High level of HSF1 associates with aggressive endometrial carcinoma and suggests potential for HSP90 inhibitors.

BACKGROUND: Recent identification of a specific role of HSF1 in cancer progression has led to new relevance of HSF1 as both a prognostic and a predictive marker. The role of HSF1 in endometrial cancer has so far been unexplored. METHODS: A total of 823 lesions from endometrial carcinoma precursors, primary tumours and metastases were prospectively collected and explored for HSF1 protein expression in relation to established markers for aggressive disease and survival. Transcriptional alterations related to HSF1 protein level were investigated by microarray analysis for 224 freshly frozen samples in parallel. RESULTS: High expression of HSF1 protein in endometrial carcinoma is significantly associated with aggressive disease and poor survival (all P-values ≤ 0.02), also among ERα-positive patients presumed to have good prognosis. The HSF1-related gene signatures increase during disease progression and were also found to have prognostic value. Gene expression analyses identified HSP90 inhibition as a potential novel therapeutic approach for cases with high protein expression of HSF1. CONCLUSIONS: We demonstrate for the first time in endometrial cancer that high expression of HSF1 and measures for transcriptional activation of HSF1 associate with poor outcome and disease progression. The HSP90 inhibitors are suggested as new targeted therapeutics for patients with high HSF1 levels in tumour in particular.

Endometrial Carcinoma Recurrence Score (ECARS) validates to identify aggressive disease and associates with markers of epithelial-mesenchymal transition and PI3K alterations.

PURPOSE: Our previously reported 29-gene expression signature identified an aggressive subgroup of endometrial cancer patients with PI3K activation. We here wanted to validate these findings by independent patient series. PATIENTS AND METHODS: The 29-gene expression signature was assessed in fresh frozen tumor tissue from 280 primary endometrial carcinomas (three independent cohorts), 19 metastatic lesions and in 333 primary endometrial carcinomas using TCGA data, and expression was related to clinico-pathologic features and survival. The 29-gene signature was assessed by real-time quantitative PCR, DNA oligonucleotide microarrays, or RNA sequencing. PI3K alterations were assessed by immunohistochemistry, DNA microarrays, DNA sequencing, SNP arrays or fluorescence in situ hybridization. A panel of markers of epithelial-mesenchymal transition (EMT) was also correlated to the 29-gene signature score. RESULTS: High 29-gene Endometrial Carcinoma Recurrence Score (ECARS) values consistently validated to identify patients with aggressive clinico-pathologic phenotype and reduced survival. Within the presumed favorable subgroups of low grade, endometrioid tumors confined to the uterus, high ECARS still predicted a poor prognosis. The score was higher in metastatic compared to primary lesions (P<0.001) and was significantly associated with potential measures of PI3K activation, markers of EMT and vascular invasion as an indicator of metastatic spread (all P<0.001). CONCLUSIONS: ECARS validates to identify aggressive endometrial carcinomas in multiple, independent patients cohorts. The higher signature score in metastatic compared to primary lesions, and the potential link to PI3K activation and EMT, support further studies of ECARS in relation to response to PI3K and EMT inhibitors in clinical trials of metastatic endometrial carcinoma.

Loss of GPER identifies new targets for therapy among a subgroup of ERα-positive endometrial cancer patients with poor outcome.

