Evaluation of polymorphisms in predicted target sites for micro RNAs differentially expressed in endometriosis

Previous microarray analyses identified 22 microRNAs (miRNAs) differentially expressed in paired ectopic and eutopic endometrium of women with and without endometriosis. To investigate further the role of these miRNAs in women with endometriosis, we conducted an association study aiming to explore the relationship between endometriosis risk and single-nucleotide polymorphisms (SNPs) in miRNA target sites for these differentially expressed miRNAs. A panel of 102 SNPs in the predicted miRNA binding sites were evaluated for an endometriosis association study and an ingenuity pathway analysis was performed. Fourteen rare variants were identified in this study. We found SNP rs14647 in the Wolf-Hirschhorn syndrome candidate gene1 (WHSC1) 3'UTR (untranslated region) was associated with endometriosis-related infertility presenting an odds ratio of 12.2 (95% confidence interval = 2.4-60.7, P = 9.03 x 10(-5)). SNP haplotype AGG in the solute carrier family 22, member 23 (SLC22A23) 3'UTR was associated with endometriosis-related infertility and more severe disease. With the individual genotyping data, ingenuity pathways analysis identified the tumour necrosis factor and cyclin-dependant kinase inhibitor as major factors in the molecular pathways. Significant associations between WHSC1 alleles and endometriosis-related infertility and SLC22A23 haplotypes and the disease severe stage were identified. These findings may help focus future research on subphenotypes of this disease. Replication studies in independent large sample sets to confirm and characterize the involvement of the gene variation in the pathogenesis of endometriosis are needed.

The role of AIP and CDKN1B/p27Kip1 in endocrine neoplasia

Identification of genes predisposing to tumor syndromes has raised general awareness of tumorigenesis. Genetic testing of tumor susceptibility genes aids the recognition of individuals at increased risk of tumors. Identification of novel predisposing genes enables further studies concerning the classification of potential associated tumors and the definition of target patient group. Pituitary adenomas are common, benign neoplasms accounting for approximately 15% of all intracranial tumors. Accurate incidence estimation is challenging since a great portion of these adenomas are small and asymptomatic. Clinically relevant adenomas, that cause symptoms due to the expansion of the cell mass or the over-secretion of normally produced hormones, occur in approximately one of 1 000 individuals. Although the majority of pituitary adenomas are sporadic, a minority occur as components of familial syndromes, such as Multiple Endocrine Neoplasia type 1 (MEN1) and Carney complex (CNC). MEN1 syndrome is caused by germ-line mutations in the MEN1 gene, whereas most of the CNC patients carry the mutated protein kinase A (PKA) regulatory subunit-1-α (PRKAR1A) gene. Recently, other conditions predisposing to endocrine tumors have been identified: Pituitary Adenoma Predisposition (PAP) and MEN type 4 (MEN4). PAP was originally identified in a genetically homogeneous Finnish population. In a population based cohort from Northern Finland, aryl hydrocarbon receptor-interacting protein (AIP) gene mutations were found in 16% of all patients diagnosed with growth hormone (GH) producing pituitary adenoma, and in 40% of the subset of patients who were diagnosed under the age of 35 years. Since AIP mutations were originally described in a defined, homogeneous population from Northern Finland, it was relevant to study whether mutations also occur in more heterogeneous populations. In patient cohorts with different ethnic origins and variable clinical phenotypes, germ-line AIP mutations were detectable at low frequencies (range 0.8-7.4%). AIP mutation-positive patients were often diagnosed with a GH-producing adenoma at a young age, and usually had no family history of endocrine tumors. The low frequency of AIP mutations in randomly selected patients, and the lack of any family history of pituitary adenomas create a challenge for the identification of PAP patients. Our preliminary study suggests that AIP immunohistochemistry may serve as a pre-screening tool to distinguish between the AIP mutation-negative and the mutation-positive tumors. Tumors of various endocrine glands are components of MEN1 and CNC syndromes. Somatic MEN1 and PRKAR1A mutations in sporadic pituitary adenomas are rare, but occur in some of the other tumors related to these syndromes. The role of AIP mutations in endocrine neoplasia was studied and our results indicated that somatic AIP mutations are rare or non-existent in sporadic tumors of endocrine glands (0 of 111). Furthermore, germ-line AIP mutations in prolactin producing adenomas (2 of 9) confirmed the role of this pituitary tumor type in the PAP phenotype. Thyroid disorders are common in the general population, and the majority of them are sporadic. Interestingly, it has been suggested that thyroid disorders might be more common in PAP families. For this reason we studied germ-line AIP mutations in 93 index cases from familial non-medullary thyroid cancer (NMTC) families. The underlying gene or genes for familial NMTC have not been identified yet. None of the patients had any potentially pathogenic AIP mutation. This suggests that AIP is unlikely to play a role in familial NMTCs. A novel multiple endocrine syndrome was originally described in rats with phenotypic features of human MEN type 1 and 2. Germ-line mutations of cyclin-dependent kinase inhibitor 1B (CDKN1B also known as p27Kip1) gene were reported later in these rats and a germ-line mutation was also identified in one human family with MEN1-like phenotype (later named MEN4). To confirm the importance of this gene’s mutations in humans, we performed a mutation screening in MEN-like patients and in patients with pituitary adenoma. Our results indicate that CDKN1B/p27Kip1 mutations appear in a small portion of MEN1-like patients (one of 36), and that such mutations are rare or non-existent in both familial (0 of 19) and sporadic pituitary adenoma patients (0 of 50). In conclusion, this work strengthens the tumor susceptibility role of AIP and CDKN1B/p27Kip1 in endocrine neoplasia. Clarifying the PAP phenotype facilitates the identification of potential AIP mutation carriers. Genetic counseling can be offered to the relatives and follow-up of the mutation carriers can be organized, hence an earlier diagnosis is feasible.

