Increasing evidence showed that Cadmium (Cd) can accumulate in the body and damage cells, resulting in cancerigenesis of the prostate with complex mechanisms. In the present study, we aimed to explore the possible key genes, pathways and therapeutic drugs using bioinformatics methods. Microarray-based data were retrieved and analyzed to screen differentially expressed genes (DEGs) between Cd-treated prostate cells and controls. Then, functions of the DEGs were annotated and hub genes were screened. Next, key genes were selected from the hub genes via validation in a prostate cancer cohort from The Cancer Genome Atlas (TCGA). Afterward, potential drugs were further predicted. Consequently, a gene expression profile, GSE9951, was retrieved. Then, 361 up-regulated and 30 down-regulated DEGs were screened out, which were enriched in various pathways. Among the DEGs, seven hub genes (HSPA5, HSP90AB1, RHOA, HSPD1, MAD2L1, SKP2, and CCT2) were dysregulated in prostate cancer compared to normal controls, and two of them (HSPD1 and CCT2) might influence the prostate cancer prognosis. Lastly, ionomycin was predicted to be a potential agent reversing Cd-induced prostate cell malignant transformation. In summary, the present study provided novel evidence regarding the mechanisms of Cd-induced prostate cell malignant transformation, and identified ionomycin as a potential small molecule against Cd toxicity.

Hsp90ab1 is upregulated in numerous solid tumors, which is thought to induce the angiogenesis and promote cancer metastasis. However, it's actions in gastric cancer (GC) has not been exhibited. In this study, Hsp90ab1 was demonstrated to be overexpressed and correlated with the poor prognosis, proliferation and invasion of GC. Ectopic expression of Hsp90ab1 promoted the proliferation and metastasis of GC cells both in vitro in cell line models of GC and in vivo using two different xenograft mouse models, while opposite effects were observed in Hsp90ab1 silenced cells. Moreover, the underlining molecular mechanism was explored by the co-immunoprecipitation, immunofluorescence, GST pull-down and in vitro ubiquitination assay. Namely, Hsp90ab1 exerted these functions via the interaction of LRP5 and inhibited ubiquitin-mediated degradation of LRP5, an indispensable coreceptor of the Wnt/β-catenin signaling pathway. In addition, the crosstalk between Hsp90ab1 and LRP5 contributed to the upregulation of multiple mesenchymal markers, which are also targets of Wnt/β-catenin. Collectively, this study uncovers the details of the Hsp90ab1-LRP5 axis, providing novel insights into the role and mechanism of invasion and metastasis in GC.

BACKGROUND: Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described.
METHODS: We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0-6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression.
RESULTS: We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases.
CONCLUSIONS: A crucial step of melanoma progression does occur at melanoma intermediate -stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement.

Understanding the biological factors that are characteristic of metastasis in melanoma remains a key approach to improving treatment. In this study, we seek to identify a gene signature of metastatic melanoma. We configured a new network-based computational pipeline, combined with a machine learning method, to mine publicly available transcriptomic data from melanoma patient samples. Our method is unbiased and scans a genome-wide protein-protein interaction network using a novel formulation for network scoring. Using this, we identify the most influential, differentially expressed nodes in metastatic as compared to primary melanoma. We evaluated the shortlisted genes by a machine learning method to rank them by their discriminatory capacities. From this, we identified a panel of 6 genes, ALDH1A1, HSP90AB1, KIT, KRT16, SPRR3 and TMEM45B whose expression values discriminated metastatic from primary melanoma (87% classification accuracy). In an independent transcriptomic data set derived from 703 primary melanomas, we showed that all six genes were significant in predicting melanoma specific survival (MSS) in a univariate analysis, which was also consistent with AJCC staging. Further, 3 of these genes, HSP90AB1, SPRR3 and KRT16 remained significant predictors of MSS in a joint analysis (HR = 2.3, P = 0.03) although, HSP90AB1 (HR = 1.9, P = 2 × 10

Dasatinib and radotinib are oral BCR-ABL tyrosine kinase inhibitors that were developed as drugs for the treatment of chronic myeloid leukemia. We report here that the c-KIT (CD117) targeting with dasatinib and radotinib promotes acute myeloid leukemia (AML) cell death, and c-KIT endocytosis is essential for triggering c-KIT-positive AML cell death by dasatinib and radotinib during the early stages. In addition, dasatinib and radotinib reduce heat shock protein 90β (HSP90β) expression and release Apaf-1 in c-KIT-positive AML cells. Finally, this activates a caspase-dependent apoptotic pathway in c-KIT-positive AML cells. Moreover, the inhibition of c-KIT endocytosis by dynamin inhibitor (DY) reversed cell viability and c-KIT expression by dasatinib and radotinib. HSP90β expression was recovered by DY in c-KIT-positive AML cells as well. Furthermore, the effect of radotinib on c-KIT and HSP90β showed the same pattern in a xenograft animal model using HEL92.1.7 cells. Therefore, dasatinib and radotinib promote AML cell death by targeting c-KIT. Taken together, these results indicate that dasatinib and radotinib treatment have a potential role in anti-leukemic therapy on c-KIT-positive AML cells.

