Homologous Deletion Research Articles

Downregulation of miR-221/222 by a microRNA sponge promotes apoptosis in oral squamous cell carcinoma cells through upregulation of PTEN.

MicroRNA-221 and microRNA-222 (miR-221/222) have been identified as oncogenes and confirmed to be overexpressed in various types of cancer. However, the regulation mechanism of miR-221/222 in oral squamous cell carcinoma (OSCC) remains to be fully elucidated. Previously, an miR-221/222 sponge was successfully constructed and its effect on the downregulation of miR-221/222 expression was investigated. In the present study, the dual luciferase reporter assay revealed a phosphatase and tensin homolog (PTEN) deletion on chromosome 10 to be a target gene of miR-221/222. It was also demonstrated that miR-221/222 suppression by transfection with an miR-221/222 sponge in vitro resulted in upregulation of PTEN. Notably, the proliferation and invasiveness of the miR-221/222 sponge-transfected cells was significantly inhibited, while apoptosis was promoted, when determined by Cell Counting Kit-8, Transwell assays and flow cytometry. The results of the present study prove that miR-221/222 may downregulate the expression of PTEN in OSCC cells and function as oncogenes, providing a novel insight into the underlying mechanism of OSCC tumorigenesis. The present study suggests that upregulating the expression of PTEN by downregulation of miR-221/222 may be a potential treatment for OSCC.

Establishment and antitumor effects of dasatinib and PKI-587 in BD-138T, a patient-derived muscle invasive bladder cancer preclinical platform with concomitant EGFR amplification and PTEN deletion.

Muscle-invasive bladder cancer (MIBC) consists of a heterogeneous group of tumors with a high rate of metastasis and mortality. To facilitate the in-depth investigation and validation of tailored strategies for MIBC treatment, we have developed an integrated approach using advanced high-throughput drug screening and a clinically relevant patient-derived preclinical platform. We isolated patient-derived tumor cells (PDCs) from a rare MIBC case (BD-138T) that harbors concomitant epidermal growth factor receptor (EGFR) amplification and phosphatase and tensin homolog (PTEN) deletion. High-throughput in vitro drug screening demonstrated that dasatinib, a SRC inhibitor, and PKI-587, a dual PI3K/mTOR inhibitor, exhibited targeted anti-proliferative and pro-apoptotic effects against BD-138T PDCs. Using established patient-derived xenograft models that successfully retain the genomic and molecular characteristics of the parental tumor, we confirmed that these anti-tumor responses occurred through the inhibition of SRC and PI3K/AKT/mTOR signaling pathways. Taken together, these experimental results demonstrate that dasatinib and PKI-587 might serve as promising anticancer drug candidates for treating MIBC with combined EGFR gene amplification and PTEN deletion.

GRIM-19, a gene associated with retinoid-interferon-induced mortality, affects endometrial receptivity and embryo implantation.

GRIM-19 is associated with apoptosis, abnormal proliferation, immune tolerance and malignant transformation, and it also plays an important role in early embryonic development. Although the homologous deletion of GRIM-19 causes embryonic lethality in mice, the precise role of GRIM-19 in embryo implantation has not been elucidated. Here we show that GRIM-19 plays an important role in endometrial receptivity and embryo implantation. Day 1 to Day 6 pregnant mouse uteri were collected. Immunohistochemistry studies revealed the presence of GRIM-19 on the luminal epithelium and glandular epithelium throughout the implantation period in pregnant mice. The protein and mRNA levels of GRIM-19 were markedly decreased on Day 4 of pregnancy in pregnant mice, but there was no change in GRIM-19 levels in a group of pseudopregnant mice. Overexpression of GRIM-19 decreased the adhesion rate of RL95-2-BeWo co-cultured spheroids and increased apoptosis. Furthermore, STAT3 and IL-11 mRNA and protein levels were reduced by overexpressing GRIM-19, but protein and mRNA levels of TNF-α were increased. These findings indicate the involvement of GRIM-19 in the embryo implantation process by regulating adhesion, apoptosis and immune tolerance.

The association of Phosphatase and tensin homolog (PTEN) deletion and prostate cancer risk: A meta-analysis.