Role of cell cycle regulators in development of the inner ear

The inner ear originates from an ectodermal thickening called the otic placode. The otic placode invaginates and closes to an otic vesicle, the otocyst. The otocyst epithelium undergoes morphogenetic changes and cell differentiation, leading to the formation of the labyrinth-like mature inner ear. Epithelial-mesenchymal interactions control inner ear morphogenesis, but the modes and molecules are largely unresolved. The expressions of negative cell cycle regulators in the epithelium of the early-developing inner ear have also not been elucidated. The mature inner ear comprises the hearing (cochlea) and balance (vestibular) organs that contain the nonsensory and sensory cells. In mammals, the inner ear sensory cells, called hair cells, exit the cell cycle during embryogenesis and are mitotically quiescent during late-embryonic differentiation stages and postnatally. The mechanisms that maintain this hair cell quiescense are largely unresolved. In this work I examined 1) the epithelial-mesenchymal interactions involved in inner ear morphogenesis, 2) expression of negative cell cycle regulators in the epithelium of the early developing inner ear and 3) the molecular mechanisms that maintain the postmitotic state of inner ear sensory cells. We observed that during otocyst stages, epithelial fibroblast growth factor 9 (Fgf9) communicates with the surrounding mesenchyme, where its receptors are expressed. Fgf9 inactivation leads to reduced proliferation of the surrounding vestibular mesenchyme and to the absence of semicircular canals. Semicircular canal development is blocked, since fusion plates do not form. These results show that the mesenchyme directs fusion plate formation and give direct evidence for the existence of reciprocal epithelial-mesenchymal interactions in the developing inner ear. Cyclin-dependent kinase inhibitors (CKIs) are negative regulators of proliferation. We show that the members of the Cip/Kip family of CKIs (p21Cip1, p27Kip1 and p57Kip2) are expressed in the early-developing inner ear. Our expression data suggest that CKIs divide the otic epithelium into proliferative and nonproliferative compartments that may underlie shaping of the otocyst. At later stages, CKIs regulate proliferation of the vestibular appendages, and this may regulate their continual growth. In addition to restricting proliferation, CKIs may play a role in regional differentiation of various epithelial cells. Differentiating and adult inner ear hair cells are postmitotic and do not proliferate in response to serum or mitogenic growth factors. In our study, we show that this is the result of the activity of negative cell cycle regulators. Based on expression profiles, we first focused on the retinoblastoma (Rb) gene, which functions downstream of the CKIs. Analysis of the inner ear phenotype of Rb mutant mice show, that the retinoblastoma protein regulates the postmitotic state of hair cells. Rb inactivation leads to hyperplasia of vestibular and cochlear sensory epithelia that is a result of abnormal cell cycle entry of differentiated hair cells and of delayed cell cycle exit of the hair cell precursor cells. In addition, we show that p21Cip1 and p19Ink4d cooperate in maintaining the postmitotic state of postnatal auditory hair cells. Whereas inactivation of p19Ink4d alone leads to low-level S-phase entry (Chen et al., 2003) and p21Cip1 null mutant mice have a normal inner ear phenotype, codeletion of p19Ink4d and p21Cip1 triggers high-level S-phase entry of auditory hair cells during early postnatal life, which leads to supernumerary hair cells. The ectopic hair cells undergo apoptosis in all of the mutant mice studied, DNA damage being the immediate cause of this death. These findings demonstrate that the maintenance of the postmitotic state of hair cells is regulated by Rb and several CKIs, and that these cell cycle regulators are critical for the lifelong survival of hair cells. These data have implications for the future design of therapies to induce hair cell regrowth.