BACKGROUND: The G protein-coupled oestrogen receptor, GPER, has been suggested as an alternative oestrogen receptor. Our purpose was to investigate the potential of GPER as a prognostic and predictive marker in endometrial carcinoma and to search for new drug candidates to improve treatment of aggressive disease. MATERIALS AND METHOD: A total of 767 primary endometrial carcinomas derived from three patient series, including an external dataset, were studied for protein and mRNA expression levels to investigate and validate if GPER loss identifies poor prognosis and new targets for therapy in endometrial carcinoma. Gene expression levels, according to ERα/GPER status, were used to search the connectivity map database for small molecular inhibitors with potential for treatment of metastatic disease for receptor status subgroups. RESULTS: Loss of GPER protein is significantly correlated with low GPER mRNA, high FIGO stage, non-endometrioid histology, high grade, aneuploidy and ERα loss (all P-values ≤0.05). Loss of GPER among ERα-positive patients identifies a subgroup with poor prognosis that until now has been unrecognised, with reduced 5-year survival from 93% to 76% (P=0.003). Additional loss of GPER from primary to metastatic lesion counterparts further supports that loss of GPER is associated with disease progression. CONCLUSION: These results support that GPER status adds clinically relevant information to ERα status in endometrial carcinoma and suggest a potential for new inhibitors in the treatment of metastatic endometrial cancers with ERα expression and GPER loss.

KRAS gene amplification and overexpression but not mutation associates with aggressive and metastatic endometrial cancer.

BACKGROUND: Three quarter of endometrial carcinomas are treated at early stage. Still, 15 to 20% of these patients experience recurrence, with little effect from systemic therapies. Homo sapiens v-Ki-ras2 Kirsten rat sarcoma viral oncogenes homologue (KRAS) mutations have been reported to have an important role in tumorigenesis for human cancers, but there is limited knowledge regarding clinical relevance of KRAS status in endometrial carcinomas. METHODS: We have performed a comprehensive and integrated characterisation of genome-wide expression related to KRAS mutations and copy-number alterations in primary- and metastatic endometrial carcinoma lesions in relation to clinical and histopathological data. A primary investigation set and clinical validation set was applied, consisting of 414 primary tumours and 61 metastatic lesions totally. RESULTS: Amplification and gain of KRAS present in 3% of the primary lesions and 18% of metastatic lesions correlated significantly with poor outcome, high International Federation of Gynaecology and Obstetrics stage, non-endometrioid subtype, high grade, aneuploidy, receptor loss and high KRAS mRNA levels, also found to be associated with aggressive phenotype. In contrast, KRAS mutations were present in 14.7% of primary lesions with no increase in metastatic lesions, and did not influence outcome, but was significantly associated with endometrioid subtype, low grade and obesity. CONCLUSION: These results support that KRAS amplification and KRAS mRNA expression, both increasing from primary to metastatic lesions, are relevant for endometrial carcinoma disease progression.

Integrated genomic profiling of endometrial carcinoma associates aggressive tumors with indicators of PI3 kinase activation.

Although 75% of endometrial cancers are treated at an early stage, 15% to 20% of these recur. We performed an integrated analysis of genome-wide expression and copy-number data for primary endometrial carcinomas with extensive clinical and histopathological data to detect features predictive of recurrent disease. Unsupervised analysis of the expression data distinguished 2 major clusters with strikingly different phenotypes, including significant differences in disease-free survival. To identify possible mechanisms for these differences, we performed a global genomic survey of amplifications, deletions, and loss of heterozygosity, which identified 11 significantly amplified and 13 significantly deleted regions. Amplifications of 3q26.32 harboring the oncogene PIK3CA were associated with poor prognosis and segregated with the aggressive transcriptional cluster. Moreover, samples with PIK3CA amplification carried signatures associated with in vitro activation of PI3 kinase (PI3K), a signature that was shared by aggressive tumors without PIK3CA amplification. Tumors with loss of PTEN expression or PIK3CA overexpression that did not have PIK3CA amplification also shared the PI3K activation signature, high protein expression of the PI3K pathway member STMN1, and an aggressive phenotype in test and validation datasets. However, mutations of PTEN or PIK3CA were not associated with the same expression profile or aggressive phenotype. STMN1 expression had independent prognostic value. The results affirm the utility of systematic characterization of the cancer genome in clinically annotated specimens and suggest the particular importance of the PI3K pathway in patients who have aggressive endometrial cancer.

High BMI is significantly associated with positive progesterone receptor status and clinico-pathological markers for non-aggressive disease in endometrial cancer.

