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.

Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X Ten.

BACKGROUND: Comprehensive genome-wide DNA methylation profiling is critical to gain insights into epigenetic reprogramming during development and disease processes. Among the different genome-wide DNA methylation technologies, whole genome bisulphite sequencing (WGBS) is considered the gold standard for assaying genome-wide DNA methylation at single base resolution. However, the high sequencing cost to achieve the optimal depth of coverage limits its application in both basic and clinical research. To achieve 15× coverage of the human methylome, using WGBS, requires approximately three lanes of 100-bp-paired-end Illumina HiSeq 2500 sequencing. It is important, therefore, for advances in sequencing technologies to be developed to enable cost-effective high-coverage sequencing. RESULTS: In this study, we provide an optimised WGBS methodology, from library preparation to sequencing and data processing, to enable 16-20× genome-wide coverage per single lane of HiSeq X Ten, HCS 3.3.76. To process and analyse the data, we developed a WGBS pipeline (METH10X) that is fast and can call SNPs. We performed WGBS on both high-quality intact DNA and degraded DNA from formalin-fixed paraffin-embedded tissue. First, we compared different library preparation methods on the HiSeq 2500 platform to identify the best method for sequencing on the HiSeq X Ten. Second, we optimised the PhiX and genome spike-ins to achieve higher quality and coverage of WGBS data on the HiSeq X Ten. Third, we performed integrated whole genome sequencing (WGS) and WGBS of the same DNA sample in a single lane of HiSeq X Ten to improve data output. Finally, we compared methylation data from the HiSeq 2500 and HiSeq X Ten and found high concordance (Pearson r > 0.9×). CONCLUSIONS: Together we provide a systematic, efficient and complete approach to perform and analyse WGBS on the HiSeq X Ten. Our protocol allows for large-scale WGBS studies at reasonable processing time and cost on the HiSeq X Ten platform.

Is cell culture a risky business? Risk analysis based on scientist survey data.

Cell culture is a technique that requires vigilance from the researcher. Common cell culture problems, including contamination with microorganisms or cells from other cultures, can place the reliability and reproducibility of cell culture work at risk. Here we use survey data, contributed by research scientists based in Australia and New Zealand, to assess common cell culture risks and how these risks are managed in practice. Respondents show that sharing of cell lines between laboratories continues to be widespread. Arrangements for mycoplasma and authentication testing are increasingly in place, although scientists are often uncertain how to perform authentication testing. Additional risks are identified for preparation of frozen stocks, storage and shipping.

Coordinated epigenetic remodelling of transcriptional networks occurs during early breast carcinogenesis.

BACKGROUND: Dysregulation of the epigenome is a common event in malignancy; however, deciphering the earliest cancer-associated epigenetic events remains a challenge. Cancer epigenome studies to date have primarily utilised cancer cell lines or clinical samples, where it is difficult to identify the initial epigenetic lesions from those that occur over time. Here, we analysed the epigenome of human mammary epithelial cells (HMEC) and a matched variant cell population (vHMEC) that have spontaneously escaped senescence and undergone partial carcinogenic transformation. Using this model of basal-like breast carcinogenesis, we provide striking new insights into the very first epigenetic changes that occur during the initial stages of malignancy. RESULTS: The first phase of malignancy is defined by coordinated changes in the epigenome. At the chromatin level, this is embodied in long-range epigenetic deregulation, which involves the concomitant but atypical acquisition or loss of active and repressive histone modifications across large regional blocks. Changes in DNA methylation also occurs in a highly coordinated manner. We identified differentially methylated regions (DMRs) in the very earliest passages of vHMECs. Notably, we find that differential methylation targets loci regulated by key transcription factors including p53, AHR and E2F family members suggesting that epigenetic deregulation of transcription factor binding is a key event in breast carcinogenesis. Interestingly, DMRs identified in vHMEC are extensively methylated in breast cancer, with hypermethylation frequently encroaching into neighbouring regions. A subset of vHMEC DMRs exhibited a strong basal-like cancer specific hypermethylation. CONCLUSIONS: Here, we generated epigenome-wide maps of the earliest phase of breast malignancy and show long-range epigenetic deregulation and coordinated DNA hypermethylation targets loci regulated by key transcription factors. These findings support a model where induction of breast cancer occurs through epigenetic disruption of transcription factor binding leading to deregulation of cancer-associated transcriptional networks. With their stability and very early occurrence, vHMECs hypermethylated loci could serve as excellent biomarkers for the initial detection of basal breast cancer.