Functional Analysis of MrpL55, Vig and Mat1 Acting at the Crossroad of Cell Cycle, Transcription and Metabolism Regulation

Cell proliferation, transcription and metabolism are regulated by complex partly overlapping signaling networks involving proteins in various subcellular compartments. The objective of this study was to increase our knowledge on such regulatory networks and their interrelationships through analysis of MrpL55, Vig, and Mat1 representing three gene products implicated in regulation of cell cycle, transcription, and metabolism. Genome-wide and biochemical in vitro studies have previously revealed MrpL55 as a component of the large subunit of the mitochondrial ribosome and demonstrated a possible role for the protein in cell cycle regulation. Vig has been implicated in heterochromatin formation and identified as a constituent of the RNAi-induced silencing complex (RISC) involved in cell cycle regulation and RNAi-directed transcriptional gene silencing (TGS) coupled to RNA polymerase II (RNAPII) transcription. Mat1 has been characterized as a regulatory subunit of cyclin-dependent kinase 7 (Cdk7) complex phosphorylating and regulating critical targets involved in cell cycle progression, energy metabolism and transcription by RNAPII. The first part of the study explored whether mRpL55 is required for cell viability or involved in a regulation of energy metabolism and cell proliferation. The results revealed a dynamic requirement of the essential Drosophila mRpL55 gene during development and suggested a function of MrpL55 in cell cycle control either at the G1/S or G2/M transition prior to cell differentiation. This first in vivo characterization of a metazoan-specific constituent of the large subunit of mitochondrial ribosome also demonstrated forth compelling evidence of the interconnection of nuclear and mitochondrial genomes as well as complex functions of the evolutionarily young metazoan-specific mitochondrial ribosomal proteins. In studies on the Drosophila RISC complex regulation, it was noted that Vig, a protein involved in heterochromatin formation, unlike other analyzed RISC associated proteins Argonaute2 and R2D2, is dynamically phosphorylated in a dsRNA-independent manner. Vig displays similarity with a known in vivo substrate for protein kinase C (PKC), human chromatin remodeling factor Ki-1/57, and is efficiently phosphorylated by PKC on multiple sites in vitro. These results suggest that function of the RISC complex protein Vig in RNAi-directed TGS and chromatin modification may be regulated through dsRNA-independent phosphorylation by PKC. In the third part of this study the role of Mat1 in regulating RNAPII transcription was investigated using cultured murine immortal fibroblasts with a conditional allele of Mat1. The results demonstrated that phosphorylation of the carboxy-terminal domain (CTD) of the large subunit of RNAPII in the heptapeptide YSPTSPS repeat in Mat-/- cells was over 10-fold reduced on Serine-5 and subsequently on Serine-2. Occupancy of the hypophosphorylated RNAPII in gene bodies was detectably decreased, whereas capping, splicing, histone methylation and mRNA levels were generally not affected. However, a subset of transcripts in absence of Mat1 was repressed and associated with decreased occupancy of RNAPII at promoters as well as defective capping. The results identify the Cdk7-CycH-Mat1 kinase submodule of TFIIH as a stimulatory non-essential regulator of transcriptional elongation and a genespecific essential factor for stable binding of RNAPII at the promoter region and capping. The results of these studies suggest important roles for both MrpL55 and Mat1 in cell cycle progression and their possible interplay at the G2/M stage in undifferentiated cells. The identified function of Mat1 and of TFIIH kinase complex in gene-specific transcriptional repression is challenging for further studies in regard to a possible link to Vig and RISC-mediated transcriptional gene silencing.

