Ascorbate content of clinical glioma tissues is related to tumour grade and to global levels of 5-hydroxymethyl cytosine.

Gliomas are incurable brain cancers with poor prognosis, with epigenetic dysregulation being a distinctive feature. 5-hydroxymethylcytosine (5-hmC), an intermediate generated in the demethylation of 5-methylcytosine, is present at reduced levels in glioma tissue compared with normal brain, and that higher levels of 5-hmC are associated with improved patient survival. DNA demethylation is enzymatically driven by the ten-eleven translocation (TET) dioxygenases that require ascorbate as an essential cofactor. There is limited data on ascorbate in gliomas and the relationship between ascorbate and 5-hmC in gliomas has never been reported. Clinical glioma samples (11 low-grade, 26 high-grade) were analysed for ascorbate, global DNA methylation and hydroxymethylation, and methylation status of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter. Low-grade gliomas contained significantly higher levels of ascorbate than high-grade gliomas (p = 0.026). Levels of 5-hmC were significantly higher in low-grade than high-grade glioma (p = 0.0013). There was a strong association between higher ascorbate and higher 5-hmC (p = 0.004). Gliomas with unmethylated and methylated MGMT promoters had similar ascorbate levels (p = 0.96). One mechanism by which epigenetic modifications could occur is through ascorbate-mediated optimisation of TET activity in gliomas. These findings open the door to clinical intervention trials in patients with glioma to provide both mechanistic information and potential avenues for adjuvant ascorbate therapy.

Increased Ascorbate Content of Glioblastoma Is Associated With a Suppressed Hypoxic Response and Improved Patient Survival.

Glioblastoma multiforme is a challenging disease with limited treatment options and poor survival. Glioblastoma tumours are characterised by hypoxia that activates the hypoxia inducible factor (HIF) pathway and controls a myriad of genes that drive cancer progression. HIF transcription factors are regulated at the post-translation level via HIF-hydroxylases. These hydroxylases require oxygen and 2-oxoglutarate as substrates, and ferrous iron and ascorbate as cofactors. In this retrospective observational study, we aimed to determine whether ascorbate played a role in the hypoxic response of glioblastoma, and whether this affected patient outcome. We measured the ascorbate content and members of the HIF-pathway of clinical glioblastoma samples, and assessed their association with clinicopathological features and patient survival. In 37 samples (37 patients), median ascorbate content was 7.6 µg ascorbate/100 mg tissue, range 0.8 - 20.4 µg ascorbate/100 mg tissue. In tumours with above median ascorbate content, HIF-pathway activity as a whole was significantly suppressed (p = 0.005), and several members of the pathway showed decreased expression (carbonic anhydrase-9 and glucose transporter-1, both p < 0.01). Patients with either lower tumour HIF-pathway activity or higher tumour ascorbate content survived significantly longer than patients with higher HIF-pathway or lower ascorbate levels (p = 0.011, p = 0.043, respectively). Median survival for the low HIF-pathway score group was 362 days compared to 203 days for the high HIF-pathway score group, and median survival for the above median ascorbate group was 390 days, compared to the below median ascorbate group with 219 days. The apparent survival advantage associated with higher tumour ascorbate was more prominent for the first 8 months following surgery. These associations are promising, suggesting an important role for ascorbate-regulated HIF-pathway activity in glioblastoma that may impact on patient survival.

The Role of 2-Oxoglutarate Dependent Dioxygenases in Gliomas and Glioblastomas: A Review of Epigenetic Reprogramming and Hypoxic Response.

Gliomas are a heterogeneous group of cancers that predominantly arise from glial cells in the brain, but may also arise from neural stem cells, encompassing low-grade glioma and high-grade glioblastoma. Whereas better diagnosis and new treatments have improved patient survival for many cancers, glioblastomas remain challenging with a highly unfavorable prognosis. This review discusses a super-family of enzymes, the 2-oxoglutarate dependent dioxygenase enzymes (2-OGDD) that control numerous processes including epigenetic modifications and oxygen sensing, and considers their many roles in the pathology of gliomas. We specifically describe in more detail the DNA and histone demethylases, and the hypoxia-inducible factor hydroxylases in the context of glioma, and discuss the substrate and cofactor requirements of the 2-OGDD enzymes. Better understanding of how these enzymes contribute to gliomas could lead to the development of new treatment strategies.