BACKGROUND: Endometrial cancer incidence is increasing in industrialised countries. High body mass index (BMI, kg m(-2)) is associated with higher risk for disease. We wanted to investigate if BMI is related to clinico-pathological characteristics, hormone receptor status in primary tumour, and disease outcome in endometrial cancer. PATIENTS AND METHODS: In total, 1129 women primarily treated for endometrial carcinoma at Haukeland University Hospital during 1981-2009 were studied. Body mass index was available for 949 patients and related to comprehensive clinical and histopathological data, hormone receptor status in tumour, treatment, and follow-up. RESULTS: High BMI was significantly associated with low International Federation of Gynaecology and Obstetrics (FIGO) stage, endometrioid histology, low/intermediate grade, and high level of progesterone receptor (PR) mRNA by qPCR (n=150; P=0.02) and protein expression by immunohistochemistry (n=433; P=0.003). In contrast, oestrogen receptor (ERα) status was not associated with BMI. Overweight/obese women had significantly better disease-specific survival (DSS) than normal/underweight women in univariate analysis (P=0.035). In multivariate analysis of DSS adjusting for age, FIGO stage, histological subtype, and grade, BMI showed no independent prognostic impact. CONCLUSION: High BMI was significantly associated with markers of non-aggressive disease and positive PR status in a large population-based study of endometrial carcinoma. Women with high BMI had significantly better prognosis in univariate analysis of DSS, an effect that disappeared in multivariate analysis adjusting for established prognostic markers. The role of PR in endometrial carcinogenesis needs to be further studied.

Highly infiltrative brain tumours show reduced chemosensitivity associated with a stem cell-like phenotype.

AIMS: Cancer stem-like cells might have important functions in chemoresistance. We have developed a model where highly infiltrative brain tumours with a stem-like phenotype were established by orthotopic transplantation of human glioblastomas to immunodeficient rats. Serial passaging gradually transformed the tumours into a less invasive and more angiogenic phenotype (high-generation tumours). The invasive phenotype (low-generation tumours) was characterized by an increase in stem cell markers and increased phosphorylation of kinases in the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. These markers were reduced in the serially passaged vascular tumours. The present study was aimed at investigating how the two phenotypes responded in vitro to doxorubicin, a clinically potent cytotoxic drug for solid tumours. METHODS: Biopsy spheroids were implanted and passaged intracranially in nude rats. Gene expression and protein analyses were performed, and drug sensitivity was assessed. RESULTS: Microarray analysis revealed gene ontology categories connected to developmental aspects and negative regulators of differentiation, especially in the infiltrative stem cell-like tumours. The highly invasive stem-like phenotype was chemoresistant compared with the angiogenic phenotype. By interfering with the PI3K it was possible to sensitize tumour spheroids to chemotherapy. Real-time quantitative polymerase chain reaction showed downregulation of the stem cell markers Nestin and Musashi-1 in low-generation biopsy spheroids following PI3K inhibition. CONCLUSIONS: Highly invasive tumours with a stem-like phenotype are more chemoresistant than angiogenic tumours derived from the same patients. We suggest that treatment resistance in glioblastomas can be related to PI3K/AKT activity in stem-like tumour cells, and that targeted interference with the PI3K/AKT pathway might differentiate and sensitize this subpopulation to chemotherapy.

Low BMI-1 expression is associated with an activated BMI-1-driven signature, vascular invasion, and hormone receptor loss in endometrial carcinoma.