Extramedullary myeloma (EMM) occurs when myeloma develops outside the bone marrow; it often develops after chemotherapy and is associated with the acquisition of chemo-resistance and a fatal course. The mechanisms underlying extramedullary spread have not yet been fully elucidated. MALAT1 is a highly abundantly and ubiquitously expressed long non-coding RNA that plays important roles in cancer metastasis. The aims of this study were to clarify the association of MALAT1 with EMM and to elucidate the underlying mechanism of EMM formation under chemotherapeutic pressure. MALAT1 expression was significantly higher in multiple myeloma (MM) than in monoclonal gammopathy of undetermined significance. Furthermore, MALAT1 expression was markedly higher in EMM compared with that in corresponding intramedullary myeloma cells. A higher MALAT1 level was associated with shorter overall and progression-free survival. MALAT1 expression level was positively correlated with expression of HSP90AA1, HSP90AB1 and HSP90B1 but not with TP53 expression. MALAT1 was significantly upregulated by bortezomib and doxorubicin. Considering the known functions of MALAT1, our results suggest that it acts as a stress response gene that is upregulated by chemotherapy, thereby linking chemotherapy to EMM formation. Elucidating the biological implication of long non-coding RNA contributes to deeper understanding concerning the pathogenesis and investigation of novel therapeutic targets for MM.

Recent advances in mass spectrometry (MS) have enabled extensive analysis of cancer proteomes. Here, we employed quantitative proteomics to profile protein expression across 24 breast cancer patient-derived xenograft (PDX) models. Integrated proteogenomic analysis shows positive correlation between expression measurements from transcriptomic and proteomic analyses; further, gene expression-based intrinsic subtypes are largely re-capitulated using non-stromal protein markers. Proteogenomic analysis also validates a number of predicted genomic targets in multiple receptor tyrosine kinases. However, several protein/phosphoprotein events such as overexpression of AKT proteins and ARAF, BRAF, HSP90AB1 phosphosites are not readily explainable by genomic analysis, suggesting that druggable translational and/or post-translational regulatory events may be uniquely diagnosed by MS. Drug treatment experiments targeting HER2 and components of the PI3K pathway supported proteogenomic response predictions in seven xenograft models. Our study demonstrates that MS-based proteomics can identify therapeutic targets and highlights the potential of PDX drug response evaluation to annotate MS-based pathway activities.

BACKGROUND: Tumour metastasis remains the major cause of death in cancer patients and, to date, the mechanism and signalling pathways governing this process are not completely understood. The TGF-β pathway is the most commonly mutated pathway in cancer, however its role in cancer progression is controversial as it can function as both a promoter and a suppressor of metastasis. Although previous studies have suggested a role for the molecular chaperone Hsp90 in regulating the TGF-β pathway, the level at which this occurs as well as the consequences in terms of colon cancer metastasis are unknown.
METHODS: The paired SW480 and SW620 colon cancer cell lines, derived from a primary tumour and its lymph node metastasis, respectively, were used as an in vitro model to study key cellular processes required for metastasis. The status of the TGF-β pathway was examined in these cells using ELISA, flow cytometry, western blot analysis and confocal microscopy. Furthermore, the effect of addition or inhibition of the TGF-β pathway and Hsp90 on adhesion, migration and anchorage-independent growth, was determined in the cell lines.
RESULTS: When comparing the canonical TGF-β1 pathway in the genetically paired cell lines our data suggests that this pathway may be constitutively active in the SW620 metastasis-derived cell line and not the SW480 primary tumour-derived line. In addition, we report that, when present in combination, TGF-β1 and Hsp90β stimulate anchorage-independent growth, reduce adhesion and stimulate migration. This effect is potentiated by inhibition of the TGF-β1 receptor and occurs via an alternate TGF-β1 pathway, mediated by αvβ6 integrin. Interestingly, in the SW620 cells, activation of this alternate TGF-β1 signalling machinery does not appear to require inhibition of the canonical TGF-β1 receptor, which would allow them to respond more effectively to the pro-metastasis stimulus of a combination of Hsp90β and TGF-β1 and this could account for the increased migratory capacity of these cells.
CONCLUSIONS: In this study we report an apparent synergy between TGF-β1 and Hsp90β in stimulating migratory behaviour of colon cancer cells when signalling occurs via αvβ6 integrin as opposed to the canonical TGF-β1 pathway.

