Gastric cancer is a leading cause of cancer deaths, but analysis of its molecular and clinical characteristics has been complicated by histological and aetiological heterogeneity. Here we describe a comprehensive molecular evaluation of 295 primary gastric adenocarcinomas as part of The Cancer Genome Atlas (TCGA) project. We propose a molecular classification dividing gastric cancer into four subtypes: tumours positive for Epstein–Barr virus, which display recurrent PIK3CA mutations, extreme DNA hypermethylation, and amplification of JAK2, CD274 (also known as PD-L1) and PDCD1LG2 (also knownasPD-L2); microsatellite unstable tumours, which show elevated mutation rates, including mutations of genes encoding targetable oncogenic signalling proteins; genomically stable tumours, which are enriched for the diffuse histological variant and mutations of RHOA or fusions involving RHO-family GTPase-activating proteins; and tumours with chromosomal instability, which show marked aneuploidy and focal amplification of receptor tyrosine kinases. Identification of these subtypes provides a roadmap for patient stratification and trials of targeted therapies.
Reverse phase protein arrays (RPPA) are an efficient, high-throughput, cost-effective method for the quantification of specific proteins in complex biological samples. The quality of RPPA data may be affected by various sources of error. One of these, spatial variation, is caused by uneven exposure of different parts of an RPPA slide to the reagents used in protein detection. We present a method for the determination and correction of systematic spatial variation in RPPA slides using positive control spots printed on each slide. The method uses a simple bi-linear interpolation technique to obtain a surface representing the spatial variation occurring across the dimensions of a slide. This surface is used to calculate correction factors that can normalize the relative protein concentrations of the samples on each slide. The adoption of the method results in increased agreement between technical and biological replicates of various tumor and cell-line derived samples. Further, in data from a study of the melanoma cell-line SKMEL-133, several slides that had previously been rejected because they had a coefficient of variation (CV) greater than 15%, are rescued by reduction of CV below this threshold in each case. The method is implemented in the R statistical programing language. It is compatible with MicroVigene and SuperCurve, packages commonly used in RPPA data analysis. The method is made available, along with suggestions for implementation, at http://bitbucket.org/rppa_preprocess/rppa_preprocess/src.
Protein levels and function are poorly predicted by genomic and transcriptomic analysis of patient tumors. Therefore, direct study of the functional proteome has the potential to provide a wealth of information that complements and extends genomic, epigenomic and transcriptomic analysis in The Cancer Genome Atlas (TCGA) projects. Here we use reverse-phase protein arrays to analyze 3,467 patient samples from 11 TCGA “Pan-Cancer” diseases, using 181 high-quality antibodies that target 128 total proteins and 53 post-translationally modified proteins. The resultant proteomic data is integrated with genomic and transcriptomic analyses of the same samples to identify commonalities, differences, emergent pathways and network biology within and across tumor lineages. In addition, tissue-specific signals are reduced computationally to enhance biomarker and target discovery spanning multiple tumor lineages. This integrative analysis, with an emphasis on pathways and potentially actionable proteins, provides a framework for determining the prognostic, predictive and therapeutic relevance of the functional proteome.
Proteomics; TCGA; Pan-Cancer; protein expression; protein networks
Loading control (LC) and variance stabilization of reverse-phase protein array (RPPA) data have been challenging mainly due to the small number of proteins in an experiment and the lack of reliable inherent control markers. In this study, we compare eight different normalization methods for LC and variance stabilization. The invariant marker set concept was first applied to the normalization of high-throughput gene expression data. A set of “invariant” markers are selected to create a virtual reference sample. Then all the samples are normalized to the virtual reference. We propose a variant of this method in the context of RPPA data normalization and compare it with seven other normalization methods previously reported in the literature. The invariant marker set method performs well with respect to LC, variance stabilization and association with the immunohistochemistry/florescence in situ hybridization data for three key markers in breast tumor samples, while the other methods have inferior performance. The proposed method is a promising approach for improving the quality of RPPA data.