Intronic TP53 Polymorphisms Are Associated with Increased Δ133TP53 Transcript, Immune Infiltration and Cancer Risk.

We investigated the influence of selected TP53 SNPs in exon 4 and intron 4 on cancer risk, clinicopathological features and expression of TP53 isoforms. The intron 4 SNPs were significantly over-represented in cohorts of mixed cancers compared to three ethnically matched controls, suggesting they confer increased cancer risk. Further analysis showed that heterozygosity at rs1042522(GC) and either of the two intronic SNPs rs9895829(TC) and rs2909430(AG) confer a 2.34-5.35-fold greater risk of developing cancer. These SNP combinations were found to be associated with shorter patient survival for glioblastoma and prostate cancer. Additionally, these SNPs were associated with tumor-promoting inflammation as evidenced by high levels of infiltrating immune cells and expression of the Δ133TP53 and TP53ß transcripts. We propose that these SNP combinations allow increased expression of the Δ133p53 isoforms to promote the recruitment of immune cells that create an immunosuppressive environment leading to cancer progression.

The oncogene BCL6 is up-regulated in glioblastoma in response to DNA damage, and drives survival after therapy.

The prognosis for people with the high-grade brain tumor glioblastoma is very poor, due largely to low cell death in response to genotoxic therapy. The transcription factor BCL6, a protein that normally suppresses the DNA damage response during immune cell maturation, and a known driver of B-cell lymphoma, was shown to mediate the survival of glioblastoma cells. Expression was observed in glioblastoma tumor specimens and cell lines. When BCL6 expression or activity was reduced in these lines, increased apoptosis and a profound loss of proliferation was observed, consistent with gene expression signatures suggestive of anti-apoptotic and pro-survival signaling role for BCL6 in glioblastoma. Further, treatment with the standard therapies for glioblastoma-ionizing radiation and temozolomide-both induced BCL6 expression in vitro, and an in vivo orthotopic animal model of glioblastoma. Importantly, inhibition of BCL6 in combination with genotoxic therapies enhanced the therapeutic effect. Together these data demonstrate that BCL6 is an active transcription factor in glioblastoma, that it drives survival of cells, and that it increased with DNA damage, which increased the survival rate of therapy-treated cells. This makes BCL6 an excellent therapeutic target in glioblastoma-by increasing sensitivity to standard DNA damaging therapy, BCL6 inhibitors have real potential to improve the outcome for people with this disease.

Elevation of the TP53 isoform Δ133p53ß in glioblastomas: an alternative to mutant p53 in promoting tumor development.

As tumor protein 53 (p53) isoforms have tumor-promoting, migration, and inflammatory properties, this study investigated whether p53 isoforms contributed to glioblastoma progression. The expression levels of full-length TP53α (TAp53α) and six TP53 isoforms were quantitated by RT-qPCR in 89 glioblastomas and correlated with TP53 mutation status, tumor-associated macrophage content, and various immune cell markers. Elevated levels of Δ133p53ß mRNA characterised glioblastomas with increased CD163-positive macrophages and wild-type TP53. In situ-based analyses found Δ133p53ß expression localised to malignant cells in areas with increased hypoxia, and in cells with the monocyte chemoattractant protein C-C motif chemokine ligand 2 (CCL2) expressed. Tumors with increased Δ133p53ß had increased numbers of cells positive for macrophage colony-stimulating factor 1 receptor (CSF1R) and programmed death ligand 1 (PDL1). In addition, cells expressing a murine 'mimic' of Δ133p53 (Δ122p53) were resistant to temozolomide treatment and oxidative stress. Our findings suggest that elevated Δ133p53ß is an alternative pathway to TP53 mutation in glioblastoma that aids tumor progression by promoting an immunosuppressive and chemoresistant environment. Adding Δ133p53ß to a TP53 signature along with TP53 mutation status will better predict treatment resistance in glioblastoma. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Telomere profiles and tumor-associated macrophages with different immune signatures affect prognosis in glioblastoma.