ATRX represses alternative lengthening of telomeres.

The unlimited proliferation of cancer cells requires a mechanism to prevent telomere shortening. Alternative Lengthening of Telomeres (ALT) is an homologous recombination-mediated mechanism of telomere elongation used in tumors, including osteosarcomas, soft tissue sarcoma subtypes, and glial brain tumors. Mutations in the ATRX/DAXX chromatin remodeling complex have been reported in tumors and cell lines that use the ALT mechanism, suggesting that ATRX may be an ALT repressor. We show here that knockout or knockdown of ATRX in mortal cells or immortal telomerase-positive cells is insufficient to activate ALT. Notably, however, in SV40-transformed mortal fibroblasts ATRX loss results in either a significant increase in the proportion of cell lines activating ALT (instead of telomerase) or in a significant decrease in the time prior to ALT activation. These data indicate that loss of ATRX function cooperates with one or more as-yet unidentified genetic or epigenetic alterations to activate ALT. Moreover, transient ATRX expression in ALT-positive/ATRX-negative cells represses ALT activity. These data provide the first direct, functional evidence that ATRX represses ALT.

STC1 expression is associated with tumor growth and metastasis in breast cancer.

Stanniocalcin-1 (STC1) is a secreted glycoprotein implicated in several pathologies including retinal degeneration, cerebral ischemia, angiogenesis and inflammation. Aberrant STC1 expression has been reported in breast cancer but the significance of this is not clear. High levels of STC1 expression were found in the aggressive 4T1 murine mammary tumor cells and in the MDA-MB-231 human breast cancer line. To investigate its significance, stable clones with STC1 down-regulation using shRNA were generated in both tumor models. The consequences of STC1 down-regulation on cell proliferation, chemotactic invasion, tumor growth and metastasis were assessed. Down-regulation of STC1 in the 4T1 murine mammary tumor cells had a major impact on mammary tumor growth. This observation was replicated in a second tumor model with the MDA-MB-231 human breast cancer line, with a significant reduction in primary tumor formation and a major inhibition of metastasis as well. Interestingly, in both models, proliferation in vitro was not affected. Subsequent microarray gene expression profiling identified 30 genes to be significantly altered by STC1 down-regulation, the majority of which are associated with known hallmarks of carcinogenesis. Furthermore, bioinformatic analysis of breast cancer datasets revealed that high expression of STC1 is associated with poor survival. This is the first study to show definitively that STC1 plays an oncogenic role in breast cancer, and indicates that STC1 could be a potential therapeutic target for treatment of breast cancer patients.

Acquired convergence of hormone signaling in breast cancer: ER and PR transition from functionally distinct in normal breast to predictors of metastatic disease.

Cumulative exposure to estrogen (E) and progesterone (P) over the menstrual cycle significantly influences the risk of developing breast cancer. Despite the dogma that PR in the breast merely serves as a marker of an active estrogen receptor (ER), and as an inhibitor of the proliferative actions of E, it is now clear that in the breast P increases proliferation independently of E action. We show here that the progesterone receptor (PR) and ER are expressed in different epithelial populations, and target non-overlapping pathways in the normal human breast. In breast cancer, PR becomes highly correlated with ER, and this convergence is associated with signaling pathways predictive of disease metastasis. These data challenge the established paradigm that ER and PR function co-operatively in normal breast, and have significant implications not only for our understanding of normal breast biology, but also for diagnosis, prognosis and/or treatment options in breast cancer patients.

Molecular characterization of collaborator of ARF (CARF) as a DNA damage response and cell cycle checkpoint regulatory protein.