Phosphatase and tensin homolog (PTEN) deleted on chromosome 10, a tumor suppressor that negatively regulates the phosphoinositide-3-kinase(PI3K) which has been implicated in a number of human malignancies including prostate cancer. However the prognostic value of PTEN deletion in prostate cancer patient's diagnosis and the mechanism of PTEN deletion in prostate cancer development still remain unclear. A meta-analysis of 26 published studies including 8097 prostate cancer patients was performed. Compared to PTEN normal patients, PTEN deletion patients showed a higher aggressive Gleason score(OR: 1.284, 95%CI=1.145-1.439) and pathological stage(OR: 1.628, 95%CI=1.270-2.087) which generally had a higher risk in prostate replace(HR: 1.738, 95%CI=1.264-2.390). Significant association between PTEN deletion and ERG rearrangements in prostate cancer development was also proved that compared to PTEN normal patients, patients with PTEN deletion showed a higher risk in ERG rearrangements(OR: 1.345, 95%CI=1.102-1.788). This study indicated that patients with PTEN deletion were associated with higher pathological stage or Gleason score and a higher risk in prostate cancer replace potentially represent a novel clinically relevant event to identify individuals at increased risk for the occurrence, progression and prognosis of prostate cancer. Prostate cancer patients with PTEN deletion usually had a higher risk in ERG rearrangements than other patients may be a potential new area for identifying poor prognosis patients and selecting patients for targeted therapies which required confirmation through adequately designed prospective studies.

211. Combination of Forced Transduction of P53 and an Agent That Blocks MDM2-p53 Interactions Produces Synergistic Cytotoxicity on Mesothelioma Defective of the INK4A/ARF Region

A majority of malignant mesothelioma specimens possesses the wild-type p53 gene with a homologous deletion in the INK4A/ARF locus which encodes the p14ARF and the p16INK4A genes. Consequently, the p53 downstream pathways are functionally inactivated since the p14 defect augments activities of MDM2 which mediates p53 degradation through the ubiquitination processes. Furthermore, a loss of p16 induces pRb phosphorylation and uninhibited cell cycle progression through up-regulated CKD activities. We then examined possible anti-tumor effects of MDM2 inhibitors, Nutlin-3a and RITA, on human mesothelioma cells in combination of adenoviruses expressing the p53 gene (Ad-p53). Nutlin-3a, inhibiting the MDM2-p53 interactions, increased p53 phosphorylation levels and suppressed viability of mesothelioma cells in a p53-dependent manner. RITA, although blocked the MDM2-p53 interactions and phosphorylated p53, inhibited viability of mesothelioma cells independently of the p53 geneotype. Transduction of mesothelioma cells with Ad-p53 induced apoptotic cell death irrespective of the genotype. Moreover p21 induced by Ad-p53 dephosphorylated pRb and subsequently inhibited cell cycle progression. A combinatory use of Ad-p53 and Nutlin-3a or RITA, produced synergistic cytotoxicity of mesothelioma irrespective of the p53 geneotype. In addition, we demonstrated that the combination produced anti-tumor effects in an orthotopic animal model, mesothelioma developed in the pleural cavity in nude mice. In contrast, heat shock protein 90 inhibitors, blocking functions of MDM4, a homologous gene product of MDM2 without ubiquitination activities, did not produce the synergistic cytotoxicity with Ad-p53. These data collectively showed that MDM2 inhibitors enhanced expression levels of endogenous and exogenously transduced p53 through increased stability of the gene produce, and consequently activated the p53-mediated apoptosis. The p53-directed therapy is thus a possible therapeutic strategy for mesothelioma with the INK4A/ARF deletion.

Glandular epithelial AR inactivation enhances PTEN deletion-induced uterine pathology.