Telomere maintenance is a hallmark of cancer and likely to be targeted in future treatments. In glioblastoma established methods of identifying telomerase and alternative lengthening of telomeres leave a significant proportion of tumors with no defined telomere maintenance mechanism. This study investigated the composition of these tumors using RNA-Seq. Glioblastomas with an indeterminate telomere maintenance mechanism had an increased immune signature compared with alternative lengthening of telomeres and telomerase-positive tumors. Immunohistochemistry for CD163 confirmed that the majority (80%) of tumors with an indeterminate telomere maintenance mechanism had a high presence of tumor-associated macrophages. The RNA-Seq and immunostaining data separated tumors with no defined telomere maintenance mechanism into three subgroups: alternative lengthening of telomeres like tumors with a high presence of tumor-associated macrophages and telomerase like tumors with a high presence of tumor-associated macrophages. The third subgroup had no increase in tumor-associated macrophages and may represent a distinct category. The presence of tumor-associated macrophages conferred a worse prognosis with reduced patient survival times (alternative lengthening of telomeres with and without macrophages P=0.0004, and telomerase with and without macrophages P=0.013). The immune signatures obtained from RNA-Seq were significantly different between telomere maintenance mechanisms. Alternative lengthening of telomeres like tumors with macrophages had increased expression of interferon-induced proteins with tetratricopeptide repeats (IFIT1-3). Telomerase-positive tumors with macrophages had increased expression of macrophage receptor with collagenous structure (MARCO), CXCL12 and sushi-repeat containing protein x-linked 2 (SRPX2). Telomerase-positive tumors with macrophages were also associated with a reduced frequency of total/near total resections (44% vs >76% for all other subtypes, P=0.014). In summary, different immune signatures are found among telomere maintenance mechanism-based subgroups in glioblastoma. The reduced extent of surgical resection of telomerase-positive tumors with macrophages suggests that some tumor-associated macrophages are more unfavorable.

A clinicopathological study of episomal papillomavirus infection of the human placenta and pregnancy complications.

Viral infections are known to adversely affect pregnancy, but scant attention has been given to human papilloma virus (HPV) infection. We aimed to determine the molecular and histopathological features of placental HPV infection, in association with pregnancy complications including fetal growth restriction, pre-maturity, pre-eclampsia, and diabetes. Three hundred and thirty-nine placentae were selected based on the presence or absence of pregnancy complications. Five independent methods were used to identify HPV in the placenta, namely, immunohistochemistry for L1 viral capsid, in situ hybridization to high-risk HPV DNA, PCR, western blotting, and transmission electron microscopy. Pregnancy complications and uterine cervical smear screening results were correlated with placental HPV histopathology. In this study, which was deliberately biased towards complications, HPV was found in the decidua of 75% of placentae (253/339) and was statistically associated with histological acute chorioamnionitis (P<0.05). In 14% (35/253) of the HPV positive cases, HPV L1 immunoreactivity also occurred in the villous trophoblast where it was associated with a lymphohistiocytic villitis (HPV-LHV), and was exclusively of high-risk HPV type. HPV-LHV significantly associated with fetal growth restriction, preterm delivery, and pre-eclampsia (all P<0.05). All cases of pre-eclampsia (20/20) in our cohort had high-risk placental HPV. A further 55 cases (22%, 55/253) of HPV positive placentae had minimal villous trophoblast HPV L1 immunoreactivity, but a sclerosing pauci-immune villitis, statistically associated with diabetes (49.1%, 27/55, P<0.05). For women with placental HPV, 33% (69/207) had an HPV-related positive smear result before pregnancy compared with (9.4% 8/85) of women with HPV-negative placentae (P=0.0001). Our findings support further investigations to determine if vaccination of women and men will improve pregnancy outcomes.

Alpha-enolase is upregulated on the cell surface and responds to plasminogen activation in mice expressing a ∆133p53α mimic.

