Patient survival time generally reflects the tumor progression and represents a key clinical parameter. In this study, we aimed to comprehensively characterize the prognosis-associated molecular alterations in hepatocellular carcinoma (HCC). In this study, copy-number changes, gene mutations, mRNA expression, and reverse phase protein arrays data in HCC samples profiled by The Cancer Genome Atlas (TCGA) were obtained. Tumors were then stratified into two groups based on the clinical outcome and identified genomic, transcriptomic, and proteomic traits associated to HCC prognosis. We found that several copy number amplifications and deletions can discriminate HCC patients with poor prognosis from those with better prognosis. Mutated DNAH8 showed a worse prognosis-specific pattern and correlated with a reduced disease-free survival in HCC. By integrating RNA sequencing data, we found that HCC samples with poor prognosis are consistently associated with the up-regulation of cell cycle process, such as chromosome separation, DNA replication, cytokinesis, and etc. At the proteomic level, seven proteins were significantly enriched in samples with poor prognosis, including acetylated α-Tubulin, p62-LCK-ligand, ARID1 A, MSH6, B-Raf, Cyclin B1, and PEA15. Acetylated α-Tubulin was frequently expressed in HCC tissues and acted as a promising prognostic factor for HCC. These alterations lay a foundation for developing relevant therapeutic strategies and improve our knowledge of the pathogenesis of HCC.

PURPOSE: To investigate the role and molecular mechanism of miR-421 in the development of colorectal cancer (CRC), providing a theoretical basis for the search for new CRC therapeutic targets.
METHODS: 30 pairs of human CRC and cancer-adjacent normal tissue samples were collected. The expression of miR-421 was detected in CRC tissues and cells. On-line target gene prediction software was applied to screen metastasis-associated protein 1 (MTA1), the potential downstream target gene of miR-421. The role of miR-421 in regulating MTA1 and its effect on the expression of epithelial-mesenchymal transition (EMT) markers (E-cadherinand Vimentin) were detected.
RESULTS: Compared with that in adjacent normal tissues and normal human intestinal epithelial cells, the miR-421expression level in CRC tissues and cells was significantly reduced. The potential target of miR-421 was analyzed by three public databases, in which we found that MTA1 was a direct target of miR-421, and miR-421 inhibited the proliferation, invasion, migration and EMT of CRC cells through the targeted regulation of the expression of target gene MTA1, thus effectively suppressing the ability of CRC cells.
CONCLUSIONS: This research demonstrated the suppressive function of miR-421 in CRC. Therefore, the miR-421/MAT1 axis is expected to be one of the targets of CRC targeted therapy.

Liver metastasis is one of the major causes of death in patients with colorectal cancer, and although treatment has improved recently, the long‑term survival rate of patients has not improved significantly. In the present study, we used immunohistochemistry to determine that phosphoprotein enriched in astrocytes‑15 kDa (PEA15) was highly expressed in colorectal cancer tissues and liver metastatic cancer tissues. It was also highly expressed in metastatic colorectal cancer patients compared to non‑metastatic patients. Through clinicopathological data of patients with liver metastasis of colorectal cancer, we found that high expression of PEA15 was positively correlated with TNM staging, liver metastasis and poor prognosis of colorectal cancer patients. Using confocal immunofluorescence microscopy, western blotting and cell proliferation, migration and invasion assays, we also determined that PEA15 could promote cancer cell proliferation in vitro and in vivo, epithelial mesenchymal transition (EMT) and the characteristics of cancer stem cells in vitro, thus promoting the abilities of invasion and migration. In addition, we revealed that PEA15 promoted the liver metastasis of colorectal cancer cells in a xenograft tumor metastasis model. In addition, concerning the mechanism, we used gene chip analysis to determine that PEA15 upregulated the ERK/MAPK signaling pathway in colorectal cancer cells. Therefore, we concluded that PEA15 may be a potential biomarker for liver metastasis of colorectal cancer therapy. Collectively, PEA15 promoted the development of liver metastasis of colorectal cancer through the ERK/MAPK signaling pathway.