INTRODUCTION: Prediction of response to preoperative breast cancer chemotherapy may offer a substantial optimization of medical management of this disease. The most efficient prediction would be done a priori, before the start of chemotherapy and based on the biological features of patient and tumor. Numerous markers have been proposed but none of them has been applied as a routine. The role of MKI67 and HSP90 expression has been recently suggested to predict treatment sensitivity in HER2-positive breast cancer. The aim of this study was to validate the utility of proliferation based markers (MKI67 and CDK1) and heat shock proteins (namely HSP90) to predict response to chemotherapy in cohort of breast cancer patients treated preoperatively.
MATERIAL AND METHODS: Ninety-three patients with breast cancer, all females, mean age 42.2 years, among them 32% T1-T2 patients, 49% T3 patients and 13% with T4 tumor stage, 27% N0, 42% N1, 16% N2, 15% N3 were subjected to initial chemotherapy. The majority of patients (86%) received anthracycline and taxane chemotherapy. Among the patients there were 9 individuals with metastatic disease (M1) at initial presentation, and 11 patients were not treated surgically after initial chemotherapy (no sufficient disease response). From 82 patients operated on, 20 patients (24%) showed pathological complete response (pCR), while in 62 patients there was no pCR. 42% of patients were hormone-sensitive HER2-negative, 20% hormone-sensitive HER2-positive, 9% only HER-positive and 29% with triple negative breast cancer. Four gene transcripts (MKI67, cyclin-dependent kinase 1 [CDK1], heat shock proteins HSP90AA1 and HSP- 90AB1) were analyzed in total RNA isolated from single core obtained during preoperative core needle biopsy by quantitative real-time PCR with fluorescent probes (Universal Probe Library, Roche). Results were normalized to the panel of reference genes.
RESULTS: There were no statistically significant differences in MKI67 and CDK1 expression between pCR and no pCR groups (p = 0.099 and 0.35, respectively), although the median expression of both genes was slightly higher in pCR group. In contrast, both HSP90AA1 and HSP90AB1 transcripts showed decreased expression in pCR group (medians 0.77 and 0.55) when compared to no p CR group (median 0.86 and 0.73), statistically significant for HSP90AA1 (p = 0.031) and of borderline significance for HSP90AB1 (p = 0.054). The most significant predictor of pCR was the ratio of CDK1 transcript to HSP90AA transcript. This ratio was significantly higher in CR group (median 0.99) than in no CR group (median 0.68, p = 0.0023), and showed a potential diagnostic utility (area under receiver operating characteristic [ROC] curve 0.72).
CONCLUSIONS: HSP90AA1 and AB1 genes exhibit low expression in breast cancers highly sensitive to chemotherapy and may indicate the patients with higher probability of pathological complete response. The ratio of HSP90AA1 to proliferation-related markers (CDK1 or MKI67) may be even better predictor of pCR chance, with higher expression of proliferation genes and lower stress response in patients sensitive to chemotherapy.

Inhibition of the HSP90 chaperone results in depletion of many signaling proteins that drive tumorigenesis, such as downstream effectors of KRAS, the most commonly mutated human oncogene. As a consequence, several small-molecule HSP90 inhibitors are being evaluated in clinical trials as anticancer agents. To prospectively identify mechanisms through which HSP90-dependent cancer cells evade pharmacologic HSP90 blockade, we generated multiple mutant KRAS-driven cancer cell lines with acquired resistance to the purine-scaffold HSP90 inhibitor PU-H71. All cell lines retained dependence on HSP90 function, as evidenced by sensitivity to short hairpin RNA-mediated suppression of HSP90AA1 or HSP90AB1 (also called HSP90α and HSP90β, respectively), and exhibited two types of genomic alterations that interfere with the effects of PU-H71 on cell viability and proliferation: (i) a Y142N missense mutation in the ATP-binding domain of HSP90α that co-occurred with amplification of the HSP90AA1 locus, (ii) genomic amplification and overexpression of the ABCB1 gene encoding the MDR1 drug efflux pump. In support of a functional role for these alterations, exogenous expression of HSP90α Y142N conferred PU-H71 resistance to HSP90-dependent cells, and pharmacologic MDR1 inhibition with tariquidar or lowering ABCB1 expression restored sensitivity to PU-H71 in ABCB1-amplified cells. Finally, comparison with structurally distinct HSP90 inhibitors currently in clinical development revealed that PU-H71 resistance could be overcome, in part, by ganetespib (also known as STA9090) but not tanespimycin (also known as 17-AAG). Together, these data identify potential mechanisms of acquired resistance to small molecules targeting HSP90 that may warrant proactive screening for additional HSP90 inhibitors or rational combination therapies.

BACKGROUND: Genes in inflammatory pathways play a pivotal role in the development of colorectal cancer. We conducted a two-stage case-control study and aimed at screening the colorectal cancer-associated genetic variations in inflammatory genes.
METHODS: Twenty-three candidate variants were genotyped in 952 primary colorectal cancer cases and 875 cancer-free controls from eastern China. Promising single nucleotide polymorphisms were further genotyped in 518 cases and 554 controls from middle China. Expression quantitative trait loci and differential gene expression analyses were performed for the associated gene.
RESULTS: rs2282151 presented consistently significant associations with the risk of colorectal cancer in both stages (odds ratio (95 % confidence interval) = 1.30 (1.16-1.46), risk allele = C, P
CONCLUSIONS: Our findings indicate that the C allele of the novel colorectal cancer-associated variant rs2282151 is associated with increased expression levels of HSP90AB1, which is expressed higher in colorectal tumor tissues than in normal tissues.

Both intracellular and extracellular heat shock protein-90 (Hsp90) family proteins (α and β) have been shown to support tumour progression. The tumour-supporting activity of the intracellular Hsp90 is attributed to their N-terminal ATPase-driven chaperone function. What molecular entity determines the extracellular function of secreted Hsp90 and the distinction between Hsp90α and Hsp90β was unclear. Here we demonstrate that CRISPR/Case9 knocking out Hsp90α nullifies tumour cells' ability to migrate, invade and metastasize without affecting the cell survival and growth. Knocking out Hsp90β leads to tumour cell death. Extracellular supplementation with recombinant Hsp90α, but not Hsp90β, protein recovers tumourigenicity of the Hsp90α-knockout cells. Sequential mutagenesis identifies two evolutionarily conserved lysine residues, lys-270 and lys-277, in the Hsp90α subfamily that determine the extracellular Hsp90α function. Hsp90β subfamily lacks the dual lysine motif and the extracellular function. Substitutions of gly-262 and thr-269 in Hsp90β with lysines convert Hsp90β to a Hsp90α-like protein. Newly constructed monoclonal antibody, 1G6-D7, against the dual lysine region of secreted Hsp90α inhibits both de novo tumour formation and expansion of already formed tumours in mice. This study suggests an alternative therapeutic approach to target Hsp90 in cancer, that is, the tumour-secreted Hsp90α, instead of the intracellular Hsp90α and Hsp90β.