The p53 protein is a master regulator of the stress response. It acts as a tumor suppressor by inducing transcriptional activation of p53 target genes, with roles in apoptosis, cell cycle arrest and metabolism. The discovery of at least 12 isoforms of p53, some of which have tumor-promoting properties, has opened new avenues of research. Our previous work studied tumor phenotypes in four mouse models with different p53 backgrounds: wild-type p53, p53 null, mutant p53 lacking the proline domain (mΔpro), and a mimic for the human Δ133p53α p53 isoform (Δ122p53). To identify the major proteins affected by p53 function early in the response to DNA damage, the current study investigated the entire proteome of bone marrow, thymus, and lung in the four p53 models. Protein extracts from untreated controls and those treated with amsacrine were analyzed using two-dimensional fluorescence difference gel electrophoresis. In the bone marrow, reactive proteins were universally decreased by wild-type p53, including α-enolase. Further analysis of α-enolase in the p53 models revealed that it was instead increased in Δ122p53 hematopoietic and tumor cell cytosol and on the cell surface. Alpha-enolase on the surface of Δ122p53 cells acted as a plasminogen receptor, with tumor necrosis factor alpha induced upon plasminogen stimulation. Taken together, these data identified new proteins associated with p53 function. One of these proteins, α-enolase, is regulated differently by wild-type p53 and Δ122p53 cells, with reduced abundance as part of a wild-type p53 response and increased abundance with Δ122p53 function. Increased cell surface α-enolase on Δ122p53 cells provides a possible explanation for the model's pro-inflammatory features and suggests that p53 isoforms may direct an inflammatory response by increasing the amount of α-enolase on the cell surface.

Loss of ATRX and DAXX expression identifies poor prognosis for smooth muscle tumours of uncertain malignant potential and early stage uterine leiomyosarcoma.

Uterine smooth muscle tumours of uncertain malignant potential (STUMP) are diagnostically and clinically challenging. The alternative lengthening of telomeres (ALT) telomere maintenance mechanism is associated with poor survival in soft tissue leiomyosarcoma. Time to first recurrence and survival were known for 18 STUMP and 43 leiomyosarcomata (LMS). These were screened for ALT telomere maintenance by the presence of ALT-associated PML bodies (APBs) and for changes associated with the ALT phenotype, namely aberrant p53 expression, isocitrate dehydrogenase 1 mutation (R132H substitution) expression, mutant ATRX (αthalassemia/mental retardation syndrome X-linked) expression and mutant DAXX (death-domain-associated protein) expression by immunohistochemistry (IHC). Overexpression of p16(INK4A) was examined immunohistologically in a subset of cases. Many of the tumours associated with death or recurrence demonstrated APBs commensurate with ALT telomere maintenance. However, all uterine STUMP (4/4), and vaginal STUMP (2/2) patients, and almost all LMS patients (88.4%, 23/26, including 90% (9/10) of stage 1 LMS cases), who had died of disease or who had recurrent disease, displayed loss of ATRX or DAXX expression. Loss of ATRX or DAXX expression identified poor prognosis (95% CI 2.1 to 40.8, p < 0.003), in the LMS group. Thus, loss of ATRX or DAXX expression in uterine smooth muscle tumours identifies a clinically aggressive molecular subtype of early stage LMS and when histopathological features are problematic such as in STUMP. As ATRX and DAXX IHC is readily performed in diagnostic laboratories these are potentially useful for routine histopathological classification and management.

The rs11515 Polymorphism Is More Frequent and Associated With Aggressive Breast Tumors with Increased ANRIL and Decreased p16 (INK4a) Expression.

Chromosome position 9p21 encodes three-tumor suppressors p16(INK4a), p14(ARF), and p15(INK4b) and the long non-coding RNA ANRIL (antisense non-coding RNA in the INK4 locus). The rs11515 single-nucleotide polymorphism in the p16 (INK4a) /p14 (ARF) 3'-untranslated region is associated with glioblastoma, melanoma, and other cancers. This study investigated the frequency and effect of rs11515 genotypes in breast cancer. Genomic DNA samples from 400 women (200 with and 200 without a diagnosis of breast cancer) were genotyped for the rs11515 major (C) and minor (G) alleles. The rs11515 polymorphism was also investigated in 108 heart tissues to test for tissue-specific effects. Four 9p21 transcripts, p16 (INK4a) , p14 (ARF) , p15 (INK4b) , and ANRIL were measured in breast tumors and myocardium using quantitative PCR. Heterozygotes (CG genotype) were more frequent in women with breast cancer compared to the control population (P = 0.0039). In those with breast cancer, the CG genotype was associated with an older age (P = 0.016) and increased lymph node involvement (P = 0.007) compared to homozygotes for the major allele (CC genotype). In breast tumors, the CG genotype had higher ANRIL (P = 0.031) and lower p16 (INK4a) (P = 0.006) expression compared to the CC genotype. The CG genotype was not associated with altered 9p21 transcripts in heart tissue. In breast cancer, the rs11515 CG genotype is more frequent and associated with a more aggressive tumor that could be due to increased ANRIL and reduced p16 (INK4a) expression. The absence of association between rs11515 genotypes and 9p21 transcripts in heart tissue suggests this polymorphism has tissue- or disease-specific functions.