Metastasis-associated gene 1 (MTA1) is correlated with prognosis of many tumors. However, little is known about the role of MAT1 in endometriosis and its relationship with the recurrence of endometriosis.The expression of MTA1 in normal, eutopic and ectopic endometrium was detected by immunohistochemistry and RT-PCR, respectively. The relationship of MTA1 expression with the recurrence of endometriosis was evaluated.In the normal endometrium, eutopic endometrium and ectopic endometrium, the positive rates of MTA1 expression showed a gradually increasing trend. In addition, the MTA1 expression difference between each two groups was significant (P < .0125). However, there was no significant difference between proliferative phase and secretory phase in each group (P > .05). In the ectopic endometrium, MTA1 expression in the severe phases (III-IV) was significantly higher than that in mild phases (I-II) (P < .05), indicating the expression of MTA1 correlates with r-AFS staging (P < .05). Additionally, the MTA1 mRNA level was also closely related to the stages of r-AFS, but not to the proliferative phase or secretory phase of endometrium. Logistic regression analysis showed that r-AFS stage and MTA1 overexpression were risk factors for the recurrence of endometriosis. While, postoperative pregnancy was a protective factor for its relapse.MTA1 is closely associated with the occurrence and development of Ems. Thus, MTA1 level may be used as a new indicator to predict the progression of endometriosis.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Up to 50% of primary UM cases will develop distant metastasis, but no effective therapies are currently available. The present study aimed to characterize the expression profile of the long noncoding RNAs (lncRNAs) and screen the potential metastasis-associated lncRNAs in UM. A genome-wide analysis of the transcriptome was performed on 11 primary UM tissues (6 metastasized and 5 nonmetastasized) through RNA sequencing. A total of 40,878 lncRNAs were detected in UM, 4,983 of which were novel candidates. We identified 329 differentially expressed lncRNAs (DELs) and 802 differentially expressed mRNAs (DEMs) by comparing the transcriptome profile between metastasized and nonmetastasized UM group. The DEL-DEM coexpression network revealed that the RP11-551L14.4, TCONS_00004101, and TCONS_00004845 DELs had the highest connectivity with the DEMs, coexpressed with 225, 28, and 10 DEMs, respectively, whereas the SPOCD1, PEA15, and SLC44A3 DEMs were most closely connected with the DELs, and were coexpressed with 89, 27, and 22 DELs, respectively. Moreover, 17 and 743 DEMs were targeted by the DELs through cis- or trans-action, respectively. These targeted DEMs were significantly enriched in D-Arginine and D-ornithine metabolism and glycerolipid metabolism of Kyoto Encyclopedia of Genes and Genomes pathways, and enriched in bradykinin receptor activity and haptoglobin binding of gene ontology biological processes. Quantitative real-time PCR confirmed the sequencing data. These findings have provided new insights into the molecular mechanism of UM metastasis and paved the way for further investigations regarding lncRNA in UM.

Hepatocellular carcinoma (HCC) is the third-leading cause of cancer-related death with limited treatment options and frequent resistance to sorafenib, the only drug currently approved for first-line therapy. Therefore, better understanding of HCC tumor biology and its resistance to treatment is urgently needed. Here, we analyzed the role of phosphoprotein enriched in diabetes (PED) in HCC. PED has been shown to regulate cell proliferation, apoptosis and migration in several types of cancer. However, its function in HCC has not been addressed yet. Our study revealed that both transcript and protein levels of PED were significantly high in HCC compared with non-tumoral tissue. Clinico-pathological correlation revealed that PED

The Pancreatic Expression Database (PED, http://www.pancreasexpression.org) continues to be a major resource for mining pancreatic -omics data a decade after its initial release. Here, we present recent updates to PED and describe its evolution into a comprehensive resource for extracting, analysing and integrating publicly available multi-omics datasets. A new analytical module has been implemented to run in parallel with the existing literature mining functions. This analytical module has been created using rich data content derived from pancreas-related specimens available through the major data repositories (GEO, ArrayExpress) and international initiatives (TCGA, GENIE, CCLE). Researchers have access to a host of functions to tailor analyses to meet their needs. Results are presented using interactive graphics that allow the molecular data to be visualized in a user-friendly manner. Furthermore, researchers are provided with the means to superimpose layers of molecular information to gain greater insight into alterations and the relationships between them. The literature-mining module has been improved with a redesigned web appearance, restructured query platforms and updated annotations. These updates to PED are in preparation for its integration with the Pancreatic Cancer Research Fund Tissue Bank (PCRFTB), a vital resource of pancreas cancer tissue for researchers to support and promote cutting-edge research.

BACKGROUND: The directed study of the functional proteome in colorectal cancer (CRC) has identified critical protein markers and signaling pathways; however, the prognostic relevance of many of these proteins remains unclear.
METHODS: We determined the prognostic implications of the functional proteome in 263 CRC tumor samples from patients treated at MD Anderson Cancer Center (MDACC) and 462 patients from The Cancer Genome Atlas (TCGA) to identify patterns of protein expression that drive tumorigenesis. A total of 163 validated proteins were analyzed by reverse phase protein array (RPPA). Unsupervised hierarchical clustering of the tumor proteins from the MDACC cohort was performed, and clustering was validated using RPPA data from TCGA CRC. Cox regression was used to identify predictors of tumor recurrence.
RESULTS: Clustering revealed dichotomization, with subtype A notable for a high epithelial-mesenchymal transition (EMT) protein signature, while subtype B was notable for high Akt/TSC/mTOR pathway components. Survival data were only available for the MDACC cohort and were used to evaluate prognostic relevance of these protein signatures. Group B demonstrated worse relapse-free survival (hazard ratio 2.11, 95% confidence interval 1.04-4.27, p = 0.039), although there was no difference in known genomic drivers between the two proteomic groups. Proteomic grouping and stage were significant predictors of recurrence on multivariate analysis. Eight proteins were found to be significant predictors of tumor recurrence on multivariate analysis: Collagen VI, FOXO3a, INPP4B, LcK, phospho-PEA15, phospho-PRAS40, Rad51, phospho-S6.
CONCLUSION: CRC can be classified into distinct subtypes by proteomic features independent of common oncogenic driver mutations. Proteomic analysis has identified key biomarkers with prognostic importance, however these findings require further validation in an independent cohort.