What's the hype about CDK5RAP2?

Cyclin dependent kinase 5 regulatory subunit-associated protein 2 (CDK5RAP2) has gained attention in the last years following the discovery, in 2005, that recessive mutations cause primary autosomal recessive microcephaly. This disease is seen as an isolated developmental defect of the brain, particularly of the cerebral cortex, and was thus historically also referred to as microcephalia vera. Unraveling the pathomechanisms leading to this human disease is fascinating scientists because it can convey insight into basic mechanisms of physiologic brain development (particularly of cortex formation). It also finds itself in the spotlight because of its implication in trends in mammalian evolution with a massive increase in the size of the cerebral cortex in primates. Here, we provide a timely overview of the current knowledge on the function of CDK5RAP2 and mechanisms that might lead to disease in humans when the function of this protein is disturbed.

The repertoire of protein kinases encoded in the draft version of the human genome: atypical variations and uncommon domain combinations

Background: Phosphorylation by protein kinases is central to cellular signal transduction. Abnormal functioning of kinases has been implicated in developmental disorders and malignancies. Their activity is regulated by second messengers and by the binding of associated domains, which are also influential in translocating the catalytic component to their substrate sites, in mediating interaction with other proteins and carrying out their biological roles. Results: Using sensitive profile-search methods and manual analysis, the human genome has been surveyed for protein kinases. A set of 448 sequences, which show significant similarity to protein kinases and contain the critical residues essential for kinase function, have been selected for an analysis of domain combinations after classifying the kinase domains into subfamilies. The unusual domain combinations in particular kinases suggest their involvement in ubiquitination pathways and alternative modes of regulation for mitogen-activated protein kinase kinases (MAPKKs) and cyclin-dependent kinase (CDK)-like kinases. Previously unexplored kinases have been implicated in osteoblast differentiation and embryonic development on the basis of homology with kinases of known functions from other organisms. Kinases potentially unique to vertebrates are involved in highly evolved processes such as apoptosis, protein translation and tyrosine kinase signaling. In addition to coevolution with the kinase domain, duplication and recruitment of non-catalytic domains is apparent in signaling domains such as the PH, DAG-PE, SH2 and SH3 domains. Conclusions: Expansion of the functional repertoire and possible existence of alternative modes of regulation of certain kinases is suggested by their uncommon domain combinations. Experimental verification of the predicted implications of these kinases could enhance our understanding of their biological roles.

Novel Alternative Splice Variants of Mouse Cdk5rap2

Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopmental disorder characterized by a pronounced reduction of brain volume and intellectual disability. A current model for the microcephaly phenotype invokes a stem cell proliferation and differentiation defect, which has moved the disease into the spotlight of stem cell biology and neurodevelopmental science. Homozygous mutations of the Cyclin-dependent kinase-5 regulatory subunit-associated protein 2 gene CDK5RAP2 are one genetic cause of MCPH. To further characterize the pathomechanism underlying MCPH, we generated a conditional Cdk5rap2 LoxP/hCMV Cre mutant mouse. Further analysis, initiated on account of a lack of a microcephaly phenotype in these mutant mice, revealed the presence of previously unknown splice variants of the Cdk5rap2 gene that are at least in part accountable for the lack of microcephaly in the mice.