reverse-phase protein array; RPPA; normalization; proteomics
Despite numerous therapies that effectively inhibit estrogen signaling in breast cancer, a significant proportion of patients with estrogen receptor (ER)-positive malignancy will succumb to their disease. Herein we demonstrate that long-term estrogen deprivation (LTED) therapy among ER-positive breast cancer cells results in the adaptive increase in ER expression and subsequent activation of multiple tyrosine kinases. Combination therapy with the ER down-regulator fulvestrant and dasatinib, a broad kinase inhibitor, exhibits synergistic activity against LTED cells, by reduction of cell proliferation, cell survival, cell invasion and mammary acinar formation. Screening kinase phosphorylation using protein arrays and functional proteomic analysis demonstrates that the combination of fulvestrant and dasatinib inhibits multiple tyrosine kinases and cancer-related pathways that are constitutively activated in LTED cells. Because LTED cells display increased insulin receptor (InsR)/insulin-like growth factor 1 receptor (IGF-1R) signaling, we added an ant-IGF-1 antibody to the combination with fulvestrant and dasatinib in an effort to further increase the inhibition. However, adding MK0646 only modestly increased the inhibition of cell growth in monolayer culture, but neither suppressed acinar formation nor inhibited cell migration in vitro and invasion in vivo. Therefore, combinations of fulvestrant and dasatinib, but not MK0646, may benefit patients with tyrosine-kinase-activated, endocrine therapy-resistant breast cancer.
breast cancer; targeting therapy; dasatinib; fulvestrant; MK0646
Lysophosphatidic acid (LPA) acts through high affinity G protein-coupled receptors to mediate a plethora of physiological and pathological activities associated with tumorigenesis. LPA receptors and autotaxin (ATX/LysoPLD), the primary enzyme producing LPA, are aberrantly expressed in multiple cancer lineages. However, the role of ATX and LPA receptors in the initiation and progression of breast cancer has not been evaluated. We demonstrate that expression of ATX or each Edg-family LPA receptor in mammary epithelium of transgenic mice is sufficient to induce a high frequency of late-onset, estrogen receptor (ER) positive, invasive and metastatic mammary cancer. Thus ATX and LPA receptors can contribute to the initiation and progression of breast cancer.
LPA; ATX; Transgenic mouse model; Breast cancer; Metastasis
Restricted Boolean networks are simplified Boolean networks that are required for either negative or positive regulations between genes. Higa et al. (BMC Proc 5:S5, 2011) proposed a three-rule algorithm to infer a restricted Boolean network from time-series data. However, the algorithm suffers from a major drawback, namely, it is very sensitive to noise. In this paper, we systematically analyze the regulatory relationships between genes based on the state switch of the target gene and propose an algorithm with which restricted Boolean networks may be inferred from time-series data. We compare the proposed algorithm with the three-rule algorithm and the best-fit algorithm based on both synthetic networks and a well-studied budding yeast cell cycle network. The performance of the algorithms is evaluated by three distance metrics: the normalized-edge Hamming distance μhame, the normalized Hamming distance of state transition μhamst, and the steady-state distribution distance μssd. Results show that the proposed algorithm outperforms the others according to both μhame and μhamst, whereas its performance according to μssd is intermediate between best-fit and the three-rule algorithms. Thus, our new algorithm is more appropriate for inferring interactions between genes from time-series data.
Restricted Boolean network; Inference; Budding yeast cell cycle
With the development of space science, it is important to analyze the relationship between the space environment and genome variations that might cause phenotypic changes in microbes. Klebsiella pneumoniae is commonly found on the human body and is resistant to multiple drugs. To study space-environment-induced genome variations and drug resistance changes, K. pneumoniae was carried into outer space by the Shenzhou VIII spacecraft.