Nucleotide excision repair (NER) pathway plays critical roles in repairing DNA disorders caused by platinum. To comprehensively understand the association between variants of NER and clinical outcomes of platinum-based chemotherapy, 173 SNPs in 27 genes were selected to evaluate association with toxicities and efficiency in 1004 patients with advanced non-small cell lung cancer. The results showed that consecutive significant signals were observed in XPA, RPA1, POLD1, POLD3. Further subgroup analysis showed that GTF2H4 presented consecutive significant signals in clinical benefit among adenocarcimoma. In squamous cell carcinoma, rs4150558, rs2290280, rs8067195 were significantly associated with anemia, rs3786136 was significantly related to thrombocytopenia, ERCC5 presented consecutive significant signals in response rate. In patients receiving TP regimen, significant association presented in neutropenia, thrombocytopenia and gastrointestinal toxicity. Association with anemia and neutropenia were found in GP regimen. rs4150558 showed significant association with anemia in NP regimen. In patients > 58, ERCC5 showed consecutive significant signals in gastrointestinal toxicity. Survival analysis showed SNPs in POLD2, XPA, ERCC6 and POLE were significantly associated with progression free survival, SNPs in GTF2H4, ERCC6, GTF2HA, MAT1, POLD1 were significantly associated with overall survival. This study suggests SNPs in NER pathway could be potential predictors for clinical outcomes of platinum-based chemotherapy among NSCLC.

Background: The first generation CDK2/7/9 inhibitor seliciclib (CYC202) causes multipolar anaphase and apoptosis in lung cancer cells with supernumerary centrosomes (known as anaphase catastrophe). We investigated a new and potent CDK2/9 inhibitor, CCT68127 (Cyclacel).
Methods: CCT68127 was studied in lung cancer cells (three murine and five human) and control murine pulmonary epithelial and human immortalized bronchial epithelial cells. Robotic CCT68127 cell-based proliferation screens were used. Cells undergoing multipolar anaphase and inhibited centrosome clustering were scored. Reverse phase protein arrays (RPPAs) assessed CCT68127 effects on signaling pathways. The function of PEA15, a growth regulator highlighted by RPPAs, was analyzed. Syngeneic murine lung cancer xenografts (n = 4/group) determined CCT68127 effects on tumorigenicity and circulating tumor cell levels. All statistical tests were two-sided.
Results: CCT68127 inhibited growth up to 88.5% (SD = 6.4%, P < .003) at 1 μM, induced apoptosis up to 42.6% (SD = 5.5%, P < .001) at 2 μM, and caused G1 or G2/M arrest in lung cancer cells with minimal effects on control cells (growth inhibition at 1 μM: 10.6%, SD = 3.6%, P = .32; apoptosis at 2 μM: 8.2%, SD = 1.0%, P = .22). A robotic screen found that lung cancer cells with KRAS mutation were particularly sensitive to CCT68127 ( P = .02 for IC 50 ). CCT68127 inhibited supernumerary centrosome clustering and caused anaphase catastrophe by 14.1% (SD = 3.6%, P < .009 at 1 μM). CCT68127 reduced PEA15 phosphorylation by 70% (SD = 3.0%, P = .003). The gain of PEA15 expression antagonized and its loss enhanced CCT68127-mediated growth inhibition. CCT68127 reduced lung cancer growth in vivo ( P < .001) and circulating tumor cells ( P = .004). Findings were confirmed with another CDK2/9 inhibitor, CYC065.
Conclusions: Next-generation CDK2/9 inhibition elicits marked antineoplastic effects in lung cancer via anaphase catastrophe and reduced PEA15 phosphorylation.

DNA damage is a serious threat to cellular homeostasis. Damaged DNA leads to genomic instability, mutation, senescence, and/or cell death. DNA damage triggers a cellular response called the DNA damage response (DDR), followed by activation of the DNA repair machinery. DDR both maintains cellular homeostasis and prevents cancer development. Germ line mutation of DDR-associated genes can lead to cancer-susceptible syndromes. Somatic mutation of DDR-associated genes has also been reported in various tumors, including hematological malignancies. Therapeutic approaches that target the DDR and DNA repair are thus now being developed. Understanding the mechanism(s) underlying DDR and DNA repair will increase our knowledge of cancer etiology and facilitate development of cancer therapies.

Paediatric chronic myeloid leukaemia (ped-CML) is rare and ped-CML with fibre accumulation in the bone marrow (MF) is thought to be even rarer. In adults (ad-CML), fibrosis represents an adverse prognostic factor. So far, the pro-fibrotic changes in the bone marrow microenvironment have not been investigated in detail in ped-CML. From a total of 66 ped-CML in chronic phase, biopsies were analysable and 10 had MF1/2 (MF1, n=8/10; MF2, n=2/10). We randomly selected 16 ped-CML and 16 ad-CML cases with and without fibrosis (each n=8) as well as 18 non-neoplastic controls. Bone marrow samples were analysed with a real-time PCR-based assay (including 127 genes for paediatric cases) and by immunohistochemistry. We found increased expression of megakaryocytic genes in ped-CML. The number of megakaryocytes and pro-platelets are increased in CML patients, but the most significant increase was noted for ped-CML-MF1/2. Anti-fibrotic MMP9 expression was lower in children than in adults. Cell mobilisation-related CXCL12 was decreased in young and adult patients with CML but not the corresponding receptor CXCR4. In summary, fibre accumulation in ped-CML-MF1/2 is associated with increased megakaryocytic proliferation and increased interstitial pro-platelet deposition. Deregulated expression of matrix-modulating factors shifts the bone marrow microenvironment towards fibrosis.

