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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Enhanced anti-cancer effect of a phosphatidylinositol-3 kinase inhibitor and doxorubicin on human breast epithelial cell lines with different p53 and oestrogen receptor status.

New efforts are being focused on signalling pathways as targets for cancer therapy. This particular study was designed to investigate whether blockade of the phosphatidylinositol 3OH-kinase (PI3K) pathway (a survival/anti-apoptosis pathway, overexpressed in various tumours) could sensitise human breast cancer cells to the effect of chemotherapeutics. Doxorubicin (Dox) and LY294002 (LY, a PI3K inhibitor) were used individually or in combination on MDA-MB-231 (p53 mutant, ER-), T47D (p53 mutant, ER+), and MCF-7 (p53 wildtype, ER+) human breast cancer cell lines, and on 184A1, a nonmalignant human breast epithelial cell line (p53 wildtype, ER-). Each drug showed time- and dose-dependent growth inhibition of cell proliferation on all 4 cell lines. The combination of Dox+LY resulted in enhanced cell growth inhibition in MDA-MB-231 and T47D cells, and additive inhibition in MCF-7 and 184A1 cells. Cell cycle analysis showed that Dox+LY enhanced the arrest of MDA-MB-231 and T47D cells in G2 with the appearance of a sub-G1 peak indicating apoptosis/necrosis, a notion supported by enhanced depolarisation of mitochondrial membrane potential in these cell types. The combination also caused a greater additive increase in Cyclin B1. Thus, the synergistic effect of the combination on cell proliferation in some, but not all, breast cancer cells may be through enhanced induction of both G2 arrest and apoptosis, in which p53 may play a role. Substantially lower doses of doxorubicin could be used with low doses of inhibitors of the PI3K pathway, without compromising the anti-cancer effect, but also lowering detrimental side-effects of doxorubicin. This study supports the notion that survival signalling pathways offer special targets for chemotherapy in cancer.

Metastatic susceptibility locus, an 8p hot-spot for tumour progression disrupted in colorectal liver metastases: 13 candidate genes examined at the DNA, mRNA and protein level.

BACKGROUND: Mortality from colorectal cancer is mainly due to metastatic liver disease. Improved understanding of the molecular events underlying metastasis is crucial for the development of new methods for early detection and treatment of colorectal cancer. Loss of chromosome 8p is frequently seen in colorectal cancer and implicated in later stage disease and metastasis, although a single metastasis suppressor gene has yet to be identified. We therefore examined 8p for genes involved in colorectal cancer progression. METHODS: Loss of heterozygosity analyses were used to map genetic loss in colorectal liver metastases. Candidate genes in the region of loss were investigated in clinical samples from 44 patients, including 6 with matched colon normal, colon tumour and liver metastasis. We investigated gene disruption at the level of DNA, mRNA and protein using a combination of mutation, semi-quantitative real-time PCR, western blotting and immunohistochemical analyses. RESULTS: We mapped a 2 Mb region of 8p21-22 with loss of heterozygosity in 73% of samples; 8/11 liver metastasis samples had loss which was not present in the corresponding matched primary colon tumour. 13 candidate genes were identified for further analysis. Both up and down-regulation of 8p21-22 gene expression was associated with metastasis. ADAMDEC1 mRNA and protein expression decreased during both tumourigenesis and tumour progression. Increased STC1 and LOXL2 mRNA expression occurred during tumourigenesis. Liver metastases with low DcR1/TNFRSF10C mRNA expression were more likely to present with extrahepatic metastases (p = 0.005). A novel germline truncating mutation of DR5/TNFRSF10B was identified, and DR4/TNFRSF10A SNP rs4872077 was associated with the development of liver metastases (p = 0.02). CONCLUSION: Our data confirm that genes on 8p21-22 are dysregulated during colorectal cancer progression. Interestingly, however, instead of harbouring a single candidate colorectal metastasis suppressor 8p21-22 appears to be a hot-spot for tumour progression, encoding at least 13 genes with a putative role in carcinoma development. Thus, we propose that this region of 8p comprises a metastatic susceptibility locus involved in tumour progression whose disruption increases metastatic potential.