The K. pneumoniae strain LCT-KP289 was selected after spaceflight based on its phenotypic differences compared to the ground-control strain. Analysis of genomic structural variations revealed one inversion, 25 deletions, fifty-nine insertions, two translocations and six translocations with inversions. In addition, 155 and 400 unique genes were observed in LCT-KP214 and LCT-KP289, respectively, including the gene encoding dihydroxyacetone kinase, which generates the ATP and NADH required for microbial growth. Furthermore, a large number of mutant genes were related to transport and metabolism. Phylogenetic analysis revealed that most genes in these two strains had a dN/dS value greater than 1, indicating that the strain diversity increased after spaceflight. Analysis of drug-resistance phenotypes revealed that the K. pneumoniae strain LCT-KP289 was resistant to sulfamethoxazole, whereas the control strain, LCT-KP214, was not; both strains were resistant to benzylpenicillin, ampicillin, lincomycin, vancomycin, chloramphenicol and streptomycin. The sulfamethoxazole resistance may be associated with sequences in Scaffold7 in LCT-KP289, which were not observed in LCT-K214; this scaffold contained the gene sul1. In the strain LCT-KP289, we also observed a drug-resistance integron containing emrE (confers multidrug resistance) and ant (confers resistance to spectinomycin, streptomycin, tobramycin, kanamycin, sisomicin, dibekacin, and gentamicin). The gene ampC (confers resistance to penicillin, cephalosporin-ii and cephalosporin-i) was present near the integron. In addition, 30 and 26 drug-resistance genes were observed in LCT-KP289 and LCT-KP214, respectively.
Comparison of a K. pneumoniae strain obtained after spaceflight with the ground-control strain revealed genome variations and phenotypic changes and elucidated the genomic basis of the acquired drug resistance. These data pave the way for future studies on the effects of spaceflight.
Klebsiella pneumoniae; Comparative genomic analysis; Virulence gene; Resistance gene
Aberrations in oncogenes and tumor suppressors frequently affect the activity of critical signal transduction pathways. To analyze systematically the relationship between the activation status of protein networks and other characteristics of cancer cells, we performed reverse phase protein array (RPPA) profiling of the NCI60 cell lines for total protein expression and activation-specific markers of critical signaling pathways. To extend the scope of the study, we merged those data with previously published RPPA results for the NCI60. Integrative analysis of the expanded RPPA data set revealed 5 major clusters of cell lines and 5 principal proteomic signatures. Comparison of mutations in the NCI60 cell lines with patterns of protein expression demonstrated significant associations for PTEN, PIK3CA, BRAF and APC mutations with proteomic clusters. PIK3CA and PTEN mutation enrichment were not cell lineage-specific but were associated with dominant yet distinct groups of proteins. The five RPPA-defined clusters were strongly associated with sensitivity to standard anti-cancer agents. RPPA analysis identified 27 protein features significantly associated with sensitivity to paclitaxel. The functional status of those proteins was interrogated in a paclitaxel whole genome siRNA library synthetic lethality screen, and confirmed the predicted associations with drug sensitivity. These studies expand our understanding of the activation status of protein networks in the NCI60 cancer cell lines, demonstrate the importance of the direct study of protein expression and activation, and provide a basis for further studies integrating the information with other molecular and pharmacological characteristics of cancer.
NCI60; reverse phase protein arrays; signal transduction
Riemerella anatipestifer is one of the most important pathogens of ducks. However, the molecular mechanisms of R. anatipestifer infection are poorly understood. In particular, the lack of genomic information from a variety of R. anatipestifer strains has proved severely limiting.
In this study, we present the complete genomes of two R. anatipestifer strains, RA-CH-1 (2,309,519 bp, Genbank accession CP003787) and RA-CH-2 (2,166,321 bp, Genbank accession CP004020). Both strains are from isolates taken from two different sick ducks in the SiChuang province of China. A comparative genomics approach was used to identify similarities and key differences between RA-CH-1 and RA-CH-2 and the previously sequenced strain RA-GD, a clinical isolate from GuangDong, China, and ATCC11845.
The genomes of RA-CH-2 and RA-GD were extremely similar, while RA-CH-1 was significantly different than ATCC11845. RA-CH-1 is 140,000 bp larger than the three other strains and has 16 unique gene families. Evolutionary analysis shows that RA-CH-1 and RA-CH-2 are closed and in a branch with ATCC11845, while RA-GD is located in another branch. Additionally, the detection of several iron/heme-transport related proteins and motility mechanisms will be useful in elucidating factors important in pathogenicity. This information will allow a better understanding of the phenotype of different R. anatipestifer strains and molecular mechanisms of infection.