Among the various areas of recent investigation in the field of human MHC class I antigens, the following have been selected for discussion in this review: (1) classical HLA class I genes: are they ubiquitously expressed?, what are the special features of their polymorphism?, are HLA-C molecules functional?, (2) non-classical HLA class I gene products: how restricted is their tissue distribution?, do they exhibit a little polymorphism?, what is their function, if any? (3) non-HLA genes recently detected in the HLA class I chromosomal region: are some of them involved in immunological function and development?, (4) other novel coding sequences present, or possibly present, in the region: the hemochromatosis gene, grc region and associated tumor suppressor genes, housekeeping genes, human equivalent of the murine H-2M region and Ped gene; (5) transcriptional regulation: are there cis-regulatory elements, including locus control region(s). located elsewhere than in the promoters? are CpG methylation, gene imprinting, chromatin structure, DNA rearrangement also implicated? what are the transcription factors involved and how do they interact with each other? is there HLA class I locus-, allele-, or isoform-specific regulation? is class I gene expression dysregulated in human tumors? The answers to these questions are crucial for the development of the future directions for research.

Poly (ADP-ribose) polymerase (PARP) is an indispensable component of the DNA repair machinery. PARP inhibitors are used as cutting-edge treatments for patients with homologous recombination repair (HRR)-defective breast cancers harboring mutations in BRCA1 or BRCA2. Other tumors defective in HRR, including some hematological malignancies, are predicted to be good candidates for treatment with PARP inhibitors. Screening of leukemia-derived cell lines revealed that lymphoid lineage-derived leukemia cell lines, except for those derived from mature B cells and KMT2A (MLL)-rearranged B-cell precursors, were relatively sensitive to PARP inhibitors. By contrast, acute myelogenous leukemia cell lines, except for RUNX1-RUNXT1 (AML1-ETO)-positive lines, were relatively resistant. Intriguingly, TCF3 (E2A)-HLF-positive leukemia was sensitive to PARP inhibitors. TCF3-HLF expression suppressed HRR activity, suggesting that PARP inhibitor treatment induced synthetic lethality. Furthermore, TCF3-HLF expression decreased levels of MCPH1, which regulates the expression of BRCA1, resulting in attenuation of HRR activity. The PARP inhibitor olaparib was also effective in an in vivo xenograft model. Our results suggest a novel therapeutic approach for treating refractory leukemia, particularly the TCF3-HLF-positive subtype.

PURPOSE: CDK-activating kinase (CAK) is required for the regulation of the cell cycle and is a trimeric complex consisting of cyclin-dependent kinase 7 (CDK7), Cyclin H, and the accessory protein, MAT1. CDK7 also plays a critical role in regulating transcription, primarily by phosphorylating RNA polymerase II, as well as transcription factors such as estrogen receptor-α (ER). Deregulation of cell cycle and transcriptional control are general features of tumor cells, highlighting the potential for the use of CDK7 inhibitors as novel cancer therapeutics.
EXPERIMENTAL DESIGN: mRNA and protein expression of CDK7 and its essential cofactors cyclin H and MAT1 were evaluated in breast cancer samples to determine if their levels are altered in cancer. Immunohistochemical staining of >900 breast cancers was used to determine the association with clinicopathologic features and patient outcome.
RESULTS: We show that expressions of CDK7, cyclin H, and MAT1 are all closely linked at the mRNA and protein level, and their expression is elevated in breast cancer compared with the normal breast tissue. Intriguingly, CDK7 expression was inversely proportional to tumor grade and size, and outcome analysis showed an association between CAK levels and better outcome. Moreover, CDK7 expression was positively associated with ER expression and in particular with phosphorylation of ER at serine 118, a site important for ER transcriptional activity.
CONCLUSIONS: Expressions of components of the CAK complex, CDK7, MAT1, and Cyclin H are elevated in breast cancer and correlate with ER. Like ER, CDK7 expression is inversely proportional to poor prognostic factors and survival. Clin Cancer Res; 22(23); 5929-38. ©2016 AACR.