We studied the expression of polycomb group (PcG) protein BMI-1 in a large population-based patient series of endometrial carcinomas in relation to clinical and molecular phenotype. Also, 57 fresh frozen endometrial carcinomas were studied for the relationship between BMI-1 protein expression, BMI-1 mRNA level, and activation of an 11-gene signature reported to represent a BMI-1-driven pathway. BMI-1 protein expression was significantly weaker in tumours with vascular invasion (P<0.0001), deep myometrial infiltration (P=0.004), and loss of oestrogen receptor (ER) (P<0.0001) and progesterone receptors (PR) (P=0.03). Low BMI-1 protein expression was highly associated with low BMI-1 mRNA expression (P=0.002), and similarly low BMI-1 mRNA expression correlated significantly with vascular invasion, ER and PR loss, and histologic grade 3. In contrast, activation of the reported 11-gene signature, supposed to represent a BMI-1-driven pathway, correlated with low mRNA expression of BMI-1 (P<0.001), hormone receptor loss, presence of vascular invasion, and poor prognosis. We conclude that BMI-1 protein and mRNA expression are significantly correlated and that BMI-1 expression is inversely associated with activation of the 11-gene signature. Loss of BMI-1 seems to be associated with an aggressive phenotype in endometrial carcinomas.

Early molecular replication of human immunodeficiency virus type 1 in cultured-blood-derived T helper dendritic cells.

The rate and efficiency of key steps in the life cycle of the human immunodeficiency virus type 1 was examined in three primary cell types, T cells, monocytes, and T helper dendritic cells using the same quantity of virus involved and same cell number. The results show that viral DNA synthesis proceeds much more rapidly and efficiently in primary T helper dendritic cell populations than in primary T cell and monocyte populations. The increased rate of virus DNA synthesis is attributable either to an increase in the efficiency and the rate of uptake of the virus particles by the T helper dendritic cells, as compared with that in other cell types, or to an increased efficiency and rate of viral DNA synthesis in the T helper dendritic cells. In the subsequent phase of viral expression the appearance of spliced viral mRNA products also occur more rapidly in cultures of primary-blood-derived T helper dendritic cells than is the case in primary T cells and monocytes. The increased efficiency of the early steps of HIV-1 replication in primary-blood-derived T helper dendritic cells than in other blood-derived mononuclear cells raises the possibility that these cells play a central role in HIV-1 infection and pathogenesis.

The human immunodeficiency virus type 1 Rev protein shuttles between the cytoplasm and nuclear compartments.

A retroviral regulatory protein, Rev (regulator of virion protein expression), is made in cells infected by human immunodeficiency virus (HIV). Rev is essential for the completion of the retroviral life cycle and interacts with the host cell at some posttranscriptional step in order to express the incompletely spliced HIV mRNAs from which HIV structural proteins are translated. Neither the host cell components nor the mechanisms responsible for this important regulation have been defined. We now report that Rev is a nucleocytoplasmic shuttle protein which is continuously transported between the cytoplasm, the nucleoli, and nucleoplasmic speckles enriched in RNA splicing and processing factors. The results show that Rev has the potential to interfere specifically with the splicing of the HIV pre-mRNA in the nucleoplasm and, next, guide such mRNAs to the cytoplasm for translation.

Labeling of RNA transcripts of eukaryotic cells in culture with BrUTP using a liposome transfection reagent (DOTAP).

Bromouridine-triphosphate (BrUTP), when introduced into eukaryotic cells in culture, substitutes for UTP during transcription, thereby labeling pre-mRNA for detection by immunochemical methods. In earlier studies, BrUTP was internalized by means of microinjection or by exposing isolated nuclei or permeable cells to BrUTP. We describe here a simple method for the extensive uptake of BrUTP into monolayers of growing cells using a cationic liposome transfectant (DOTAP). Within minutes, DOTAP mediates uptake of BrUTP both at 37 degrees and 4 degrees C. This is followed by incorporation into RNA in the nucleus upon further incubation under culture conditions. In this way, large numbers of actively growing cells may be subjected to biochemical experiments.

Viral RNA species in cell lines persistently and lytically infected with measles virus.

Identical measles viral mRNA species were present in similar amounts in the persistently infected cell lines LU106, HEpPi and MaSSPE, and in lytically infected cells as determined from Northern blots. The attenuation of transcription with the gene order did not vary significantly between different infected systems. A previously described selective restriction of F protein production in Lu106 cells could not be explained by defective transcription of F mRNA. RNA synthesis also continued unimpeded at restrictive temperatures for the temperature-sensitive viruses in Lu106 and HEpPi cells. Northern blotting revealed a prominent band in HEpPi RNA and a weak band in Lu106 RNA with the characteristics of incomplete genomes. In all infected cells, previously unrecognized small RNA species, hybridizing with the F- and H-specific probes, were discovered.