Network-based strategies provided by systems biology are attractive tools for cancer therapy. Modulation of cancer networks by anticancer drugs may alter the response of malignant cells and/or drive network re-organization into the inhibition of cancer progression. Previously, using systems biology approach and cancer signaling networks, we identified top-5 highly expressed and connected proteins (HSP90AB1, CSNK2B, TK1, YWHAB and VIM) in the invasive MDA-MB-231 breast cancer cell line. Here, we have knocked down the expression of these proteins, individually or together using siRNAs. The transfected cell lines were assessed for in vitro cell growth, colony formation, migration and invasion relative to control transfected MDA-MB-231, the non-invasive MCF-7 breast carcinoma cell line and the non-tumoral mammary epithelial cell line MCF-10A. The knockdown of the top-5 upregulated connectivity hubs successfully inhibited the in vitro proliferation, colony formation, anchorage independence, migration and invasion in MDA-MB-231 cells; with minimal effects in the control transfected MDA-MB-231 cells or MCF-7 and MCF-10A cells. The in vitro validation of bioinformatics predictions regarding optimized multi-target selection for therapy suggests that protein expression levels together with protein-protein interaction network analysis may provide an optimized combinatorial target selection for a highly effective anti-metastatic precision therapy in triple-negative breast cancer. This approach increases the ability to identify not only druggable hubs as essential targets for cancer survival, but also interactions most susceptible to synergistic drug action. The data provided in this report constitute a preliminary step toward the personalized clinical application of our strategy to optimize the therapeutic use of anti-cancer drugs.

BACKGROUND: Isocitrate dehydrogenase (IDH) mutations have been reported as biomarkers associated with tumorigenesis and prognosis in gliomas. However, genes affected by these mutations are still under investigation. The purpose of this study is to identify new molecular biomarkers associated with IDH mutation and prognosis in astrocytic tumors, which account for the largest proportion of gliomas.
MATERIALS AND METHODS: NanoString analysis was conducted on 40 astrocytic tumors. In total, 69 genes and 6 fusion genes were selected for screening. Quantitative real-time polymerase chain reaction and immunohistochemistry were used to validate the selected discriminatory genes. Kaplan-Meier survival curves and log-rank test were used to analyze the overall survival and progression-free survival.
RESULTS: mRNA levels of NTRK3, ERCC1, JAK2, AXL, BCL2, ESR1, HSP90AB1, TUBB3, RET, and ABCG2 were elevated in the IDH mutant group, whereas levels of POSTN and ERBB2 were elevated in the IDH wild-type group. Genes more highly expressed in the better prognosis group included NTRK3, ERCC1, ROS1, ERBB4, BCL2, CDKN2A, AXL, PI3KCA, HSP90AB1, ABCG2, JAK2, and RET. In the worse prognosis group, TIMP1, POSTN, and ERBB2 showed increased expressions. The elevated expression of HSP90AB1 was correlated with IDH mutation, long survival, and secondary glioblastomas. Elevated TIMP1 expression was related to high tumor grade and short patient survival. The results of NanoString were confirmed with quantitative real-time polymerase chain reaction and immunohistochemistry.
CONCLUSIONS: HSP90AB1 is related to IDH mutation and the expressions of HSP90AB1 and TIMP1 can predict prognosis in astrocytic tumors. The NanoString analysis system is a precise and reliable method to detect mRNA expression in formalin-fixed paraffin-embedded samples.

Exosomes are small membrane vesicles that have important roles in transporting a great variety of bioactive molecules between epithelial compartment and their microenvironment during tumor formation including colorectal adenoma-carcinoma sequence. We tested the mRNA expression of the top 25 exosome-related markers based on ExoCharta database in healthy (n=49), adenoma (n=49) and colorectal carcinoma (n=49) patients using Affymetrix HGU133 Plus2.0 microarrays. Most related genes showed significantly elevated expression including PGK1, PKM, ANXA5, ENO1, HSP90AB1 and MSN during adenoma-carcinoma sequence. Surprisingly, the expression of ALIX (ALG 2-interacting protein X), involved in multivesicular body (MVB) and exosome formation, was significantly reduced in normal vs adenoma (P=5.02 × 10(-13)) and in normal vs colorectal carcinoma comparisons (P=1.51 × 10(-10)). ALIX also showed significant reduction (P<0.05) at the in situ protein level in the epithelial compartment of adenoma (n=35) and colorectal carcinoma (n=37) patients compared with 27 healthy individuals. Furthermore, significantly reduced ALIX protein levels were accompanied by their gradual transition from diffuse cytoplasmic expression to granular signals, which fell into the 0.6-2 μm diameter size range of MVBs. These ALIX-positive particles were seen in the tumor nests, including tumor-stroma border, which suggest their exosome function. MVB-like structures were also detected in tumor microenvironment including α-smooth muscle actin-positive stromal cells, budding off cancer cells in the tumor front as well as in cancer cells entrapped within lymphoid vessels. In conclusion, we determined the top aberrantly expressed exosome-associated markers and revealed the transition of diffuse ALIX protein signals into a MVB-like pattern during adenoma-carcinoma sequence. These tumor-associated particles seen both in the carcinoma and the surrounding microenvironment can potentially mediate epithelial-stromal interactions involved in the regulation of tumor growth, metastatic invasion and therapy response.