BACKGROUND: The development of intrinsic and acquired resistance to antineoplastic agents is a major obstacle to successful chemotherapy in ovarian cancers. Identification and characterisation of chemoresponse-associated biomarkers are of paramount importance for novel therapeutic development.
METHODS: Global RNA expression profiles were obtained by high-throughput microarray analysis. Cell cycle, proliferation rate, and paclitaxel sensitivity of ovarian cancer cells harbouring cyclin A1-inducible expression construct were compared with and without tetracycline induction, as well as when the cyclin A1 expression was suppressed by short inhibiting RNA (siRNA). Cellular senescence was evaluated by β-galactosidase activity staining.
RESULTS: Global RNA expression profiling and subsequent correlation studies of gene expression level and drug response has identified that elevated expression of cyclin A1 (CCNA1) was significantly associated with cellular resistance to paclitaxel, doxorubicin and 5-fluorouracil. The role of cyclin A1 in paclitaxel resistance was confirmed in ovarian cancer cells that harbour an inducible cyclin A1 expression construct, which showed reduced paclitaxel-mediated growth inhibition and apoptosis when cyclin A1 expression was induced, whereas downregulation of cyclin A1 expression in the same cell lines using cyclin A1-specific siRNAs sensitised the cells to paclitaxel toxicity. However, ovarian cancer cells with ectopic expression of cyclin A1 demonstrated slowdown of proliferation and senescence-associated β-galactosidase activity.
CONCLUSIONS: Our profiling and correlation studies have identified cyclin A1 as one chemoresistance-associated biomarker in ovarian cancer. The results of the characterisation studies suggest that cyclin A1 functions as an oncogene that controls proliferative and survival activities in tumourigenesis and chemoresistance of ovarian cancer.

Identifying therapeutic targets in rare cancers remains challenging due to the paucity of established models to perform preclinical studies. As a proof-of-concept, we developed a patient-derived cancer cell line, CLF-PED-015-T, from a paediatric patient with a rare undifferentiated sarcoma. Here, we confirm that this cell line recapitulates the histology and harbours the majority of the somatic genetic alterations found in a metastatic lesion isolated at first relapse. We then perform pooled CRISPR-Cas9 and RNAi loss-of-function screens and a small-molecule screen focused on druggable cancer targets. Integrating these three complementary and orthogonal methods, we identify CDK4 and XPO1 as potential therapeutic targets in this cancer, which has no known alterations in these genes. These observations establish an approach that integrates new patient-derived models, functional genomics and chemical screens to facilitate the discovery of targets in rare cancers.

BACKGROUND: The control of breast cell survival is of critical importance for preventing breast cancer initiation and progression. The activity of many proteins which regulate cell survival is controlled by reversible phosphorylation, so that the relevant kinases and phosphatases play crucial roles in determining cell fate. Several protein kinases act as oncoproteins in breast cancer and changes in their activities contribute to the process of transformation. Through counteracting the activity of oncogenic kinases, the protein phosphatases are also likely to be important players in breast cancer development, but this class of molecules is relatively poorly understood. Here we have investigated the role of the serine/threonine protein phosphatase 4 in the control of cell survival of breast cancer cells.
METHODS: The breast cancer cell lines, MCF7 and MDA-MB-231, were transfected with expression vectors encoding the catalytic subunit of protein phosphatase 4 (PP4c) or with PP4c siRNAs. Culture viability, apoptosis, cell migration and cell cycle were assessed. The involvement of phosphoprotein enriched in astrocytes 15kDa (PEA15) in PP4c action was investigated by immunoblotting approaches and by siRNA-mediated silencing of PEA15.
RESULTS: In this study we showed that PP4c over-expression inhibited cell proliferation, enhanced spontaneous apoptosis and decreased the migratory and colony forming abilities of breast cancer cells. Moreover, PP4c down-regulation produced complementary effects. PP4c is demonstrated to regulate the phosphorylation of PEA15, and PEA15 itself regulates the apoptosis of breast cancer cells. The inhibitory effects of PP4c on breast cancer cell survival and growth were lost in PEA15 knockdown cells, confirming that PP4c action is mediated, at least in part, through the de-phosphorylation of apoptosis regulator PEA15.
CONCLUSION: Our work shows that PP4 regulates breast cancer cell survival and identifies a novel PP4c-PEA15 signalling axis in the control of breast cancer cell survival. The dysfunction of this axis may be important in the development and progression of breast cancer.

Gliomas are highly malignant tumors, the most common of which are astrocytomas. A growing number of studies suggest that dysregulation of miRNAs is a frequent event contributing to the pathogenesis of gliomas. In this study, we found that over-expression of miR-132 inhibited cell proliferation and migration and triggered apoptosis, while knockdown of miR-132 showed opposite effects. PEA-15 was identified as a direct target of miR-132. Reintroduction of PEA-15 without 3'UTR region reversed the inhibitory effects of miR-132 on cell proliferation, migration, and apoptosis. MiR-132 was inversely correlated with the PEA-15 expression. CREB (cAMP response element binding protein) and KLF (Krüppel-like factor 8) were conformed as transcription factors of miR-132, which bidirectionally regulate the expression of miR-132. Our study suggests that miR-132 is an important tumor suppressor of astrocytoma progression by targeting PEA-15, while CREB and KLF can modulate the expression of miR-132, thus providing new insight into the molecular mechanisms underlying astrocytoma progression in vitro.