Riemerella anatipestifer; Comparative genomics; Structural variation
DNA methylation is involved in tissue-specific and developmentally regulated gene expression. Here, we screened a novel methylation gene Sox30, whose methylation might contribute to its regulation and testis development in mice. Sox30 is a member of Sox transcription factors, and is considered to be involved in spermatogonial differentiation and spermatogenesis. However, the precise function and regulatory expression pattern remain unclear. In the present study, we found that Sox30 is highly expressed in adult testes but not in ovaries. Sox30 expression begins in early development, and in the testes, it is specifically increased coincidentally with development until adulthood. Moreover, Sox30 is expressed not only in testis germ cells, but also in sertoli cells. Sox30 is hypo-methylated in testis, epididymis and lung of adult mice, in which Sox30 is expressed. By contrast, Sox30 is hypermethylated in ovary, heart, brain, liver, kidney, spleen, pancreas, muscle, intestine, pituitary gland, blood and hippocampus of adult mice, in which the Sox30 is absent. Importantly, decreased methylation at CpG islands of Sox30 is observed in mouse developmental testes after birth, which is associated with enhanced Sox30 expression. However, the hypermethylated status of Sox30 is maintained in ovaries that does not express Sox30 during this period. Further, following demethylation treatment using 5-aza-dC, Sox30 expression is restored in GC2, TM3 and TM4 cell lines. This observation convincingly confirms that methylation really contributes to Sox30 silencing. In summary, we show that Sox30 expression is under the control of DNA methylation status, and this expression pattern is associated with testis development in mice.
Blunt snout bream (Megalobrama amblycephala) are susceptible to hepatic steatosis when maintained in modern intensive culture systems. The aim of this study was to investigate the potential roles of microRNAs (miRNAs) in diet-induced hepatic steatosis in this species. MiRNAs, small non-coding RNAs that regulate gene expression at the posttranscriptional level, are involved in diverse biological processes, including lipid metabolism. Deep sequencing of hepatic small RNA libraries from blunt snout bream fed normal-fat and high-fat diets identified 202 (193 known and 9 novel) miRNAs, of which 12 were differentially expressed between the normal-fat and high-fat diet groups. Quantitative stem-loop reverse transcriptase-polymerase chain reaction analyses confirmed the upregulation of miR-30c and miR-30e-3p and the downregulation of miR-145 and miR-15a-5p in high-fat diet-fed fish. Bioinformatics tools were used to predict the targets of these verified miRNAs and to explore potential downstream gene ontology biological process categories and Kyoto Encyclopedia of Genes and Genomes pathways. Six putative lipid metabolism-related target genes (fetuin-B, Cyp7a1, NADH dehydrogenase (ubiquinone) 1 beta subcomplex subunit 2, 3-oxoacid CoA transferase 1b, stearoyl-CoA desaturase, and fatty-acid synthase) were identified as having potential important roles in the development of diet-induced hepatic steatosis in blunt snout bream. The results presented here are a foundation for future studies of miRNA-controlled lipid metabolism regulatory networks in blunt snout bream.
The Rheocricotopus (Psilocricotopus) chalybeatus species group from China is reviewed. Three new species, R. (P.) brochus
sp. n., R. (P.) rotundus
sp. n. and R. (P.) serratus
sp. n. are described as adult males. R. (P.) imperfectus Makarchenko & Makarchenko, 2005, R. (P.) robacki (Beck & Beck, 1964) and R. (P.) valgus Chaudhuri & Sinharay, 1983 are recorded from China for the first time and annotated. The diagnosis for the species group is emended and a key to adult males of the species group in China is presented.
Chironomidae; Rheocricotopus; chalybeatus species group; new species; key; China
Hematologic disorders are very common in Systmic lupus erythematosus (SLE).First presentation of SLE with severe aplastic anemia (SAA) is extremely rare. We report a patient with the diagnosis of secondary SAA associated with SLE. Conventional therapy was not effective. She received Rituximab (RTX) and Antithymocyte globulin (ATG) therapy, her response was satisfactory finally. Her hematologic parameters were within normal ranges until last follow-up, eight months and six months after therapy was initiated with RTX and ATG, respectively. This is the first time RTX and ATG were successfully used in the treatment of SAA secondary to SLE.