Phosphatase and tensin homolog (PTEN) deletion induces uterine pathology, whereas androgen actions via androgen receptor (AR) support uterine growth and therefore may modify uterine cancer risk. We hypothesized that the androgen actions mediated via uterine glandular epithelial AR could modify PTEN deletion-induced uterine pathology. To test our hypothesis, we developed uterine glandular epithelium-specific PTEN and/or AR knockout mouse models comparing the uterine pathology among wild-type (WT), glandular epithelium-specific AR inactivation (ugeARKO), PTEN deletion (ugePTENKO), and the combined PTEN and AR knockout (ugePTENARKO) female mice. The double knockout restricted to glandular epithelium showed that AR inactivation enhanced PTEN deletion-induced uterine pathology with development of intraepithelial neoplasia by 20 weeks of age. In ugePTENARKO, 6/10 (60%) developed intraepithelial neoplasia, whereas 3/10 (30%) developed only glandular hyperplasia in ugePTENKO uterus. No uterine pathology was observed in WT (n=8) and ugeARKO (n=7) uteri. Uterine weight was significantly (P=0.002) increased in ugePTENARKO (374±97 mg (mean±s.e.)) compared with WT (97±6 mg), ugeARKO (94±12 mg), and ugePTENKO (205±33 mg). Estrogen receptor alpha (ERα) and P-AKT expression was modified by uterine pathology but did not differ between ugePTENKO and ugePTENARKO, suggesting that its expressions are not directly affected by androgens. However, progesterone receptor (PR) expression was reduced in ugePTENARKO compared to ugePTENKO uterus, suggesting that PR expression could be regulated by glandular epithelial AR inactivation. In conclusion, glandular epithelial AR inactivation (with persistent stromal AR action) enhanced PTEN deletion-induced uterine pathology possibly by downregulating PR expression in the uterus.

TRF2-RAP1 is required to protect telomeres from engaging in homologous recombination-mediated deletions and fusions.

Repressor/activator protein 1 (RAP1) is a highly conserved telomere-interacting protein. Yeast Rap1 protects telomeres from non-homologous end joining (NHEJ), plays important roles in telomere length control and is involved in transcriptional gene regulation. However, a role for mammalian RAP1 in telomere end protection remains controversial. Here we present evidence that mammalian RAP1 is essential to protect telomere from homology directed repair (HDR) of telomeres. RAP1 cooperates with the basic domain of TRF2 (TRF2B) to repress PARP1 and SLX4 localization to telomeres. Without RAP1 and TRF2B, PARP1 and SLX4 HR factors promote rapid telomere resection, resulting in catastrophic telomere loss and the generation of telomere-free chromosome fusions in both mouse and human cells. The RAP1 Myb domain is required to repress both telomere loss and formation of telomere-free fusions. Our results highlight the importance of the RAP1-TRF2 heterodimer in protecting telomeres from inappropriate processing by the HDR pathway.

(Partial) Loss of BAF250a (ARID1A) in rectovaginal deep-infiltrating endometriosis, endometriomas and involved pelvic sentinel lymph nodes.

Loss of protein BAF250a (ARID1A) expression is present in women with rectovaginal deep-infiltrating endometriosis (DIE) and endometriosis affecting the pelvic sentinel lymph nodes (PSLN). Partial loss of protein BAF250a was found in some of our patient samples, comprising all endometriosis entities, including rectovaginal DIE and endometriosis affecting the PSLN. Loss of BAF250a (BRG-associated factor 250a)/ARIDIA (AT-rich interactive domain 1A) protein expression was identified among endometriosis-associated ovarian carcinomas and ovarian endometriosis, and this phenomenon was described as a possible early event in the transformation of endometriosis into cancer. DIE affecting the bowel/rectovaginal site is the most aggressive presentation of endometriosis and its 'risk' of malignant transformation has not been studied so far. We evaluated the immunohistochemical expression of BAF250a protein in 70 samples from patients enrolled in this study who were surgically treated at a tertiary center, university Hospital. The samples submitted to investigation were from rectovaginal DIE (n= 25/30), endometriosis affecting the PSLN (n= 5/7), ovarian endometriosis (n= 20/20) and endometrium from patients without endometriosis used as controls (n= 20/20). Partial loss (i.e. in one tissue section some cells stained positive for BAF250a while other cells, usually an adjacent group, were negative) of BAF250a protein was identified in 36% (9/25) of rectovaginal DIE samples, 40% (2/5) of endometriosis lesions involving the PSLN, 30% (6/20) of endometriomas, and also in 25% (5/20) of endometrium from controls. We found no statistical correlation between occurrence of partial loss of BAF250a protein and the use or not of hormone medications (P = 0.106), cycle phase (P = 0.917) and stage of disease (P = 0.717). We only found partial loss of BAF250a protein expression, and in a small population of women, with relatively high frequency in all benign tissues assessed in the present analysis. Therefore, this finding alone should not be correlated directly with the risk of malignant transformation in these lesions. The occurrence of partial loss of BAF250a protein expression in women with rectovaginal DIE and endometriosis affecting the PSLN is described for the first time. The value of this finding as a predictor of malignant transformation in endometriosis must still be clarified and further studied in association with other molecular events, such as PTEN (phosphatase and tensin homolog) deletion and PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) mutation. We might then be able to identify in the future which patients with endometriosis are at higher risk of cancer. This study was supported by an internal Charité grant to the Endometriosis Research Center and the authors declare no conflicts of interest.