OBJECTIVE: The study aimed at the silence effect of MAT1 gene and proteins in Ishikawa cell of human endometrial carcinoma cell line through RNAi mediated by siRNA and also discussed the effect of MTA1 gene silence on the invasion and metastasis of endometrial carcinoma.
MATERIALS AND METHODS: According to the principle of designing siRNA sequence, siRNA aiming at MTA1 gene (MTA1-siRNA) and siRNA used for negative control (Control-siRNA) were designed and synthesized and Ishikawa cell was transfected by transfection reagents. RT-PCR method as well as western blot was used, respectively, to detect the MTA1 mRNA and protein expression of stably transfected cells. Transwell method and scarification experiment were adopted to detect the invasion and metastasis of Ishikawa cells.
RESULTS: The expression results of MTA1 on the levels of mRNA and protein showed that the expression level in transfected MTA1-siRNA group was obviously lower than that in non-transfected group and transfected control-siRNA group (p <0.05), while there was no significant difference between non-transfected group and transfected control-siRNA group (p > 0.05). Effective interference on the expression of MTA1 gene remarkably lowered the invasion and metastasis of endometrial carcinoma Ishikawa cells.
CONCLUSION: RNAi aiming at MTA1 can effectively inhibit the expression of MTA1 in endometrial carcinoma Ishikawa cells and the effective silence of MTA1 can weaken the invasion and metastasis of Ishikawa cells, which provides a new strategy for gene therapy of endometrial carcinoma and an experimental basis for inhibiting the invasion and metastasis of endometrial carcinoma.

We investigated the effects of isoalantolactone on cell growth inhibition and underlying cell death mechanisms in SKOV3 human ovarian cancer cells. The effects of isoalantolactone on cell proliferation and cell cycle were examined by EdU incorporation assay and DNA content assay. Western blotting was performed to determine the protein expression effects of isoalantolactone on cell cycle‑related proteins, autophagic regulators and PEA‑15. Autophagic vacuoles were observed by acridine orange staining. PEA‑15 knockdown by siRNA was used to confirm that PEA‑15 was involved in isoalantolactone‑induced autophagy of SKOV3 cells. Isoalantolactone inhibited the viability and proliferation of SKOV3 cells in a dose‑ and time‑dependent fashion. Isoalantolactone induced cell cycle arrest at G2/M phase and decreased the expression of cell cycle‑related proteins cyclin B1 and CDK1 in SKOV3 cells. Accordingly, isoalantolactone also induced SKOV3 cell autophagy via accumulation of autophagic vacuoles in the cytoplasm, increased Beclin1 protein expression, and increased LC3 cleavage. Furthermore, we observed that isoalantolactone‑induced autophagy was through increased PEA‑15 expression and the phosphorylation of ERK, whereas less change was observed to autophagy on SKOV3 cells through PEA‑15 knockdown by siRNA. Isoalantolactone‑induced autophagic cell death was further confirmed by pretreatment with the autophagy inhibitor 3‑methyladenine (3‑MA). In conclusion, isoalantolactone induced cell cycle arrest and autophagy and inhibited cell proliferation of SKOV3 cells via the upregulated PEA‑15 expression and the phosphorylation of ERK.

Death Receptor 5 (DR5) agonists demonstrate anti-tumor activity in preclinical models but have yet to demonstrate robust clinical responses. A key limitation may be the lack of patient selection strategies to identify those most likely to respond to treatment. To overcome this limitation, we screened a DR5 agonist Nanobody across >600 cell lines representing 21 tumor lineages and assessed molecular features associated with response. High expression of DR5 and Casp8 were significantly associated with sensitivity, but their expression thresholds were difficult to translate due to low dynamic ranges. To address the translational challenge of establishing thresholds of gene expression, we developed a classifier based on ratios of genes that predicted response across lineages. The ratio classifier outperformed the DR5+Casp8 classifier, as well as standard approaches for feature selection and classification using genes, instead of ratios. This classifier was independently validated using 11 primary patient-derived pancreatic xenograft models showing perfect predictions as well as a striking linearity between prediction probability and anti-tumor response. A network analysis of the genes in the ratio classifier captured important biological relationships mediating drug response, specifically identifying key positive and negative regulators of DR5 mediated apoptosis, including DR5, CASP8, BID, cFLIP, XIAP and PEA15. Importantly, the ratio classifier shows translatability across gene expression platforms (from Affymetrix microarrays to RNA-seq) and across model systems (in vitro to in vivo). Our approach of using gene expression ratios presents a robust and novel method for constructing translatable biomarkers of compound response, which can also probe the underlying biology of treatment response.

Phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) is known to sequester extracellular signal-regulated kinase (ERK) in the cytoplasm, inhibiting tumorigenesis of human breast cancer cells. Here, we describe how PEA-15 expression affects the dephosphorylation of epidermal growth factor receptor (EGFR) through endoplasmic reticulum (ER)-plasma membrane (PM) contacts in MDA-MB-468, triple-negative breast cancer (TNBC) cells. The increased intracellular calcium concentration resulting from increased cytoplasmic phosphorylated ERK facilitates movement of ER-anchored calcium sensors to the PM. The driving force of trans-localization of calcium-dependent proteins enhances the contact between the activated EGFR and ER-localized phosphatase, PTP1B. Consequently, our findings suggest a mechanism underneath the facilitation of EGFR dephosphorylation by cytoplasmic PEA-15 expression inside TNBC cells, which may be one of the dynamic mechanisms for down-regulation of activated EGFR in cancer cells.