Aplastic anemia; Rituximab; Antithymocyte globulin; Systmic lupus erythematosus
This phase I clinical trial was conducted to determine the safety, efficacy, and molecular effects of sorafenib with temsirolimus in patients with advanced melanoma.
Patients and Methods
Patients with stage IV or unresectable or recurrent stage III melanoma and ECOG performance status of 0 to 1 were eligible. Sorafenib was given orally once or twice daily and temsirolimus was given intravenously weekly, both starting on day 1, with a 4-week cycle. Responses were assessed every 2 cycles per RECIST criteria. Consenting patients with accessible tumors underwent optional tumor biopsies prior to treatment and after the second infusion of temsirolimus. Tumor biopsies were analyzed for activating mutations in BRAF and NRAS, and for expression of P-ERK and P-S6 proteins.
A total of 25 patients were accrued to the study. The MTD doses were sorafenib 400 mg qAM / 200 mg qPM daily and temsirolimus 25 mg IV weekly. Dose-limiting toxicities included thrombocytopenia, hand-foot syndrome (HFS), serum transaminase elevation and hypertriglyceridemia. There were no complete (CR) or partial (PR) responses with the combination; 10 patients achieved stabilization of disease as their best response. The median progression-free survival (PFS) was 2.1 months. Matching pre-treatment and day 15 tumor biopsies demonstrated marked inhibition of P-S6 with treatment in 3 of 4 evaluable patients, but minimal inhibition of P-ERK.
Combination therapy with sorafenib and temsirolimus resulted in significant toxicity at higher dose levels, failed to achieve any clinical responses in genetically unselected patient population, and did not inhibit P-ERK.
phase I; sorafenib; temsirolimus; metastatic melanoma; BRAF
The Klebsiella pneumoniae strain LCT-KP182 acquired hemolytic properties after space flight. Here, we present the draft genome sequence of this strain.
To characterize patient-derived xenografts (PDXs) for functional studies,
we made whole-genome comparisons with originating breast cancers representative
of the major intrinsic subtypes. Structural and copy number aberrations were
found to be retained with high fidelity. However, at the single-nucleotide
level, variable numbers of PDX-specific somatic events were documented, although
they were only rarely functionally significant. Variant allele frequencies were
often preserved in the PDXs, demonstrating that clonal representation can be
transplantable. Estrogen-receptor-positive PDXs were associated with
ESR1 ligand-binding-domain mutations, gene amplification,
or an ESR1/YAP1 translocation. These events produced different
endocrine-therapy-response phenotypes in human, cell line, and PDX
endocrine-response studies. Hence, deeply sequenced PDX models are an important
resource for the search for genome-forward treatment options and capture
endocrine-drug-resistance etiologies that are not observed in standard cell
lines. The originating tumor genome provides a benchmark for assessing genetic
drift and clonal representation after transplantation.
In the present study, the effects of metformin on the proliferation of human immortalized keratinocytes (HaCaTs) and the underlying mechanisms were investigated. HaCaT cells in the logarithmic growth phase were treated with 50 mM metformin for 24, 48 and 72 h. Cell morphology after 24 h of treatment was observed under a microscope. Cell proliferation was detected using a colorimetric cell proliferation and cytotoxicity assay kit. Western blot analyses were performed to detect the protein phosphorylation levels of adenosine monophosphate-activated protein kinase (AMPK) and extracellular signal-related kinase 1/2 (ERK1/2). Metformin treatment resulted in morphological changes of the HaCaT cells. The survival rates of HaCaT cells treated with metformin were 36.18, 12.70 and 10.12% at 24, 48 and 72 h, respectively. As the treatment time extended, the survival rates of HaCaT cells decreased. Western blot analysis results showed that the mean level of phosphorylated (p)-AMPK in the HaCaT cells without metformin treatment was 2.856±0.323. However, the mean p-AMPK level following metformin treatment for 24 h increased to 5.198±0.625, indicating a significant difference between these two groups (P<0.05). The mean absorbance ratio of p-ERK1/2 was 7.550±1.087 for the untreated cells, but the levels in cells following metformin treatment for 24 h increased to 10.430±1.217, indicating a significant difference between the two groups (P<0.05). In conclusion, metformin treatment upregulated the levels of p-AMPK and p-ERK1/2 in HaCaT cells, and significantly inhibited HaCaT cell proliferation in vitro by a mechanism associated with activation of the mitogen-activated protein kinase signaling pathway.