GFG-3a is a novel glycoprotein previously purified from the fermented mycelia of Grifola frondosa with novel sugar compositions and protein sequencing. The present study aims to investigate its effects on the cell cycle, differential proteins expression, and apoptosis of human gastric cancer SGC-7901 cells. Our findings revealed that GFG-3a induced the cell apoptosis and arrested cell cycle at S phase. GFG-3a treatment resulted in the differential expression of 21 proteins in SGC-7901 cells by upregulating 10 proteins including RBBP4 associated with cell cycle arrest and downregulating 11 proteins including RUVBL1, NPM, HSP90AB1, and GRP78 involved in apoptosis and stress response. qRT-PCR and Western blot analysis also suggested that GFG-3a could increase the expressions of Caspase-8/-3, p53, Bax, and Bad while decrease the expressions of Bcl2, Bcl-xl, PI3K, and Akt1. These results indicated that the stress response, p53-dependent mitochondrial-mediated, Caspase-8/-3-dependent, and PI3k/Akt pathways were involved in the GFG-3a-induced apoptosis process in SGC-7901 cells. These findings might provide a basis to prevent or treat human gastric cancer with GFG-3a and understand the tumor-inhibitory molecular mechanisms of mushroom glycoproteins.

Citreoviridin, one of toxic mycotoxins derived from fungal species, can suppress lung cancer cell growth by inhibiting the activity of ectopic ATP synthase, but has limited effect on normal cells. However, the mechanism of citreoviridin triggering dynamic molecular responses in cancer cells remains unclear. Here, we performed temporal phosphoproteomics to elucidate the dynamic changes after citreoviridin treatment in cells and xenograft model. We identified a total of 829 phosphoproteins and demonstrated that citreoviridin treatment affects protein folding, cell cycle, and cytoskeleton function. Furthermore, response network constructed by mathematical modeling shows the relationship between the phosphorylated heat shock protein 90 β and mitogen-activated protein kinase signaling pathway. This work describes that citreoviridin suppresses cancer cell growth and mitogen-activated protein kinase/extracellular signal-regulated kinase signaling by site-specific dephosphorylation of HSP90AB1 on Serine 255 and provides perspectives in cancer therapeutic strategies.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a powerful technique for examining gene expression changes during tumorigenesis. Target gene expression is generally normalized by a stably expressed endogenous reference gene; however, reference gene expression may differ among tissues under various circumstances. Because no valid reference genes have been documented for human breast cancer cell lines containing different cancer subtypes treated with transient transfection, we identified appropriate and reliable reference genes from thirteen candidates in a panel of 10 normal and cancerous human breast cell lines under experimental conditions with/without transfection treatments with two transfection reagents. Reference gene expression stability was calculated using four algorithms (geNorm, NormFinder, BestKeeper and comparative delta Ct), and the recommended comprehensive ranking was provided using geometric means of the ranking values using the RefFinder tool. GeNorm analysis revealed that two reference genes should be sufficient for all cases in this study. A stability analysis suggests that 18S rRNA-ACTB is the best reference gene combination across all cell lines; ACTB-GAPDH is best for basal breast cancer cell lines; and HSPCB-ACTB is best for ER+ breast cancer cells. After transfection, the stability ranking of the reference gene fluctuated, especially with Lipofectamine 2000 transfection reagent in two subtypes of basal and ER+ breast cell lines. Comparisons of relative target gene (HER2) expression revealed different expressional patterns depending on the reference genes used for normalization. We suggest that identifying the most stable and suitable reference genes is critical for studying specific cell lines under certain circumstances.

Metabolic rates of cancer cells are faster compared to normal cells. This faster rate yields aberrant protein folding and causes loss of protein function. Therefore, cancer cells need more Heat Shock Proteins (HSPs) for proper substrate- protein folding on oncogenic pathways. Pseudogenes regulate tumor suppressors and oncogenes, and pseudogenes are deregulated in cancer progression. Further, alterations in miRNA expression have been identified in different cancer types. MiRNAs also have both oncogenic and tumour-suppressive roles in breast cancer post-transcriptional gene regulation. Breast cancer is a genetic disease and we performed miRNA analysis in human breast cancer cell lines to identify miRNAs in association with HSPs and pseudogenes by employing CellMiner; a web-based suite. CellMiner integrates several databases and help analysing microarray metadata. The experimental data provide a platform for researchers to compare macromolecules' relationships in NCI-60 cell lines. Breast cancer associated miRNAs gathered from literature and analyzed by employing this suite, significantly correlated HSP genes and pseudogenes in the breast cancer are determined as; HSPA13, HSP90AB1, TRAP1, HSPB1, DNAJB4, HSPD1 and HSP90AA4P, HSPB1P1, DNAJC8P1, HSPD1P9 respectively. HSPs involved in breast cancer are regulated by several miRNAs and miRNA regulators from CellMiner data found as hsa-miR-17, hsa-miR-22, hsa-miR-93, hsa-miR-106a, hsa-miR-125b, hsa-miR-130a, and hsamiR- 141. Cross check of the determined miRNAs and target HSPs was performed by target site prediction software. Comparison of the experimental data from CellMiner and software predicted data indicate differences. CellMiner data provide a vast miRNA types compared to prediction softwares-web tools data and reported miRNAs in the literature. Therefore, reported key miRNAs in this work that are not studied earlier may help cancer researchers to uncover novel posttranslational regulation mechanisms. Cancer cells use HSP network as an escape mechanism from apoptosis, therefore inhibition of associated HSPs by modulating miRNAs may provide a novel therapy for the tumorigenesis.