Production of TGF-β Is Decreased in the Bone Marrow of Double Knockout (DKO) SMAD3-/- Fancd2-/- Mice

Homologous deletion recombinant negative DKO mice, missing action of both Fancd2 and SMAD3 proteins were derived to test the hypothesis that abrogation of TGF-β signaling would reverse Fanconi Anemia (FA) stem cell sensitivity to TGF-β. To determine whether DKO mouse tissues demonstrated detectable differences in production of the ligand for TGF-β receptor, tissues from adult six - eight week old DKO mice were compared to those from Fancd2-/- (C57BL/6J background), SMAD3-/- (129/Sv genetic background), or wild type F1 mice (129/Sv X B6) for levels of TGF-β. Tissues examined included bone marrow, intestine, spleen, liver, muscle, and brain. Tissues were homogenized, and analyzed for bone marrow protein by Luminex analysis using a TGF beta 1 multispecies kit for Luminex Platform (Life Technologies).The results demonstrated decreased but not significant production of TGF-β in the bone marrow of DKO mice (3068 ± 753 pg TGF-β/mg protein) compared to 5211 ± 1662 (p = 0.0761) for wild type F1 mice (129/Sv X B6), and 5192 ± 705 (0.1085) for Fancd2-/- mice. However, TGF-β production in DKO was significantly decreased compared to Smad3-/- (129/Sv) mice (9828 ± 1076, p = 0.0127). The lungs from DKO also had decreased TGF-β production compared to wild type F1 mice (436 ± 125 and 1159 ± 44 pg/mg, respectively, p= 0.0217). Decreased TGF-β production was also seen in the DKO liver compared to F1 wild type liver (13.2 ± 1.7 and 33.5 ± 3.6, respectively, p = 0.0072). Bone marrow stromal cell lines derived from long-term bone marrow cultures of each mouse strain were tested for production of TGF-β. SMAD3-/- bone marrow stromal cells also had an increased production of TGF-β (236 pg/mg) compared to wild type F1, Fancd2-/- and DKO cell lines (117, 136 and 144 pg/ml). Bone marrow CFU-GEMM from each mouse strain was tested for sensitivity to inhibition by increasing concentrations of TGF-β, and both fresh bone marrow from DKO and SMAD3-/- mice demonstrated no TGF-β mediated abrogation of colony formation. In contrast, fresh marrow from wild type and Fancd2-/- mice demonstrated TGF-β concentration dependent inhibition of CFU-GEMM colony formation in vitro. These data indicate that TGF-β production in DKO mice is decreased in bone marrow, lung and liver compared to that in F1 wild type, SMAD3-/- or Fancd2-/- mice, and suggest that the control of TGF-β production by abrogation of TGF-β signaling in the setting of deletion of Fancd2 may be modulated in an organ specific manner.Supported by research grant NIAID/NIH, U19A168021. DisclosuresNo relevant conflicts of interest to declare.

Oncogenic Transformation of Dendritic Cells and Their Precursors Leads to Rapid Cancer Development in Mice.