metformin; HaCaT cells; proliferation; psoriasis; adenosine monophosphate-activated protein kinase; extracellular signal-related kinase 1/2
Small cell lung cancer (SCLC) is an aggressive malignancy distinct from non-small cell lung cancer (NSCLC) in its metastatic potential and treatment response. Using an integrative proteomic and transcriptomic analysis, we investigated molecular differences contributing to the distinct clinical behavior of SCLC and NSCLC. SCLC demonstrated lower levels of several receptor tyrosine kinases and decreased activation of PI3K and Ras/MEK pathways, but significantly increased levels of E2F1-regulated factors including EZH2, thymidylate synthase, apoptosis mediators, and DNA repair proteins. Additionally, poly (ADP-ribose) polymerase 1 (PARP1), a DNA repair protein and E2F1 co-activator, was highly expressed at the mRNA and protein levels in SCLC. SCLC growth was inhibited by PARP1 and EZH2 knockdown. Furthermore, SCLC was significantly more sensitive to PARP inhibitors than NSCLC, and PARP inhibition downregulated key components of the DNA repair machinery and enhanced the efficacy of chemotherapy.
Osterix (Osx) is an osteoblast-specific transcription factor which is essential for bone formation. MicroRNAs (miRNAs) have been previously shown to be involved in osteogenesis. However, it is unclear whether Osx is involved in the regulation of miRNA expression. In this study, we have identified groups of miRNAs that are differentially expressed in calvaria of the E18.5 Osx−/− embryos compared to wild type embryos. The correlation between the levels of miRNAs and Osx expression was further verified in cultured M-Osx cells in which over-expression of Osx is inducible. Our results suggest that Osx down-regulates expression of a group of miRNAs including mir-133a and -204/211, but up-regulates expression of another group of miRNAs such as mir-141/200a. Mir-133a and -204/211 are known to target the master osteogenic transcription factor Runx2. Further assays suggest that Sost, which encodes the Wnt signaling antagonist Sclerostin, and alkaline phosphatase (ALP) are two additional targets of mir-204/211. Mir-141/200a has been known to target the transcription factor Dlx5. Thus, we postulate that during the process of Osx-controlled osteogenesis, Osx has the ability to coordinately modulate Runx2, Sclerostin, ALP and Dlx5 proteins at levels appropriate for optimal osteoblast differentiation and function, at least in part, through regulation of specific miRNAs. Our study shows a tight correlation between Osx and the miRNAs involved in bone formation, and provides new information about molecular mechanisms of Osx-controlled osteogenesis.
Protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues is challenging due to extensive molecular crosslinking that occurs upon formalin fixation. Reverse-phase protein array (RPPA) is a high-throughput technology, which can detect changes in protein levels and protein functionality in numerous tissue and cell sources. It has been used to evaluate protein expression mainly in frozen preparations or FFPE-based studies of limited scope. Reproducibility and reliability of the technique in FFPE samples has not yet been demonstrated extensively. We developed and optimized an efficient and reproducible procedure for extraction of proteins from FFPE cells and xenografts, and then applied the method to FFPE patient tissues and evaluated its performance on RPPA.
Fresh frozen and FFPE preparations from cell lines, xenografts and breast cancer and renal tissues were included in the study. Serial FFPE cell or xenograft sections were deparaffinized and extracted by six different protein extraction protocols. The yield and level of protein degradation were evaluated by SDS-PAGE and Western Blots. The most efficient protocol was used to prepare protein lysates from breast cancer and renal tissues, which were subsequently subjected to RPPA. Reproducibility was evaluated and Spearman correlation was calculated between matching fresh frozen and FFPE samples.