Omi/HtrA2 promotes cell apoptosis in human cancer cells. Early studies showed that primary hepatocellular carcinoma requires Omi/HtrA2 expression for cell apoptosis. Additionally, the Omi/HtrA2 pro-apoptotic marker demonstrated a difference in some cell types. However, how the Omi/HtrA2 pro-apoptotic marker reacts during the process of hepatocellular carcinoma cell apoptosis remains to be determined. Thus, we investigated the role and possible mechanism of Omi/HtrA2 on hepatocellular carcinoma cell apoptosis using various hepatocellular carcinoma cell lines. The results were analyzed using RT‑qPCR and western blot analysis. In the present study, we found that Omi/HtrA2 was overexpressed in hepatocellular carcinoma cell lines and induced hepatocellular carcinoma cell apoptosis. Additiionally, the only manner in which Omi/HtrA2 participated in cell death in PLC cells may be dependent on IAP-binding. Omi/HtrA2‑inducing HepG2 cell apoptosis may mainly depend on its serine protease activity while both IAP-binding and its serine protease activity participated in Hep3B cell apoptosis. This result suggested that Omi/HtrA2 pro-apoptotic marker differs in various hepatocellular carcinoma cell lines. PLC cells were also devoid of the expression of ped/pea-15 as the substrate of Omi/HtrA2 serine protease while ped/pea-15 was overexpressed in HepG2 and Hep3B cells and ped/pea-15 expression was higher in HepG2 cells than that in Hep3B cells. These results showed that Omi/HtrA2 overexpression promotes hepatocellular carcinoma cell apoptosis and the ped/pea-15 expression level causes this difference of the Omi/HtrA2 pro-apoptotic marker in the various hepatocellular carcinoma cell lines.

BACKGROUND AND PURPOSE: Paroxysmal Kinesigenic Dyskinesia (PKD) is an episodic involuntary movement disorder characterized by recurrent and brief involuntary movements. Proline-rich transmembrane protein 2 (PRRT2) has been identified as the causative gene for PKD, Benign familial infantile convulsions (BFIC) and Infantile convulsions with choreoathetosis (ICCA). As well, PRRT2 mutations have been detected in patients with PED or PNKD. To date, most of the mutations have been found to be nonsense.
METHOD: We used inhibitors of nonsense-mediated mRNA decay (NMD) pathway --emetine dihydrochloride hydrate and cycloheximide and silencing regulator of nonsense transcripts 1(UPF1) with immortalized lymphoblasts to detect whether the truncated mutations lead to NMD, a type of mRNA surveillance in every eukaryotic cell proved so far and that generally degrades mRNA containing premature translation termination codons (PTCs). In addition, we transfected the SH-SY5Y cells with wild-type and mutant PRRT2 plasmids to identify the PRRT2 protein's subcellular localization.
RESULTS: We detected, low expression of truncated PRRT2 and was further rescued by applying the inhibitor of NMD pathway, suggesting that NMD plays an important role in the pathogenesis of PKD by haplo-insufficiency. Moreover, for the small portion of undegraded mutant PRRT2 that was translated into truncated proteins, their cellular localization changed from membrane to cytoplasm and nuclear, which might lead to a functional loss.
CONCLUSION: We suggest that the NMD of truncated mutation of PRRT2 and altered cellular localization of undegraded of PRRT2, might lead to PKD.

Advanced breast cancer requires systemic treatment, therefore developing an efficient and safe strategy is urgently needed. To ensure the success of target therapy, we have developed a breast cancer-specific construct (T-VISA) composed of the human telomerase reverse transcriptase (hTERT; T) promoter and a versatile transgene amplification vector VISA (VP16-GAL4-WPRE integrated systemic amplifier) to target PEA-15 (phosphoprotein enriched in astrocytes) in advanced breast tumors. PEA-15 contains a death effector domain that sequesters extracellular signal-regulated kinase (ERK) in the cytoplasm, thereby inhibiting cell proliferation and inducing apoptosis. T-VISA-PEA-15 was found to be highly specific, selectively express PEA-15 in breast cancer cells, and induce cancer-cell killing in vitro and in vivo without affecting normal cells. Moreover, intravenous treatment with T-VISA-PEA-15 coupled with liposome nanoparticles attenuated tumor growth and prolonged survival in mice bearing advanced breast tumors. Importantly, there was virtually no severe toxicity when PEA-15 is expressed by our T-VISA system compared with cytomegalovirus (CMV) promoter. Thus, our findings demonstrate an effective cancer-targeted therapy that is worthy of development in clinical trials eradicating advanced breast cancer.