Dendritic cells (DCs) are powerful APCs that can induce Ag-specific adaptive immune responses and are increasingly recognized as important players in innate immunity to both infection and malignancy. Interestingly, although there are multiple described hematological malignancies, DC cancers are rarely observed in humans. Whether this is linked to the immunogenic potential of DCs, which might render them uniquely susceptible to immune control upon neoplastic transformation, has not been fully investigated. To address the issue, we generated a genetically engineered mouse model in which expression of Cre recombinase driven by the C-type lectin domain family 9, member a (Clec9a) locus causes expression of the Kirsten rat sarcoma viral oncogene homolog (Kras)G12D oncogenic driver and deletion of the tumor suppressor p53 within developing and differentiated DCs. We show that these Clec9aKras-G12D mice rapidly succumb from disease and display massive accumulation of transformed DCs in multiple organs. In bone marrow chimeras, the development of DC cancer could be induced by a small number of transformed cells and was not prevented by the presence of untransformed DCs. Notably, activation of transformed DCs did not happen spontaneously but could be induced upon stimulation. Although Clec9aKras-G12D mice showed altered thymic T cell development, peripheral T cells were largely unaffected during DC cancer development. Interestingly, transformed DCs were rejected upon adoptive transfer into wild-type but not lymphocyte-deficient mice, indicating that immunological control of DC cancer is in principle possible but does not occur during spontaneous generation in Clec9aKras-G12D mice. Our findings suggest that neoplastic transformation of DCs does not by default induce anti-cancer immunity and can develop unhindered by immunological barriers.

Aquaporin 5 expression is frequent in prostate cancer and shows a dichotomous correlation with tumor phenotype and PSA recurrence

Aquaporin 5 (AQP5) is an androgen-regulated member of a family of small hydrophobic integral transmembrane water channel proteins regulating cellular water homeostasis and growth signaling. To evaluate its clinical impact and relationship with key genomic alterations in prostate cancer, AQP5 expression was analyzed by immunohistochemistry on a tissue microarray containing 12427 prostate cancers. The analysis revealed weak to moderate immunostaining in normal prostate epithelium. In prostate cancers AQP5 staining levels were more variable and also included completely negative and highly overexpressing cases. Negative, weak, moderate, and strong AQP5 staining was found in 25.0%, 32.5%, 32.5%, and 10.0% of 10239 interpretable tumors. Comparison of AQP5 expression levels with tumor characteristics showed a dichotomous pattern with both high and low staining levels being linked to unfavorable tumor phenotype. AQP5 was negative in 28%, 23%, 24%, and 35% of tumors with Gleason score ≤3 + 3, 3 + 4, 4 + 3 and ≥4 + 4, while the rate of strongly positive cases continuously increased from 7.0% over 10.0% and 12.0% to 13.0% in cancers with Gleason score ≤3 + 3, 3 + 4, 4 + 3 and ≥4 + 4. AQP5 expression was also related to ERG positivity and phosphatase and tensin homolog (PTEN) deletion (P < .0001 each). Strong AQP5 positivity was seen in 15.5% of ERG-positive and 5.8% of ERG-negative cancers (P < .0001) as well as in 14.7% of cancers with PTEN deletion and 9.4% of cancers without PTEN deletion. Remarkably, both negativity and strong positivity of AQP5 were linked to unfavorable disease outcome. This was however only seen in subgroups defined by TMPRSS2-ERG fusion and/or PTEN deletion. In summary, AQP5 can be both overexpressed and lost in subgroups of prostate cancers. Both alterations are linked to unfavorable outcome in molecularly defined cancer subgroups. It is hypothesized that this dichotomous role of AQP5 is due to two highly different mechanisms as to how the protein can influence cancer cells, that is, hydraulic motility regulation and Ras/MAPK pathway activation.

Deletion of homologs of the SREBP pathway results in hyper-production of cellulases in Neurospora crassa and Trichoderma reesei.