The most effective approach from six protein extraction protocols tested enabled efficient extraction of immunoreactive protein from cell line, breast cancer and renal tissue sample sets. 85% of the total of 169 markers tested on RPPA demonstrated significant correlation between FFPE and frozen preparations (p < 0.05) in at least one cell or tissue type, with only 23 markers common in all three sample sets. In addition, FFPE preparations yielded biologically meaningful observations related to pathway signaling status in cell lines, and classification of renal tissues.
With optimized protein extraction methods, FFPE tissues can be a valuable source in generating reproducible and biologically relevant proteomic profiles using RPPA, with specific marker performance varying according to tissue type.
Formalin-fixed; Paraffin-embedded tissue; Protein extraction; Reverse phase protein array; Breast cancer; Renal cancer
Elastase-mediated cleavage of cyclin E generates low molecular weight cyclin E (LMW-E) isoforms exhibiting enhanced CDK2–associated kinase activity and resistance to inhibition by CDK inhibitors p21 and p27. Approximately 27% of breast cancers express high LMW-E protein levels, which significantly correlates with poor survival. The objective of this study was to identify the signaling pathway(s) deregulated by LMW-E expression in breast cancer patients and to identify pharmaceutical agents to effectively target this pathway. Ectopic LMW-E expression in nontumorigenic human mammary epithelial cells (hMECs) was sufficient to generate xenografts with greater tumorigenic potential than full-length cyclin E, and the tumorigenicity was augmented by in vivo passaging. However, cyclin E mutants unable to interact with CDK2 protected hMECs from tumor development. When hMECs were cultured on Matrigel, LMW-E mediated aberrant acinar morphogenesis, including enlargement of acinar structures and formation of multi-acinar complexes, as denoted by reduced BIM and elevated Ki67 expression. Similarly, inducible expression of LMW-E in transgenic mice generated hyper-proliferative terminal end buds resulting in enhanced mammary tumor development. Reverse-phase protein array assay of 276 breast tumor patient samples and cells cultured on monolayer and in three-dimensional Matrigel demonstrated that, in terms of protein expression profile, hMECs cultured in Matrigel more closely resembled patient tissues than did cells cultured on monolayer. Additionally, the b-Raf-ERK1/2-mTOR pathway was activated in LMW-E–expressing patient samples, and activation of this pathway was associated with poor disease-specific survival. Combination treatment using roscovitine (CDK inhibitor) plus either rapamycin (mTOR inhibitor) or sorafenib (a pan kinase inhibitor targeting b-Raf) effectively prevented aberrant acinar formation in LMW-E–expressing cells by inducing G1/S cell cycle arrest. LMW-E requires CDK2–associated kinase activity to induce mammary tumor formation by disrupting acinar development. The b-Raf-ERK1/2-mTOR signaling pathway is aberrantly activated in breast cancer and can be suppressed by combination treatment with roscovitine plus either rapamycin or sorafenib.
Effective cancer treatment should include targeting not only drivers of tumorigenicity but also the downstream signaling pathways that these drivers activate. Special attention has to be given to the model systems that identify these targets and interrogating if these targets are poor prognostic indicators in patients. Using cell lines cultured on plastic and extracellular matrix (Matrigel) and comparing their proteomic profiles to breast cancer tumor samples, we demonstrated that overexpression of LMW-E is concomitant with activation of the b-Raf-ERK1/2-mTOR pathway. Using mouse models, we show that induction of LMW-E is sufficient to induce mammary tumor development in vivo. Next, cells established from the tumors were treated with combination therapy targeting the LMW-E/CDK2 complex and the b-Raf-ERK1/2-mTOR pathway. Results revealed that this combination therapy effectively inhibited the altered proliferation of these cells. Most significantly, we showed that breast cancer patients whose tumors overexpress both LMW-E and different components of the b-Raf-ERK1/2-mTOR pathway have the worst prognosis. In summary, through the use of multiple in vitro and in vivo model systems and translating the findings to clinical specimens, we have identified a novel targeted therapy in breast cancer patients whose tumors overexpress LMW-E.