Expression, purification and spectral characterization of p21(Waf1/Cip1)

p21(Waf1/Cip1), best known as a broad-specificity inhibitor of cyclin/cyclin-dependent kinase complexes, can interact with various target proteins, and this ability relies on its structural plasticity. Therefore, studies on the structural properties of p(21) are very important to understand its structure-function relationship. However, detailed studies on its secondary structure and biophysical properties have been comparatively sparse. A human p(21) gene was cloned into the temperature expression vector pBV220 and transformed into Escherichia coli strain JM109.

A novel, non-apoptotic role for Scythe/BAT3: a functional switch between the pro- and anti-proliferative roles of p21 during the cell cycle.

BACKGROUND: Scythe/BAT3 is a member of the BAG protein family whose role in apoptosis has been extensively studied. However, since the developmental defects observed in Bat3-null mouse embryos cannot be explained solely by defects in apoptosis, we investigated whether BAT3 is also involved in cell-cycle progression. METHODS/PRINCIPAL FINDINGS: Using a stable-inducible Bat3-knockdown cellular system, we demonstrated that reduced BAT3 protein level causes a delay in both G1/S transition and G2/M progression. Concurrent with these changes in cell-cycle progression, we observed a reduction in the turnover and phosphorylation of the CDK inhibitor p21, which is best known as an inhibitor of DNA replication; however, phosphorylated p21 has also been shown to promote G2/M progression. Our findings indicate that in Bat3-knockdown cells, p21 continues to be synthesized during cell-cycle phases that do not normally require p21, resulting in p21 protein accumulation and a subsequent delay in cell-cycle progression. Finally, we showed that BAT3 co-localizes with p21 during the cell cycle and is required for the translocation of p21 from the cytoplasm to the nucleus during the G1/S transition and G2/M progression. CONCLUSION: Our study reveals a novel, non-apoptotic role for BAT3 in cell-cycle regulation. By maintaining a low p21 protein level during the G1/S transition, BAT3 counteracts the inhibitory effect of p21 on DNA replication and thus enables the cells to progress from G1 to S phase. Conversely, during G2/M progression, BAT3 facilitates p21 phosphorylation by cyclin A/Cdk2, an event required for G2/M progression. BAT3 modulates these pro- and anti-proliferative roles of p21 at least in part by regulating cyclin A abundance, as well as p21 translocation between the cytoplasm and the nucleus to ensure that it functions in the appropriate intracellular compartment during each phase of the cell cycle.

Ubiquitylation of p53 by the APC/C inhibitor Trim39.

Tripartite motif 39 (Trim39) is a RING domain-containing E3 ubiquitin ligase able to inhibit the anaphase-promoting complex (APC/C) directly. Through analysis of Trim39 function in p53-positive and p53-negative cells, we have found, surprisingly, that p53-positive cells lacking Trim39 could not traverse the G1/S transition. This effect did not result from disinhibition of the APC/C. Moreover, although Trim39 loss inhibited etoposide-induced apoptosis in p53-negative cells, apoptosis was enhanced by Trim39 knockdown in p53-positive cells. Furthermore, we show here that the Trim39 can directly bind and ubiquitylate p53 in vitro and in vivo, leading to p53 degradation. Depletion of Trim39 significantly increased p53 protein levels and cell growth retardation in multiple cell lines. We found that the relative importance of Trim39 and the well-characterized p53-directed E3 ligase, murine double minute 2 (MDM2), varied between cell types. In cells that were relatively insensitive to the MDM2 inhibitor, nutlin-3a, apoptosis could be markedly enhanced by siRNA directed against Trim39. As such, Trim39 may serve as a potential therapeutic target in tumors with WT p53 when MDM2 inhibition is insufficient to elevate p53 levels and apoptosis.