BackgroundThe filamentous fungus Neurospora crassa efficiently utilizes plant biomass and is a model organism for genetic, molecular and cellular biology studies. Here, a set of 567 single-gene deletion strains was assessed for cellulolytic activity as compared to the wild-type parental strain. Mutant strains included were those carrying a deletion in: (1) genes encoding proteins homologous to those implicated in the Saccharomyces cerevisiae secretion apparatus; (2) genes that are homologous to those known to differ between the Trichoderma reesei hyper-secreting strain RUT-C30 and its ancestral wild-type strain; (3) genes encoding proteins identified in the secretome of N. crassa when cultured on plant biomass and (4) genes encoding proteins predicted to traverse the secretory pathway.ResultsThe 567 single-gene deletion collection was cultured on crystalline cellulose and a comparison of levels of secreted protein and cellulase activity relative to the wild-type strain resulted in the identification of seven hyper-production and 18 hypo-production strains. Some of these deleted genes encoded proteins that are likely to act in transcription, protein synthesis and intracellular trafficking, but many encoded fungal-specific proteins of undetermined function. Characterization of several mutants peripherally linked to protein processing or secretion showed that the hyper- or hypo-production phenotypes were primarily a response to cellulose. The altered secretome of these strains was not limited to the production of cellulolytic enzymes, yet was part of the cellulosic response driven by the cellulase transcription factor CLR-2. Mutants implicated the loss of the SREBP pathway, which has been found to regulate ergosterol biosynthesis genes in response to hypoxic conditions, resulted in a hyper-production phenotype. Deletion of two SREBP pathway components in T. reesei also conferred a hyper-production phenotype under cellulolytic conditions.ConclusionsThese studies demonstrate the utility of screening the publicly available N. crassa single-gene deletion strain collection for a particular phenotype. Mutants in a predicted E3 ligase and its target SREBP transcription factor played an unanticipated role in protein production under cellulolytic conditions. Furthermore, phenotypes similar to those observed in N. crassa were seen following the targeted deletion of orthologous SREBP pathway loci in T. reesei, a fungal species commonly used in industrial enzyme production.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0297-9) contains supplementary material, which is available to authorized users.

The opportunistic marine pathogen Vibrio parahaemolyticus becomes virulent by acquiring a plasmid that expresses a deadly toxin

Acute hepatopancreatic necrosis disease (AHPND) is a severe, newly emergent penaeid shrimp disease caused by Vibrio parahaemolyticus that has already led to tremendous losses in the cultured shrimp industry. Until now, its disease-causing mechanism has remained unclear. Here we show that an AHPND-causing strain of V. parahaemolyticus contains a 70-kbp plasmid (pVA1) with a postsegregational killing system, and that the ability to cause disease is abolished by the natural absence or experimental deletion of the plasmid-encoded homologs of the Photorhabdus insect-related (Pir) toxins PirA and PirB. We determined the crystal structure of the V. parahaemolyticus PirA and PirB (PirA(vp) and PirB(vp)) proteins and found that the overall structural topology of PirA(vp)/PirB(vp) is very similar to that of the Bacillus Cry insecticidal toxin-like proteins, despite the low sequence identity (<10%). This structural similarity suggests that the putative PirAB(vp) heterodimer might emulate the functional domains of the Cry protein, and in particular its pore-forming activity. The gene organization of pVA1 further suggested that pirAB(vp) may be lost or acquired by horizontal gene transfer via transposition or homologous recombination.

Gene structure variation in segmental duplication block C of human chromosome 7q 11.23 during primate evolution

Segmental duplication, or low-copy repeat (LCR) event, occurs during primate evolution and is an important source of genomic diversity, including gain or loss of gene function. The human chromosome 7q 11.23 is related to the William–Beuren syndrome and contains large region-specific LCRs composed of blocks A, B, and C that have different copy numbers in humans and different primates. We analyzed the structure of POM121, NSUN5, FKBP6, and TRIM50 genes in the LCRs of block C. Based on computational analysis, POM121B created by a segmental duplication acquired a new exonic region, whereas NSUN5B (NSUN5C) showed structural variation by integration of HERV-K LTR after duplication from the original NSUN5 gene. The TRIM50 gene originally consists of seven exons, whereas the duplicated TRIM73 and TRIM74 genes present five exons because of homologous recombination-mediated deletion. In addition, independent duplication events of the FKBP6 gene generated two pseudogenes at different genomic locations. In summary, these clustered genes are created by segmental duplication, indicating that they show dynamic evolutionary events, leading to structure variation in the primate genome.