Increased paired box transcription factor 8 has a survival function in glioma.

BACKGROUND: The molecular basis to overcome therapeutic resistance to treat glioblastoma remains unclear. The anti-apoptotic b cell lymphoma 2 (BCL2) gene is associated with treatment resistance, and is transactivated by the paired box transcription factor 8 (PAX8). In earlier studies, we demonstrated that increased PAX8 expression in glioma cell lines was associated with the expression of telomerase. In this current study, we more extensively explored a role for PAX8 in gliomagenesis. METHODS: PAX8 expression was measured in 156 gliomas including telomerase-negative tumours, those with the alternative lengthening of telomeres (ALT) mechanism or with a non-defined telomere maintenance mechanism (NDTMM), using immunohistochemistry and quantitative PCR. We also tested the affect of PAX8 knockdown using siRNA in cell lines on cell survival and BCL2 expression. RESULTS: Seventy-two percent of glioblastomas were PAX8-positive (80% telomerase, 73% NDTMM, and 44% ALT). The majority of the low-grade gliomas and normal brain cells were PAX8-negative. The suppression of PAX8 was associated with a reduction in both cell growth and BCL2, suggesting that a reduction in PAX8 expression would sensitise tumours to cell death. CONCLUSIONS: PAX8 is increased in the majority of glioblastomas and promoted cell survival. Because PAX8 is absent in normal brain tissue, it may be a promising therapeutic target pathway for treating aggressive gliomas.

Smoking during pregnancy causes double-strand DNA break damage to the placenta.

Despite the adverse effects of smoking, many pregnancies are exposed to tobacco smoke. Recent studies have investigated whether smoking damages placental DNA by measuring DNA adducts. This study investigated whether a more severe lesion, double-strand DNA breaks, was also present in the tobacco smoking-exposed placenta. Term placentae from women who smoked during their entire pregnancies (n = 52), from those who had ceased smoking for at least 4 weeks before delivery (previous smokers, n = 34), and from nonsmoking women (n = 150) were examined using the DNA double-strand break marker phosphorylated γ H2AX. The extent of DNA damage was assessed according to cell type and additional markers were applied for cell fate (apoptosis and DNA repair), and function (human chorionic gonadotropin, human placental lactogen, and glucose transporter 1), to characterize the effect of the DNA damage on placental integrity. Marked phosphorylated γ H2AX-positive cells occurred in the villous syncytiotrophoblast and syncytial knot nuclei in placentae from smokers (P < .001). Phosphorylated γ H2AX foci did not colocalize with the DNA repair protein 53BP1, and damaged nuclei had a marked reduction in expression of human chorionic gonadotropin, human placental lactogen, and glucose transporter 1. Minimal DNA damage, similar to nonsmokers, was present in previous smokers including those that had ceased smoking for just over 4 weeks before delivery. In summary, smoking during pregnancy was associated with marked double-strand DNA break damage to the syncytiotrophoblast. We suggest that smoking cessation is important to prevent additional DNA damage and to facilitate DNA repair.

Detection of alternative lengthening of telomeres by telomere quantitative PCR.

Alternative lengthening of telomeres (ALT) is one of the two known telomere length maintenance mechanisms that are essential for the unlimited proliferation potential of cancer cells. Existing methods for detecting ALT in tumors require substantial amounts of tumor material and are labor intensive, making it difficult to study prevalence and prognostic significance of ALT in large tumor cohorts. Here, we present a novel strategy utilizing telomere quantitative PCR to diagnose ALT. The protocol is more rapid than conventional methods and scrutinizes two distinct characteristics of ALT cells concurrently: long telomeres and the presence of C-circles (partially double-stranded circles of telomeric C-strand DNA). Requiring only 30 ng of genomic DNA, this protocol will facilitate large-scale studies of ALT in tumors and can be readily adopted by clinical laboratories.