The lack of large panels of validated antibodies, tissue handling variability, and intratumoral heterogeneity potentially hamper comprehensive study of the functional proteome in non-microdissected solid tumors. The purpose of this study was to address these concerns and to demonstrate clinical utility for the functional analysis of proteins in non-microdissected breast tumors using reverse phase protein arrays (RPPA).
Herein, 82 antibodies that recognize kinase and steroid signaling proteins and effectors were validated for RPPA. Intraslide and interslide coefficients of variability were <15%. Multiple sites in non-microdissected breast tumors were analyzed using RPPA after intervals of up to 24 h on the benchtop at room temperature following surgical resection.
Twenty-one of 82 total and phosphoproteins demonstrated time-dependent instability at room temperature with most variability occurring at later time points between 6 and 24 h. However, the 82-protein functional proteomic “fingerprint” was robust in most tumors even when maintained at room temperature for 24 h before freezing. In repeat samples from each tumor, intratumoral protein levels were markedly less variable than intertumoral levels. Indeed, an independent analysis of prognostic biomarkers in tissue from multiple tumor sites accurately and reproducibly predicted patient outcomes. Significant correlations were observed between RPPA and immunohistochemistry. However, RPPA demonstrated a superior dynamic range. Classification of 128 breast cancers using RPPA identified six subgroups with markedly different patient outcomes that demonstrated a significant correlation with breast cancer subtypes identified by transcriptional profiling.
Thus, the robustness of RPPA and stability of the functional proteomic “fingerprint” facilitate the study of the functional proteome in non-microdissected breast tumors.
Functional proteome; RPPA; Breast cancer; Kinase signaling; Steroid signaling
Thermoanaerobic bacteria are of interest in cellulosic-biofuel production, due to their simultaneous pentose and hexose utilization (co-utilization) and thermophilic nature. In this study, we experimentally reconstructed the structure and dynamics of the first genome-wide carbon utilization network of thermoanaerobes. The network uncovers numerous novel pathways and identifies previously unrecognized but crucial pathway interactions and the associated key junctions. First, glucose, xylose, fructose, and cellobiose catabolism are each featured in distinct functional modules; the transport systems of hexose and pentose are apparently both regulated by transcriptional antiterminators of the BglG family, which is consistent with pentose and hexose co-utilization. Second, glucose and xylose modules cooperate in that the activity of the former promotes the activity of the latter via activating xylose transport and catabolism, while xylose delays cell lysis by sustaining coenzyme and ion metabolism. Third, the vitamin B12 pathway appears to promote ethanologenesis through ethanolamine and 1, 2-propanediol, while the arginine deiminase pathway probably contributes to cell survival in stationary phase. Moreover, by experimentally validating the distinct yet collaborative nature of glucose and xylose catabolism, we demonstrated that these novel network-derived features can be rationally exploited for product-yield enhancement via optimized timing and balanced loading of the carbon supply in a substrate-specific manner. Thus, this thermoanaerobic glycobiome reveals novel genetic features in carbon catabolism that may have immediate industrial implications and provides novel strategies and targets for fermentation and genome engineering.
Renewable liquid fuels derived from lignocellulosic biomass could alleviate global energy shortage and climate change. Cellulose and hemicellulose are the main components of lignocellulosic biomass. Therefore, the ability to simultaneously utilize pentose and hexose (i.e., co-utilization) has been a crucial challenge for industrial microbes producing lignocellulosic biofuels. Certain thermoanaerobic bacteria demonstrate this unusual talent, but the genetic foundation and molecular mechanism of this process remain unknown. In this study, we reconstructed the structure and dynamics of the first genome-wide carbon utilization network of thermoanaerobes. This transcriptome-based co-expression network reveals that glucose, xylose, fructose, and cellobiose catabolism are each featured on distinct functional modules. Furthermore, the dynamics of the network suggests a distinct yet collaborative nature between glucose and xylose catabolism. In addition, we experimentally demonstrated that these novel network-derived features can be rationally exploited for product-yield enhancement via optimized timing and balanced loading of the carbon supply in a substrate-specific manner. Thus, the newly discovered modular and precisely regulated network elucidates unique features of thermoanaerobic glycobiomes and reveals novel perturbation strategies and targets for the enhanced thermophilic production of lignocellulosic biofuels.