Clinical activity of androgen deprivation therapy in patients with metastatic/relapsed androgen receptor–positive salivary gland cancers

Androgen deprivation therapy has some clinical activity in selected salivary gland cancer histotypes, with androgen receptor expression. We retrospectively analyzed patients with androgen receptor-expressing recurrent/metastatic salivary gland cancer, treated with androgen deprivation therapy. Protein expression of androgen receptor and ErbB family members was investigated. Progression-free survival (PFS) and overall survival (OS) were the main endpoints. Seventeen patients were identified. No significant toxicities were reported. Overall response rate was 64.7%; 3-year PFS and 5-year OS were 11.8% and 19.3%, respectively. Androgen receptor overexpression may be sustained by gain of chromosome X (58%) and TP53 mutation (44%). No association between response to androgen deprivation therapy and epidermal growth factor receptor (EGFR), human epidermal growth factor receptor (HER)2, HER3 expression, PIK3CA mutations, or phosphatase and tensin homolog (PTEN) deletion was identified. We confirm the activity of androgen deprivation therapy in androgen receptor-expressing recurrent/metastatic salivary gland cancers. The hypothesis that an androgen receptor increased gene copy number may represent a possible mechanism of primary resistance should be further investigated.

VEGFR-1 overexpression identifies a small subgroup of aggressive prostate cancers in patients treated by prostatectomy.

The VEGFR-1 is suggested to promote tumor progression. In the current study we analyzed prevalence and prognostic impact of the VEGFR-1 by immunohistochemistry on a tissue microarray containing more than 3000 prostate cancer specimens. Results were compared to tumor phenotype, ETS-related gene (ERG) status, and biochemical recurrence. Membranous VEGFR-1 expression was detectable in 32.6% of 2669 interpretable cancers and considered strong in 1.7%, moderate in 6.7% and weak in 24.2% of cases. Strong VEGFR-1 expression was associated with TMPRSS2:ERG fusion status as determined by fluorescence in situ hybridization (FISH) and immunohistochemistry (p < 0.0001 each). Elevated VEGFR-1 expression was linked to high Gleason grade and advanced pT stage in TMPRSS2:ERG negative cancers (p = 0.0008 and p = 0.001), while these associations were absent in TMPRSS2:ERG positive cancers. VEGFR-1 expression was also linked to phosphatase and tensin homolog (PTEN) deletions. A comparison with prostate specific antigen (PSA) recurrence revealed that the 1.7% of prostate cancers with the highest VEGFR-1 levels had a strikingly unfavorable prognosis. This could be seen in all cancers, in the subsets of TMPRSS2:ERG positive or negative, PTEN deleted or undeleted carcinomas (p < 0.0001 each). High level VEGFR-1 expression is infrequent in prostate cancer, but identifies a subgroup of aggressive cancers, which may be candidates for anti-VEGFR-1 targeted therapy.

Functional analysis of Vibrio vulnificus RND efflux pumps homologous to Vibrio cholerae VexAB and VexCD, and to Escherichia coli AcrAB.

Resistance-nodulation-division (RND) efflux pumps are associated with multidrug resistance in many gram-negative pathogens. The genome of Vibrio vulnificus encodes 11 putative RND pumps homologous to those of Vibrio cholerae and Escherichia coli. In this study, we analyzed three putative RND efflux pumps, showing homology to V. cholerae VexAB and VexCD and to E. coli AcrAB, for their functional roles in multidrug resistance of V. vulnificus. Deletion of the vexAB homolog resulted in increased susceptibility of V. vulnificus to bile acid, acriflavine, ethidium bromide, and erythromycin, whereas deletion of acrAB homologs rendered V. vulnificus more susceptible to acriflavine only. Deletion of vexCD had no effect on susceptibility of V. vulnificus to these chemicals. Upon exposure to these antibacterial chemicals, expression of tolCV1 and tolCV2, which are putative outer membrane factors of RND efflux pumps, was induced, whereas expression levels of vexAB, vexCD, and acrAB homologs were not significantly changed. Our results show that the V. vulnificus homologs of VexAB largely contributed to in vitro antimicrobial resistance with a broad substrate specificity that was partially redundant with the AcrAB pump homologs.