Construction and characterization of complementary DNA libraries from Vero cells infected with measles virus.

Several cDNA libraries have been generated from poly(A)RNA from Vero cells infected for 24 hours with measles virus. Different protocols for cDNA library construction were compared and some critical steps were evaluated. From these libraries, a measles virus specific sequence corresponding to 885 of 1600 nucleotides of the measles virus phosphoprotein gene has been cloned. The phosphoprotein gene accounts for 1% of the total cDNA library after 24 hours of infection at 37 degrees C. The technique of differential colony hybridization was used to analyze the distribution and change of the poly(A)-RNA expression in uninfected Vero cells and in cells infected with measles virus for 24 hours.

Study of transcription in measles virus-infected Vero cells using cDNA probes prepared from poly(A)RNA from uninfected and infected cells.

From af primary plasmid cDNA library prepared from measles virus-infected Vero cell poly(A)RNA, 435 clones selected at random were used to examine the sensitivity and specificity of cDNA probes derived from total poly(A)RNA from uninfected and infected Vero cells. The correlation between the abundance level of a particular species in the cDNA probe and the hybridization signal strength generated by the corresponding cDNA clone on a filter was reliably determined only when at least three independently prepared filters were examined. Variation in the amount of target plasmid was the most important cause of spurious signals. Variation in cDNA insert length did not disturb the signal strength within certain limits. cDNA species with abundance levels down to 0.08-0.01% were able to produce a hybridization signal above background. Unspecific cross-hybridization was shown to define the sensitivity limit of mixed cDNA probes. Despite the many false signals present at different stages, cDNA probes provided valuable information: the cDNA probes were used to monitor relative RNA expression levels and to clone five different measles virus transcripts and 2 host cell transcripts more abundantly expressed in infected cells. The abundance levels of the measles virus nucleocapsid, phosphoprotein, matrix, fusion protein and haemagglutinin genes were 1.5%, 1.5%, 1%, 0.75% and 0.5%, respectively, of the total cDNA library.

Stability of the nucleotide sequence of the phosphoprotein gene of measles virus during lytic infections.

Three clones with cDNA inserts encoding large portions of the measles virus phosphoprotein mRNA were characterized and compared with a previously published sequence of the Edmonston strain of measles virus. The two cloned viruses were separated by more than 100 passages. Only one out of 1477 nucleotides differed in the two sequences reflecting a very low mutation rate of the phosphoprotein gene during dilute lytic passages. The discovery that a third reading frame in the phosphoprotein gene may code for a novel peptide chain in addition to the P and C peptides may explain some of the high stability of the gene. The new reading frame was accessed by a translational shift caused by insertion of one extra G at a particular site in one of three otherwise identical cDNA sequences. A discrepancy was also found between the presumably high error rate of viral RNA polymerases and the stability of nucleotides in which mutations would not lead to amino acid substitutions. A few errors in the previously published sequence were discovered and the corrections are presented.

Two species of the phosphoprotein mRNA with differently edited reading frames discovered in measles virus infected Vero cells. Brief report.

One cDNA clone representing the phosphoprotein mRNA sequence of the Edmonston strain of measles virus contained 4 G nucleotides at a particular position. Two other clones contained 3 G nucleotides at the same position. Otherwise the nucleotide sequence were identical. The mRNA with 3 G nucleotides codes for the 70 kD phosphoprotein. The mRNA with 4 G nucleotides may code for a putative new peptide with 231 aminoterminal amino acids in common with the P protein whereas the 68 carboxyterminal amino acids are different from any amino acid sequence of the phosphoprotein. Thus the consequence of the insertion of one additional G is a translational shift to a shorter open reading frame. There are several indications that the observation is of biological significance.