BACKGROUND: Burkitt lymphoma (BL) is the most common non-Hodgkin lymphoma in children worldwide and the most common paediatric malignancy in sub-Saharan Africa. The endemic (eBL) and sporadic (sBL) variants have distinct epidemiologic and virologic characteristics. Although gene expression studies have defined the transcriptional profiles of both, their proteomic signatures have not been studied.
METHODS: We compared the proteomic expression profiles using differential mass spectrometry-based isotope tag for relative and absolute quantitation (iTRAQ) analysis of a cell line representing Epstein-Barr virus (EBV)+ eBL, EBV+ and EBV- sBL, and EBV+/- normal B cells from healthy donors.
RESULTS: In total, there were 144 differentially expressed proteins with a statistically significant false discovery rate (FDR) of ⩽0.2. Results revealed over-expression of specific proteins with well-established links to lymphomagenesis such as TUBB2C (FDR 0.05), UCHL1 (FDR 0.05) and HSP90AB1 (FDR 0.1). Distinct characteristics based upon the epidemiologic and virologic subtypes of BL were also identified. In sBL, PCNA (FDR 0.05) and SLC3A2 (FDR 0.1) were significantly over-expressed. In eBL, C1QBP (FDR 0.1) and ENO1 (FDR 0.25) were significantly over-expressed. Comparison of EBV+ to EBV- BL cell lines and B cells revealed significant over-expression of DDX3X (FDR 0.1). Proteins were validated using Western blot analysis.
CONCLUSION: Our results suggest unique signal transduction pathways associated with EBV infection and epidemiological subtype of BL that may contribute to lymphomagenesis. These proteomic findings provide potential diagnostic, prognostic and therapeutic links to BL.

Genetic mutations are known to drive cancer progression and certain tumors have mutation signatures that reflect exposures to environmental carcinogens. Benzo[a]pyrene (BaP) has a known mutation signature and has proven capable of inducing changes to DNA sequence that drives normal pre-stasis human mammary epithelial cells (HMEC) past a first tumor suppressor barrier (stasis) and toward immortality. We analyzed normal, pre-stasis HMEC, three independent BaP-derived post-stasis HMEC strains (184Aa, 184Be, 184Ce) and two of their immortal derivatives(184A1 and 184BE1) by whole exome sequencing. The independent post-stasis strains exhibited between 93 and 233 BaP-induced mutations in exons. Seventy percent of the mutations were C:G>A:T transversions, consistent with the known mutation spectrum of BaP. Mutations predicted to impact protein function occurred in several known and putative cancer drivers including p16, PLCG1, MED12, TAF1 in 184Aa; PIK3CG, HSP90AB1, WHSC1L1, LCP1 in 184Be and FANCA, LPP in 184Ce. Biological processes that typically harbor cancer driver mutations such as cell cycle, regulation of cell death and proliferation, RNA processing, chromatin modification and DNA repair were found to have mutations predicted to impact function in each of the post-stasis strains. Spontaneously immortalized HMEC lines derived from two of the BaP-derived post-stasis strains shared greater than 95% of their BaP-induced mutations with their precursor cells. These immortal HMEC had 10 or fewer additional point mutations relative to their post-stasis precursors, but acquired chromosomal anomalies during immortalization that arose independent of BaP. The results of this study indicate that acute exposures of HMEC to high dose BaP recapitulate mutation patterns of human tumors and can induce mutations in a number of cancer driver genes.

AIM: Hsp90-β and annexin A1 have been demonstrated to be associated with tumorigenesis. However, the effect of Hsp90-β and annexin A1 in lung cancer remains poorly understood. In this research, the correlation of Hsp90-β and annexin A1 in lung cancer patients were analyzed.
METHODS: The expression levels of Hsp90-β and annexin A1 were examined by immunohistochemistry and ELISA.
RESULTS: Lung cancer tissues and serum exhibited higher co-expression of Hsp90-β and annexin A1 than control groups (p < 0.05). Hsp90-β and annexin A1 could discriminate lung cancer from the control groups (sensitivity of Hsp90-β was 80.2% in tissues and 96% in serum; specificity of Hsp90-β was 80% in tissues and 83.33% in serum; sensitivity of annexin A1 was 68.76% in tissues and 95.23% in serum; specificity of annexin A1 was 75% in tissues and 85.7% in serum) and multi-index combined detection had a better diagnostic value.
CONCLUSION: The expression levels of Hsp90-β and annexin A1 positively correlated and such co-overexpression of Hsp90-β and annexin A1 contributed to lung cancer diagnosis.