Regulation of the DNA damage response and cell cycle progression is critical for maintaining genome integrity. Here, we report that in response to DNA damage, COPS5 deubiquitinates and stabilizes PEA15 in an ATM kinase-dependent manner. PEA15 expression oscillates throughout the cell cycle, and the loss of PEA15 accelerates cell cycle progression by activating CDK6 expression via the c-JUN transcription factor. Cells lacking PEA15 exhibit a DNA damage-induced G2/M checkpoint defect due to increased CDC25C activity and, consequentially, higher cyclin-dependent kinase 1 (CDK1)/cyclin B activity, and accordingly they have an increased rate of spontaneous mutagenesis. We find that oncogenic RAS inhibits PEA15 expression and that ectopic PEA15 expression blocks RAS-mediated transformation, which can be partially rescued by ectopic expression of CDK6. Finally, we show that PEA15 expression is downregulated in colon, breast, and lung cancer samples. Collectively, our results demonstrate that tumor suppressor PEA15 is a regulator of genome integrity and is an integral component of the DNA damage response pathway that regulates cell cycle progression, the DNA-damage-induced G2/M checkpoint, and cellular transformation.

Most breast cancers are estrogen receptor-positive and treated with antiestrogens, but aberrant signaling networks can induce drug resistance. One of these networks involves the scaffolding protein BCAR1/p130CAS, which regulates cell growth and migration/invasion. A less investigated scaffolding protein that also confers antiestrogen resistance is the SH2 domain-containing protein BCAR3. BCAR1 and BCAR3 bind tightly to each other through their C-terminal domains, thus potentially connecting their associated signaling networks. However, recent studies using BCAR1 and BCAR3 interaction mutants concluded that association between the two proteins is not critical for many of their interrelated activities regulating breast cancer malignancy. We report that these previously used BCAR mutations fail to cause adequate loss-of-function of the complex. By using structure-based BCAR1 and BCAR3 mutants that lack the ability to interact, we show that BCAR3-induced antiestrogen resistance in MCF7 breast cancer cells critically depends on its ability to bind BCAR1. Interaction with BCAR3 increases the levels of phosphorylated BCAR1, ultimately potentiating BCAR1-dependent antiestrogen resistance. Furthermore, antiestrogen resistance in cells overexpressing BCAR1/BCAR3 correlates with increased ERK1/2 activity. Inhibiting ERK1/2 through overexpression of the regulatory protein PEA15 negates the resistance, revealing a key role for ERK1/2 in BCAR1/BCAR3-induced antiestrogen resistance. Reverse-phase protein array data show that PEA15 levels in invasive breast cancers correlate with patient survival, suggesting that PEA15 can override ERK1/2 activation by BCAR1/BCAR3 and other upstream regulators. We further uncovered that the BCAR3-related NSP3 can also promote antiestrogen resistance. Thus, strategies to disrupt BCAR1-BCAR3/NSP3 complexes and associated signaling networks could ultimately lead to new breast cancer therapies.

The Pancreatic Expression Database (PED, http://www.pancreasexpression.org) is the only device currently available for mining of pancreatic cancer literature data. It brings together the largest collection of multidimensional pancreatic data from the literature including genomic, proteomic, microRNA, methylomic and transcriptomic profiles. PED allows the user to ask specific questions on the observed levels of deregulation among a broad range of specimen/experimental types including healthy/patient tissue and body fluid specimens, cell lines and murine models as well as related treatments/drugs data. Here we provide an update to PED, which has been previously featured in the Database issue of this journal. Briefly, PED data content has been substantially increased and expanded to cover methylomics studies. We introduced an extensive controlled vocabulary that records specific details on the samples and added data from large-scale meta-analysis studies. The web interface has been improved/redesigned with a quick search option to rapidly extract information about a gene/protein of interest and an upload option allowing users to add their own data to PED. We added a user guide and implemented integrated graphical tools to overlay and visualize retrieved information. Interoperability with biomart-compatible data sets was significantly improved to allow integrative queries with pancreatic cancer data.

MAT1, an assembly factor and targeting subunit of both cyclin-dependent kinase-activating kinase (CAK) and general transcription factor IIH (TFIIH) kinase, regulates cell cycle and transcription. Previous studies show that expression of intact MAT1 protein is associated with expansion of human hematopoietic stem cells (HSC), whereas intrinsically programmed or retinoic acid (RA)-induced MAT1 fragmentation accompanies granulocytic differentiation of HSC or leukemic myeloblasts. Here we determined that, in humanized mouse microenvironment, MAT1 overexpression resisted intrinsic MAT1 fragmentation to sustain hematopoietic CD34+ cell expansion while preventing granulopoiesis. Conversely, we mimicked MAT1 fragmentation in vitro and in a mouse model by overexpressing a fragmented 81-aa MAT1 polypeptide (pM9) that retains the domain for assembling CAK but cannot affix CAK to TFIIH-core. Our results showed that pM9 formed ΔCAK by competing with MAT1 for CAK assembly to mimic MAT1 fragmentation-depletion of CAK. This resulting ΔCAK acted as a dominant negative to inhibit the growth and metastasis of different leukemic myeloblasts, with or without RA resistance, by concurrently suppressing CAK and TFIIH kinase activities to inhibit cell cycle and gene transcription. These findings suggest that the intrinsically programmed MAT1 expression and fragmentation regulate granulopoiesis by inversely coordinating CAK and TFIIH activities, whereas pM9 shares a mechanistic resemblance with MAT1 fragmentation in suppressing myeloid leukemogenesis.