Serum antibodies from MS patients do not recognize HTLV-I, HIV-1, HIV-2 and SIV.

A retroviral aetiology has been proposed for multiple sclerosis (MS). Although there is as yet no definitive evidence of viral involvement, there have been preliminary reports of antiretroviral antibody detection in sera from MS patients. Such sera have, for example, been found to react with HTLV-I. We here describe investigations involving various immunological techniques which attempt to confirm the virus-specific nature of these antibodies against a range of human and macaque retroviruses. Sera from 25 MS patients, 25 patients with non-associated neurological diseases and 16 patients with non-neurological conditions were tested by immunoblotting methods using lysates of HIV-1-, HIV-2-, HTLV-I- and SIV-infected cells as antigens. None of the sera reacted against any of these retroviral antigens but each serum demonstrated a distinctive and reproducible reaction pattern against cellular components of the cells in which the viruses were propagated. Further examination of the sera was carried out by ELISA using synthetic oligopeptides covering the HIV-1 Gag p24 protein as antigens. None of the sera reacted with the peptides. Our results suggest that in some MS patients the repeated seropositivity to HTLV-I may be due to the reaction with host cell proteins.

Mechanism of spontaneous loss of heat-stable toxin (STa) production in enterotoxigenic Escherichia coli.

Five strains of enterotoxigenic Escherichia coli (ETEC) showing spontaneous loss of heat-stable enterotoxin (STa) production were studied to elucidate the underlying genetic mechanisms. Southern blot analysis revealed that loss of STa production, and the corresponding lack of hybridization with the STa gene probes, were associated with deletions of DNA fragments harboring the relevant toxin genes rather than with loss of plasmids.

Immunoglobulin G subclass antibodies to rubella virus in chronic liver disease, acute rubella and healthy controls.

Ten patients with chronic liver disease, seven healthy seropositive individuals with a remote history of rubella, and three patients with acute rubella were examined for serum levels of IgG subclasses and subclass antibodies against rubella virus structural proteins. One patient with AICAH had no detectable total or rubella specific IgG3 or IgG4. The liver disease patients were hypergammaglobulinemic and had greatly raised IgG1 levels. Patients with acute rubella lacked antibodies to the rubella virus E2 protein and showed no IgG4 antibody response. The liver disease patients showed a somewhat weaker IgG4 antibody response against the core (C) protein than healthy controls. However, differences are suggested within the subclasses in antibody reactivity against the individual rubella virus antigens. It is concluded that test systems that discriminate reactivities against individual antigens have to be used for characterization of viral antibody subclass profiles.

Viral antibodies in infectious mononucleosis.

Patients with Epstein-Barr virus (EBV) infectious mononucleosis (IM) usually develop heterophilic antibodies and some autoantibodies. Antibodies to rubella, measles, adeno-, entero-, herpes simplex, cytomegalo- and varicella-zoster viruses were titrated in sera from IM patients and matched healthy controls using the complement fixation test (CFT) and the haemagglutination inhibition test. Except for herpes simplex virus and cytomegalovirus, the IM sera had significantly higher arithmetical and geometrical mean antibody titres and showed in most cases higher antibody prevalences in the CFT. The titre rise was most pronounced for rubella and measles antibodies, between 2- and 3-fold. There were no cases of very high titres occasionally seen in IM. The IM sera had higher total IgG serum levels than the controls, 17.27 g/l and 11.8 g/l, respectively (P < 0.001). The present data show that in addition to previously reported high levels of some autoantibodies and of heterophilic antibodies, there is a more general increase in IgG antibodies to commonly occurring viruses. This increase is most likely due to the polyclonal activation of B-lymphocytes following the binding of EBV to the complement receptor CR2 (CD21). When due consideration is given to the possible occasional occurrence of a false positive rubella IgM test, the raised antibody-titres will most likely not interfere with routine diagnostics.