant substrates has been obtained by applying a bioinformatics context filter. In this study we report the detailed characterization of one of these substrates, and we propose that DEP-1 modulates the RAS pathway by directly dephosphorylating Tyr-204 of ERK1/2. In addition, we show that the efficient removal of the phosphate group from Tyr-204 requires the integrity of a docking site on the ERK1/2 proteins.