Elemental biochemical analysis of the polysaccharides in the extracellular matrix of the yeast Saccharomyces cerevisiae.

In yeast multicellular aggregates, such as biofilms and colonies, cells are supported by a yeast extracellular matrix (yECM) of glycosidic nature, the composition of which is mostly unknown. Saccharomyces cerevisiae ECM was produced, extracted and partitioned. An analytical-grade pure glycoside fraction was obtained, fractionated by anionic exchange liquid chromatography and analyzed by gas chromatography-mass spectrometry and polyacrylamide gel electrophoresis. Two different molecular weight polysaccharides were found, composed of glucose, mannose and small relative amounts of galactose. One of the polysaccharides had a low molecular weight, compatible with the association with glycoproteins abundantly occurring in yECM. In addition, these polysaccharide species were separated by diaminopropane agarose gel electrophoresis and induced metachromatic shift, suggesting chemical substitution, which was corroborated by anticoagulation activity. This was shown to be associated with the double deletion of the yeast homologues of the mammalian Hedgehog modulators Hhatl and Hhat, respectively yeast Gup1 and Gup2. These results pioneer the study of the molecular biology of the ECM supporting S. cerevisiae multicellular aggregates such as biofilms.

Combination of Multiple Markers Predicts Prostate Cancer Outcome

Today we are facing a large problem of overtreatment in men with prostate cancer (PCa) due to the current lack of reliable prognostic biomarkers. Aberrations including ETS family gene rearrangements, phosphatase and tensin homolog (PTEN) deletions, enhancer of zeste homolog 2 (EZH2) overexpression and changes in the androgen receptor (AR) are commonly described in PCa and are believed to have an important role in progression of the disease. The aim of this study is to analyze if the protein expression of ERG, AR, PTEN and EZH2, and the deletion status of the PTEN, either alone or in combination can predict clinical outcome. Our cohort consists of 214 men that have undergone radical prostatectomy at the University Hospital of Orebro, Sweden between 1989-2005. Immunohistochemistry was used to detect AR, ERG, PTEN and EZH2 antigen and PTEN deletion was assessed using chromogenic in situ hybridization. The overall frequency showed AR-, ERG- and EZH2 expression in 99.5%, 52.9%, and 92.3% respectively. PTEN deletion was seen in 37.4% of the cases, where homozygous and heterozygous deletion was present in 18.1% and 19.2%, respectively. Our results show that there was a significant association between the combined ERG- and EZH2 expression and PTEN deletion with PCa specific death (p=0.035). This significant association was also seen in the group of cases that harbored both ERG expression and PTEN deletion (p=0.036). Cases expressing ERG, exhibiting PTEN loss (either hetero- or homozygous loss) and a Gleason score ≥8 showed a significantly higher rate of developing castration-resistant PCa (CRPC) and dying of PCa. The current lack of a reliable prognostic tool available for PCa is a large problem, the results from this study and others shows the great potential in using multiple biomarkers to predict PCa outcome.

Engineering validamycin production by tandem deletion of γ-butyrolactone receptor genes in Streptomyces hygroscopicus 5008

Paired homologs of γ-butyrolactone (GBL) biosynthesis gene afsA and GBL receptor gene arpA are located at different positions in genome of Streptomyces hygroscopicus 5008. Inactivation of afsA homologs dramatically decreased biosynthesis of validamycin, an important anti-fungal antibiotic and a critical substrate for antidiabetic drug synthesis, and the deletion of arpA homologs increased validamycin production by 26% (ΔshbR1) and 20% (ΔshbR3). By double deletion, the ΔshbR1/R3 mutant showed higher transcriptional levels of adpA-H (the S. hygroscopicus ortholog of the global regulatory gene adpA) and validamycin biosynthetic genes, and validamycin production increased by 55%. Furthermore, by engineering a high-producing industrial strain via tandem deletion of GBL receptor genes, validamycin production and productivity were enhanced from 19 to 24g/L (by 26%) and from 6.7 to 9.7gL−1d−1 (by 45%), respectively, which was the highest ever reported. The strategy demonstrated here may be useful to engineering other Streptomyces spp. with multiple pairs of afsA–arpA homologs.