Characterization of a thymidylate synthase (TS)-inducible cell line: a model system for studying sensitivity to TS- and non-TS-targeted chemotherapies

Thymidylate synthase (TS) is responsible for the de novo synthesis of thymidylate, which is required for DNA synthesis and repair and which is an important target for fluoropyrimidines such as 5-fluorouracil (5-FU), and antifolates such as Tomudex (TDX), ZD9331, and multitargeted antifolate (MTA). To study the importance of TS expression in determining resistance to these agents, we have developed an MDA435 breast cancer-derived cell line with tetracycline-regulated expression of TS termed MTS-5. We have demonstrated that inducible expression of TS increased the IC(50) dose of the TS-targeted therapeutic agents 5-FU, TDX, and ZD9331 by 2-, 9- and 24-fold respectively. An IC(50) dose for MTA was unobtainable when TS was overexpressed in these cells, which indicated that MTA toxicity is highly sensitive to increased TS expression levels. The growth inhibitory effects of the chemotherapeutic agents CPT-11, cisplatin, oxaliplatin, and Taxol were unaffected by TS up-regulation. Cell cycle analyses revealed that IC(50) doses of 5-FU, TDX and MTA caused an S-phase arrest in cells that did not overexpress TS, and this arrest was overcome when TS was up-regulated. Furthermore, the S-phase arrest was accompanied by 2- to 4-fold increased expression of the cell cycle regulatory genes cyclin E, cyclin A, and cyclin dependent kinase 2 (cdk2). These results indicate that acute increases in TS expression levels play a key role in determining cellular sensitivity to TS-directed chemotherapeutic drugs by modulating the degree of S-phase arrest caused by these agents. Moreover, CPT-11, cisplatin, oxaliplatin, and Taxol remain highly cytotoxic in cells that overexpress TS.

Downregulation of beta1,4-galactosyltransferase 1 inhibits CDK11(p58)-mediated apoptosis induced by cycloheximide.

Cyclin-dependent kinase 11 (CDK11; also named PITSLRE) is part of the large family of p34(cdc2)-related kinases whose functions appear to be linked with cell cycle progression, tumorigenesis, and apoptotic signaling. The mechanism that CDK11(p58) induces apoptosis is not clear. Some evidences suggested beta1,4-galactosyltransferase 1 (beta1,4-GT 1) might participate in apoptosis induced by CDK11(p58). In this study, we demonstrated that ectopically expressed beta1,4-GT 1 increased CDK11(p58)-mediated apoptosis induced by cycloheximide (CHX). In contrast, RNAi-mediated knockdown of beta1,4-GT 1 effectively inhibited apoptosis induced by CHX in CDK11(p58)-overexpressing cells. For example, the cell morphological and nuclear changes were reduced; the loss of cell viability was prevented and the number of cells in sub-G1 phase was decreased. Knock down of beta1,4-GT 1 also inhibited the release of cytochrome c from mitochondria and caspase-3 processing. Therefore, the cleavage of CDK11(p58) by caspase-3 was reduced. We proposed that beta1,4-GT 1 might contribute to the pro-apoptotic effect of CDK11(p58). This may represent a new mechanism of beta1,4-GT 1 in CHX-induced apoptosis of CDK11(p58)-overexpressing cells.

Interleukin-8 signaling promotes androgen-independent proliferation of prostate cancer cells via induction of androgen receptor expression and activation

The aim of our study was to assess the importance of the CXC chemokine and interleukin (IL)-8 in promoting the transition of prostate cancer (CaP) to the androgen-independent state. Stimulation of the androgen-dependent cell lines, LNCaP and 22Rv1, with exogenous recombinant human interleukin-8 (rh-IL-8) increased androgen receptor (AR) gene expression at the messenger RNA (mRNA) and protein level, assessed by quantitative polymerase chain reaction and immunoblotting, respectively. Using an androgen response element-luciferase construct, we demonstrated that rh-IL-8 treatment also resulted in increased AR transcriptional activity in both these cell lines, and a subsequent upregulation of prostate-specific antigen and cyclin-dependent kinase 2 mRNA transcript levels in LNCaP cells. Blockade of CXC chemokine receptor-2 signaling using a small molecule antagonist (AZ10397767) attenuated the IL-8-induced increases in AR expression and transcriptional activity. Furthermore, in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, coadministration of AZ10397767 reduced the viability of LNCaP and 22Rv1 cells exposed to bicalutamide. Our data show that IL-8 signaling increases AR expression and promotes ligand-independent activation of this receptor in two androgen-dependent cell lines, describing two mechanisms by which this chemokine may assist in promoting the transition of CaP to the androgen-independent state. In addition, our data show that IL-8-promoted regulation of the AR attenuates the effectiveness of the AR antagonist bicalutamide in reducing CaP cell viability.