The alternative lengthening of telomeres pathway may operate in non-neoplastic human cells.

The alternative lengthening of telomeres (ALT) mechanism represents an alternative to the enzyme telomerase in the maintenance of mammalian telomeres in 25-60% of sarcomas and a minority of carcinomas (about 5-15%). ALT-positive cells are distinguished by long and heterogeneous telomere length distributions by terminal restriction fragment (TRF) Southern blotting. Another diagnostic marker of ALT is discrete nuclear co-localized signals of telomeric DNA and the promyelocytic leukaemia protein (PML), referred to as ALT-associated PML bodies (APBs). Recently, we detected smaller sized co-localized PML and telomere DNA (APB-like) bodies in endothelial cells adjacent to astrocytoma tumour cells in situ. In this study, we examined a wide variety of non-neoplastic tissues, and report that co-localized signals of PML and telomere DNA are present in endothelial, stromal, and some epithelial cells. Co-localized signals of PML and telomere DNA showed an increased frequency in non-neoplastic cells with DNA damage. These results suggest that a mechanism similar to that in ALT-positive tumours also operates in non-neoplastic cells, which may be activated by DNA damage.

The CDKN2A G500 allele is more frequent in GBM patients with no defined telomere maintenance mechanism tumors and is associated with poorer survival.

Prognostic markers for glioblastoma multiforme (GBM) are important for patient management. Recent advances have identified prognostic markers for GBMs that use telomerase or the alternative lengthening of telomeres (ALT) mechanism for telomere maintenance. Approximately 40% of GBMs have no defined telomere maintenance mechanism (NDTMM), with a mixed survival for affected individuals. This study examined genetic variants in the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene that encodes the p16(INK4a) and p14(ARF) tumor suppressors, and the isocitrate dehydrogenase 1 (IDH1) gene as potential markers of survival for 40 individuals with NDTMM GBMs (telomerase negative and ALT negative by standard assays), 50 individuals with telomerase, and 17 individuals with ALT positive tumors. The analysis of CDKN2A showed NDTMM GBMs had an increased minor allele frequency for the C500G (rs11515) polymorphism compared to those with telomerase and ALT positive GBMs (p = 0.002). Patients with the G500 allele had reduced survival that was independent of age, extent of surgery, and treatment. In the NDTMM group G500 allele carriers had increased loss of CDKN2A gene dosage compared to C500 homozygotes. An analysis of IDH1 mutations showed the R132H mutation was associated with ALT positive tumors, and was largely absent in NDTMM and telomerase positive tumors. In the ALT positive tumors cohort, IDH1 mutations were associated with a younger age for the affected individual. In conclusion, the G500 CDKN2A allele was associated with NDTMM GBMs from older individuals with poorer survival. Mutations in IDH1 were not associated with NDTMM GBMs, and instead were a marker for ALT positive tumors in younger individuals.

Prognostic association of YB-1 expression in breast cancers: a matter of antibody.

The literature concerning the subcellular location of Y-box binding protein 1 (YB-1), its abundance in normal and cancer tissues, and its prognostic significance is replete with inconsistencies. An explanation for this could be due in part to the use of different antibodies in immunohistochemical and immunofluorescent labeling of cells and tissues. The inconsistencies could also be due to poor resolution of immunohistochemical data. We analyzed two cohorts of breast tumours for both abundance and subcellular location of YB-1 using three different antibodies; two targeting N-terminal epitopes (AB-a and AB-b) and another (AB-c) targeting a C-terminal epitope. We also investigated stress-induced nuclear translocation of YB-1 in cell culture. We report that both AB-a and AB-c detected increased YB-1 in the cytoplasm of high-grade breast cancers, and in those lacking estrogen and progesterone receptors; however the amount of YB-1 detected by AB-a in these cancers is significantly greater than that detected by AB-c. We confirm our previously published findings that AB-b is also detecting hnRNP A1, and cannot therefore be used to reliably detect YB-1 by immunohistochemistry. We also report that AB-a detected nuclear YB-1 in some tumour tissues and stress treated cells, whereas AB-c did not. To understand this, cancer cell lines were analyzed using native gel electrophoresis, which revealed that the antibodies detect different complexes in which YB-1 is a component. Our data suggest that different YB-1 antibodies show different staining patterns that are determined by the accessibility of epitopes, and this depends on the nature of the YB-1 complexes. It is important therefore to standardize the protocols if YB-1 is to be used reproducibly as a prognostic guide for different cancers.