Association of mutant TP53 with alternative lengthening of telomeres and favorable prognosis in glioma.

The molecular basis for alternative lengthening of telomeres (ALT), a prognostic marker for glioma patients, remains unknown. We examined TP53 status in relation to telomere maintenance mechanism (TMM) in 108 patients with glioblastoma multiforme and two patients with anaplastic astrocytoma from New Zealand and United Kingdom. Tumor samples were analyzed with respect to telomerase activity, telomere length, and ALT-associated promyelocytic leukemia nuclear bodies to determine their TMM. TP53 mutation was analyzed by direct sequencing of coding exons 2 to 11. We found an association between TP53 mutation and ALT mechanism and between wild-type TP53 and telomerase and absence of a known TMM (P < 0.0001). We suggest that TP53 deficiency plays a permissive role in the activation of ALT.

Y-box factor YB1 controls p53 apoptotic function.

Nuclear localization and high levels of the Y-box-binding protein YB1 appear to be important indicators of drug resistance and tumor prognosis. YB1 also interacts with the p53 tumor suppressor protein. In this paper, we have continued to explore YB1/p53 interactions. We report that transcriptionally active p53 is required for nuclear localization of YB1. We go on to show that nuclear YB1 regulates p53 function. Our data demonstrate that YB1 inhibits the ability of p53 to cause cell death and to transactivate cell death genes, but does not interfere with the ability of p53 to transactivate the CDKN1A gene, encoding the kinase p21(WAF1/CIP1) required for cell cycle arrest, nor the MDM2 gene. We also show that nuclear YB1 is associated with a failure to increase the level of the Bax protein in normal mammary epithelial cells after stress activation of p53. Together these data suggest that (nuclear) YB1 selectively alters p53 activity, which may in part provide an explanation for the correlation of nuclear YB1 with drug resistance and poor tumor prognosis.

A robust assay for alternative lengthening of telomeres in tumors shows the significance of alternative lengthening of telomeres in sarcomas and astrocytomas.

PURPOSE AND EXPERIMENTAL DESIGN: Telomeres of tumor cells may be maintained by telomerase or by alternative lengthening of telomeres (ALT). The standard ALT assay requires Southern analysis of high molecular weight genomic DNA. We aimed to establish and validate an ALT assay suitable for archived paraffin-embedded tumors and to use it to examine the prevalence and clinical significance of ALT in various types of tumors that are often telomerase negative. RESULTS: To assay for ALT, we detected ALT-associated promyelocytic leukemia (PML) bodies (APBs) by combined PML immunofluorescence and telomere fluorescence in situ hybridization. APBs are PML nuclear domains containing telomeric DNA and are a known hallmark of ALT in cell lines. The APB assay concurred with the standard ALT assay in 62 of 62 tumors and showed that 35% of 101 soft tissue sarcomas (STS), 47% of 58 osteosarcomas (especially younger patients), 34% of 50 astrocytomas, and 0% of 17 papillary thyroid carcinomas were ALT positive (ALT+). The prevalence of ALT varied greatly among different STS subtypes: malignant fibrous histiocytomas, 77%; leiomyosarcomas, 62%; liposarcomas, 33%; synovial sarcomas, 9%; and rhabdomyosarcomas, 6%. ALT correlated with survival in glioblastoma multiforme and occurred more often in lower-grade astrocytomas, but ALT+ and ALT- sarcomas were equally aggressive in terms of grade and clinical outcome. CONCLUSION: The APB assay for ALT is suitable for paraffin-embedded tumors. It showed that a substantial proportion of STS, osteosarcomas, and astrocytomas, but not papillary thyroid carcinomas use ALT. APB positivity correlated strongly with survival of patients with astrocytomas.

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.