The Deubiquitinating Enzyme USP17 Is Highly Expressed in Tumor Biopsies, Is Cell Cycle Regulated, and Is Required for G1-S Progression

Ubiquitination is a reversible posttranslational modification that is essential for cell cycle control, and it is becoming increasingly clear that the removal of ubiquitin from proteins by deubiquitinating enzymes (DUB) is equally important. In this study, we have identified high levels of the DUB USP17 in several tumor-derived cell lines and primary lung, colon, esophagus, and cervix tumor biopsies. We also report that USP17 is tightly regulated during the cell cycle in all the cells examined, being abundantly evident in G1 and absent in S phase. Moreover, regulated USP17 expression was necessary for cell cycle progression because its depletion significantly impaired G1-S transition and blocked cell proliferation. Previously, we have shown that USP17 regulates the intracellular translocation and activation of the GTPase Ras by controlling Ras-converting enzyme 1 (RCE1) activation. RCE1 also regulates the processing of other proteins with a CAAX motif, including Rho family GTPases. We now show that USP17 depletion blocks Ras and RhoA localization and activation. Moreover, our results confirm that USP17-depleted cells have constitutively elevated levels of the cyclin-dependent kinase inhibitors p21cip1 and p27kip1, known downstream targets of Ras and RhoA signaling. These observations clearly show that USP17 is tightly regulated during cell division and that its expression is necessary to coordinate cell cycle progression, and thus, it may be considered a promising novel cancer therapeutic target. Cancer Res; 70(8); 3329–39. ©2010 AACR.

Overexpression of endoplasmic reticulum protein 29 regulates mesenchymal-epithelial transition and suppresses xenograft tumor growth of invasive breast cancer cells

Endoplasmic reticulum protein 29 (ERp29) is a novel endoplasmic reticulum ( ER) secretion factor that facilitates the transport of secretory proteins in the early secretory pathway. Recently, it was found to be overexpressed in several cancers; however, little is known regarding its function in breast cancer progression. In this study, we show that the expression of ERp29 was reduced with tumor progression in clinical specimens of breast cancer, and that overexpression of ERp29 resulted in G(0)/G(1) arrest and inhibited cell proliferation in MDA-MB-231 cells. Importantly, overexpression of ERp29 in MDA-MB-231 cells led to a phenotypic change and mesenchymal-epithelial transition (MET) characterized by cytoskeletal reorganization with loss of stress fibers, reduction of fibronectin (FN), reactivation of epithelial cell marker E-cadherin and loss of mesenchymal cell marker vimentin. Knockdown of ERp29 by shRNA in MCF-7 cells reduced E-cadherin, but increased vimentin expression. Furthermore, ERp29 overexpression in MDA-MB-231 and SKBr3 cells decreased cell migration/invasion and reduced cell transformation, whereas silencing of ERp29 in MCF-7 cells enhanced cell aggressive behavior. Significantly, expression of ERp29 in MDA-MB-231 cells suppressed tumor formation in nude mice by repressing the cell proliferative index (Ki-67 positivity). Transcriptional profiling analysis showed that ERp29 acts as a central regulator by upregulating a group of genes with tumor suppressive function, for example, E-cadherin (CDH1), cyclin-dependent kinase inhibitor (CDKN2B) and spleen tyrosine kinase (SYK), and by downregulating a group of genes that regulate cell proliferation (eg, FN, epidermal growth factor receptor ( EGFR) and plasminogen activator receptor ( uPAR)). It is noteworthy that ERp29 significantly attenuated the overall ERK cascade, whereas the ratio of p-ERK1 to p-ERK2 was highly increased. Taken together, our results showed that ERp29 is a novel regulator leading to cell growth arrest and cell transition from a proliferative to a quiescent state, and reprogramming molecular portraits to suppress the tumor growth of MDA-MB-231 breast cancer cells. Laboratory Investigation (2009) 89, 1229-1242; doi: 10.1038/labinvest.2009.87; published online 21 September 2009