BACKGROUND/AIM: Heat-shock proteins (HSPs) are molecular chaperones which modify the structures and interactions of other proteins. The aim of our study was to investigate HSP90AA1, HSP90AB1 and HSP90B1 gene polymorphisms in patients with non-small cell lung cancer (NSCLC).
MATERIALS AND METHODS: Ninety-seven patients with NSCLC and 97 healthy controls were included in the study. Real-time polymerase chain reaction was used for genotyping.
RESULTS: The frequency of mutant CC genotype for HSP90AA1 (rs4947C/T), mutant AA genotype for HSP90AB1 (rs13296A/G) and mutant CC genotype for HSP90B1 (rs2070908 C/G) was significantly higher in the patient group than in controls (p=0.019, p=0.004 and p=0.036, respectively). The frequency of patients with homozygote mutant allele was also significantly higher than that of controls and possessing of the mutant genotype increased the risk for disease by approximately 2.9, 4.8, 1.9 for HSP90AA1, HSP90AB1 and HSP90B1, respectively. The present study appears to be the first of its kind to report data on these gene polymorphisms in patients with NSCLC in the Turkish population.

PURPOSE: The growth and survival of neurofibromatosis type 2 (NF2)-deficient cells are enhanced by the activation of multiple signaling pathways including ErbBs/IGF-1R/Met, PI3K/Akt, and Ras/Raf/Mek/Erk1/2. The chaperone protein HSP90 is essential for the stabilization of these signaling molecules. The aim of the study was to characterize the effect of HSP90 inhibition in various NF2-deficient models.
EXPERIMENTAL DESIGN: We tested efficacy of the small-molecule NXD30001, which has been shown to be a potent HSP90 inhibitor. The antiproliferative activity of NXD30001 was tested in NF2-deficient cell lines and in human primary schwannoma and meningioma cultures in vitro. The antitumor efficacy of HSP90 inhibition in vivo was verified in two allograft models and in one NF2 transgenic model. The underlying molecular alteration was further characterized by a global transcriptome approach.
RESULTS: NXD30001 induced degradation of client proteins in and suppressed proliferation of NF2-deficient cells. Differential expression analysis identified subsets of genes implicated in cell proliferation, cell survival, vascularization, and Schwann cell differentiation whose expression was altered by NXD30001 treatment. The results showed that NXD30001 in NF2-deficient schwannoma suppressed multiple pathways necessary for tumorigenesis.
CONCLUSIONS: HSP90 inhibition showing significant antitumor activity against NF2-related tumor cells in vitro and in vivo represents a promising option for novel NF2 therapies.

The mechanisms of successful epigenetic reprogramming in cancer are not well characterized as they involve coordinated removal of repressive marks and deposition of activating marks by a large number of histone and DNA modification enzymes. Here, we have used a cross-species functional genomic approach to identify conserved genetic interactions to improve therapeutic effect of the histone deacetylase inhibitor (HDACi) valproic acid, which increases survival in more than 20% of patients with advanced acute myeloid leukemia (AML). Using a bidirectional synthetic lethality screen revealing genes that increased or decreased VPA sensitivity in C. elegans, we identified novel conserved sensitizers and synthetic lethal interactors of VPA. One sensitizer identified as a conserved determinant of therapeutic success of HDACi was UTX (KDM6A), which demonstrates a functional relationship between protein acetylation and lysine-specific methylation. The synthetic lethal screen identified resistance programs that compensated for the HDACi-induced global hyper-acetylation, and confirmed MAPKAPK2, HSP90AA1, HSP90AB1 and ACTB as conserved hubs in a resistance program for HDACi that are drugable in human AML cell lines. Hence, these resistance hubs represent promising novel targets for refinement of combinatorial epigenetic anti-cancer therapy.

Pancreas cancer cells escape most treatment options. Heat shock protein (Hsp)90 is frequently over-expressed in pancreas carcinomas and protects a number of cell-cycle regulators such as the proto-oncogene Cdc25A. We show that inhibition of Hsp90 with geldanamycin (GD) destabilizes Cdc25A independent of Chk1/2, whereas the standard drug for pancreas carcinoma treatment, gemcitabine (GEM), causes Cdc25A degradation through the activation of Chk2. Both agents applied together additively inhibit the expression of Cdc25A and the proliferation of pancreas carcinoma cells thereby demonstrating that both Cdc25A-destabilizing/degrading pathways are separated. The role of Hsp90 as stabilizer of Cdc25A in pancreas carcinoma cells is further supported by two novel synthetic inhibitors 4-tosylcyclonovobiocic acid and 7-tosylcyclonovobiocic acid and specific Hsp90AB1 (Hsp90β) shRNA. Our data show that targeting Hsp90 reduced the resistance of pancreas carcinoma cells to treatment with GEM.