Hyperproliferation, cancer, and inflammation in mice expressing a Δ133p53-like isoform.

The p53 protein is a pivotal tumor suppressor that is frequently mutated in many human cancers, although precisely how p53 prevents tumors is still unclear. To add to its complexity, several isoforms of human p53 have now been reported. The Δ133p53 isoform is generated from an alternative transcription initiation site in intron 4 of the p53 gene (Tp53) and lacks the N-terminus. Elevated levels of Δ133p53 have been observed in a variety of tumors. To explore the functions of Δ133p53, we created a mouse expressing an N-terminal deletion mutant of p53 (Δ122p53) that corresponds to Δ133p53. Δ122p53 mice show decreased survival and a different and more aggressive tumor spectrum compared with p53 null mice, implying that Δ122p53 is a dominant oncogene. Consistent with this, Δ122p53 also confers a marked proliferative advantage on cells and reduced apoptosis. In addition to tumor development, Δ122p53 mice show a profound proinflammatory phenotype having increased serum concentrations of interleukin-6 and other proinflammatory cytokines and lymphocyte aggregates in the lung and liver as well as other pathologies. Based on these observations, we propose that human Δ133p53 also functions to promote cell proliferation and inflammation, one or both of which contribute to tumor development.

Presence of alternative lengthening of telomeres mechanism in patients with glioblastoma identifies a less aggressive tumor type with longer survival.

Patients with glioblastoma (GBM) have variable clinical courses, but the factors that underlie this heterogeneity are not understood. To determine whether the presence of the telomerase-independent alternative lengthening of telomeres (ALTs) mechanism is a significant prognostic factor for survival, we performed a retrospective analysis of 573 GBM patients. The presence of ALT was identified in paraffin sections using a combination of immunofluorescence for promyelocytic leukemia body and telomere fluorescence in situ hybridization. Alternative lengthening of telomere was present in 15% of the GBM patients. Patients with ALT had longer survival that was independent of age, surgery, and other treatments. Mutations in isocitrate dehydrogenase (IDH1mut) 1 frequently accompanied ALT, and in the presence of both molecular events, there was significantly longer overall survival. These data suggest that most ALT+ tumors may be less aggressive proneural GBMs, and the better prognosis may relate to the set of genetic changes associated with this tumor subtype. Despite improved overall survival of patients treated with the addition of chemotherapy to radiotherapy and surgery, ALT and chemotherapy independently provided a survival advantage, but these factors were not found to be additive. These results suggest a critical need for developing new therapies to target these specific GBM subtypes.

Normal human mammary epithelial cells proliferate rapidly in the presence of elevated levels of the tumor suppressors p53 and p21(WAF1/CIP1).

In normal cells, p53 protein is maintained at low levels, but the levels increase after stress or inappropriate growth signals to coordinate growth arrest or apoptosis. Human mammary epithelial cells (HMECs) are unusual in that they exhibit two phases of growth. The second growth phase, referred to as post-selection, follows a period of temporary growth arrest and is characterized by the absence of p16(INK4a) (also known as CDK4I and p16-INK4a) expression. Previously, we observed that post-selection HMECs have elevated levels of p53. Exogenous p16(INK4a) expression decreased levels of both p53 transcript and protein, and this effect was inhibited by nutlin-3a, indicating that p16(INK4a) can regulate p53 expression by affecting both p53 transcription and Mdm2-dependent degradation of p53. The p53 in post-selection HMECs was wild type and, as expected, increased p53 expression was associated with elevated p21(WAF1/CIP1) and Mdm2 levels; the p53 response to DNA damage seemed normal. Despite elevated levels of wild-type p53 and p21(WAF1/CIP1), post-selection cells grew more rapidly than their pre-selection HMEC precursors. We found that the post-selection HMECs contain a truncated Mdm2 protein (p60), which presumably lacks the p53 ubiquitylation domain. We propose that the increased levels of p53 in post-selection HMECs are due to the presence of an Mdm2 fragment that binds p53 but does not result in its degradation.