The mTOR complex 1 (mTORC1) kinase nucleates a pathway that promotes cell growth and proliferation and is the target of rapamycin, a drug with many clinical uses. mTORC1 regulates messenger RNA translation, but the overall translational program is poorly defined and no unifying model exists to explain how mTORC1 differentially controls the translation of specific mRNAs. Here we use high-resolution transcriptome-scale ribosome profiling to monitor translation in mouse cells acutely treated with the mTOR inhibitor Torin 1, which, unlike rapamycin, fully inhibits mTORC1 (ref. 2). Our data reveal a surprisingly simple model of the mRNA features and mechanisms that confer mTORC1-dependent translation control. The subset of mRNAs that are specifically regulated by mTORC1 consists almost entirely of transcripts with established 5' terminal oligopyrimidine (TOP) motifs, or, like Hsp90ab1 and Ybx1, with previously unrecognized TOP or related TOP-like motifs that we identified. We find no evidence to support proposals that mTORC1 preferentially regulates mRNAs with increased 5' untranslated region length or complexity. mTORC1 phosphorylates a myriad of translational regulators, but how it controls TOP mRNA translation is unknown. Remarkably, loss of just the 4E-BP family of translational repressors, arguably the best characterized mTORC1 substrates, is sufficient to render TOP and TOP-like mRNA translation resistant to Torin 1. The 4E-BPs inhibit translation initiation by interfering with the interaction between the cap-binding protein eIF4E and eIF4G1. Loss of this interaction diminishes the capacity of eIF4E to bind TOP and TOP-like mRNAs much more than other mRNAs, explaining why mTOR inhibition selectively suppresses their translation. Our results clarify the translational program controlled by mTORC1 and identify 4E-BPs and eIF4G1 as its master effectors.

INTRODUCTION: Although human epidermal growth factor receptor 2 (HER2) positive or estrogen receptor (ER) positive breast cancers are treated with clinically validated anti-HER2 or anti-estrogen therapies, intrinsic and acquired resistance to these therapies appears in a substantial proportion of breast cancer patients and new therapies are needed. Identification of additional molecular factors, especially those characterized by aggressive behavior and poor prognosis, could prioritize interventional opportunities to improve the diagnosis and treatment of breast cancer.
METHODS: We compiled a collection of 4,010 breast tumor gene expression data derived from 23 datasets that have been posted on the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database. We performed a genome-scale survival analysis using Cox-regression survival analyses, and validated using Kaplan-Meier Estimates survival and Cox Proportional-Hazards Regression survival analyses. We conducted a genome-scale analysis of chromosome alteration using 481 breast cancer samples obtained from The Cancer Genome Atlas (TCGA), from which combined expression and copy number data were available. We assessed the correlation between somatic copy number alterations and gene expression using analysis of variance (ANOVA).
RESULTS: Increased expression of each of the heat shock protein (HSP) 90 isoforms, as well as HSP transcriptional factor 1 (HSF1), was correlated with poor prognosis in different subtypes of breast cancer. High-level expression of HSP90AA1 and HSP90AB1, two cytoplasmic HSP90 isoforms, was driven by chromosome coding region amplifications and were independent factors that led to death from breast cancer among patients with triple-negative (TNBC) and HER2-/ER+ subtypes, respectively. Furthermore, amplification of HSF1 was correlated with higher HSP90AA1 and HSP90AB1 mRNA expression among the breast cancer cells without amplifications of these two genes. A collection of HSP90AA1, HSP90AB1 and HSF1 amplifications defined a subpopulation of breast cancer with up-regulated HSP90 gene expression, and up-regulated HSP90 expression independently elevated the risk of recurrence of TNBC and poor prognosis of HER2-/ER+ breast cancer.
CONCLUSIONS: Up-regulated HSP90 mRNA expression represents a confluence of genomic vulnerability that renders HER2 negative breast cancers more aggressive, resulting in poor prognosis. Targeting breast cancer with up-regulated HSP90 may potentially improve the effectiveness of clinical intervention in this disease.

The anticancer drug belinostat is a hydroxamate histone deacetylase inhibitor that has shown significant antitumour activity in various tumour models and also in clinical trials. In this study, we utilized a proteomic approach in order to evaluate the effect of this drug on protein expression in the human colon cancer cell line HCT116. Protein extracts from untreated HCT116 cells, and cells grown for 24 h in the presence of 1 and 10 muM belinostat were analysed by 2-D gel electrophoresis. Proteins were visualized by colloidal Coomassie blue staining and quantitative analysis of gel images revealed 45 unique differentially expressed proteins that were identified by LC-MSMS analysis. Among these proteins, of particular interest are the downregulated proteins nucleophosmin and stratifin, and the upregulated proteins nucleolin, gelsolin, heterogeneous nuclear ribonucleoprotein K, annexin 1, and HSP90B that all were related to the proto-oncogene proteins p53, Myc, activator protein 1, and c-fos protein. The modulation of these proteins is consistent with the observations that belinostat is able to inhibit clonogenic cell growth of HCT116 cells and the biological role of these proteins will be discussed.

Increased attention has been paid to the determination of the potential biomarker and therapeutic target for ovarian cancer in recent years. However, the normalization of quantitative real-time PCR is important to obtain accurate gene expression data. We investigated the stability of 20 reference genes in ovarian tissues under different conditions to determine the most adequate for this application. The study characterized the expression of 20 possible reference genes among 52 ovarian tissue samples involving the normal, non-malignant, and primary ovarian carcinomas. One-way analysis of variance (ANOVA) method was used to compare the candidate gene changes brought about by the disease progression. The stability and suitability of the genes with no statistic difference were further validated employing geNorm and NormFinder softwares. Results showed that the expression levels of the 20 reference genes varied, while the RPL4, RPLP0, HSPCB, TPT1, RPL13A, 18S rRNA, PPIA, TBP, and GUSB kept statistic stability despite different ovarian tissue conditions. RPL4, RPLP0, and HSPCB were demonstrated as the most stable reference genes and the combination of the RPLP0 and RPL4 should be recommended as a much more reliable normalization strategy.