CARF is an ARF-binding protein that has been shown to regulate the p53-p21-HDM2 pathway. CARF overexpression was shown to cause growth arrest of human cancer cells and premature senescence of normal cells through activation of the p53 pathway. Because replicative senescence involves permanent withdrawal from the cell cycle in response to DNA damage response-mediated signaling, in the present study we investigated the relationship between CARF and the cell cycle and whether it is involved in the DNA damage response. We demonstrate that the half-life of CARF protein is less than 60 min, and that in cycling cells CARF levels are highest in G2 and early prophase. Serially passaged normal human skin and stromal fibroblasts showed upregulation of CARF during replicative senescence. Induction of G1 growth arrest and senescence by a variety of drugs was associated with increase in CARF expression at the transcriptional and translational level and was seen to correlate with increase in DNA damage response and checkpoint proteins, ATM, ATR, CHK1, CHK2, γH2AX, p53 and p21. Induction of growth arrest by oncogenic RAS and shRNA-mediated knockdown of TRF2 in cancer cells also caused upregulation of CARF. We conclude that CARF is associated with DNA damage response and checkpoint signaling pathways.

Enhanced isolation of fibroblasts from human skin explants.

Here we describe a method for growing fibroblasts from human skin explants that increases the number of cells obtained by up to two orders of magnitude, thus increasing the amount of material available for research and diagnostic purposes and potentially for cell-based therapies. Explants can be transferred sequentially up to 80 times, if required, at which point the explants appear to be completely depleted of fibroblasts. Utilizing skin samples obtained from 16 donors, aged 18-66 years old, the first 20 transfers produced cultures with lifespan and growth characteristics that were all very similar to each other, but the cultures derived from later transfers had a decreasing replicative capacity. Final cumulative population doublings did not correlate with donor age, but correlated positively with the telomere length at early passage. We also demonstrated that explants can be transduced directly by lentiviral infection, and that cryopreserved tissue can be explanted successfully using this procedure.

Five dysfunctional telomeres predict onset of senescence in human cells.

Replicative senescence is accompanied by a telomere-specific DNA damage response (DDR). We found that DDR+ telomeres occur spontaneously in early-passage normal human cells and increase in number with increasing cumulative cell divisions. DDR+ telomeres at replicative senescence retain TRF2 and RAP1 proteins, are not associated with end-to-end fusions and mostly result from strand-independent, postreplicative dysfunction. On the basis of the calculated number of DDR+ telomeres in G1-phase cells just before senescence and after bypassing senescence by inactivation of wild-type p53 function, we conclude that the accrual of five telomeres in G1 that are DDR+ but nonfusogenic is associated with p53-dependent senescence.

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.

DNA C-circles are specific and quantifiable markers of alternative-lengthening-of-telomeres activity.

Alternative lengthening of telomeres (ALT) is likely to be an important target for anticancer treatment as approximately 10% of cancers depend on this telomere maintenance mechanism for continued growth, and inhibition of ALT can cause cellular senescence. However, no ALT inhibitors have been developed for therapeutic use because of the lack of a suitable ALT activity assay and of known ALT-specific target molecules. Here we show that partially single-stranded telomeric (CCCTAA)(n) DNA circles (C-circles) are ALT specific. We provide an assay that is rapidly and linearly responsive to ALT activity and that is suitable for screening for ALT inhibitors. We detect C-circles in blood from ALT(+) osteosarcoma patients, suggesting that the C-circle assay (CC assay) may have clinical utility for diagnosis and management of ALT(+) tumors.

Hormone-responsive model of primary human breast epithelium.

Retention of hormone responsiveness in primary culture models of human breast is essential for studies aimed at understanding the mechanisms of action of the ovarian hormones in the human breast. In this chapter we describe the development of a culture model of primary human breast that retains critical features of the tissue in vivo. We find that primary normal human breast tissue in embedded culture recapitulates the morphology, cell lineages, functional gene expression characteristics and estrogen and progesterone receptor responsiveness of the breast in vivo. The ratio of luminal to myoepithelial cells after culture recapitulates that observed in the uncultured tissue, highlighting the fact that progenitor cells capable of giving rise to both epithelial cell lineages are retained in this model system. By contrast, primary cells placed into monolayer culture, even for a single passage, lose bipotent progenitors, and the myoepithelial lineage predominates, demonstrating the rapidity with which phenotypic changes and selection occur in normal breast cells, unless cultured under conditions that prevent this outcome. Primary matrix-embedded culture of normal human breast cells provides researchers with a new opportunity to understand ovarian hormone action in the human breast.

Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers.

Basal-like breast cancers arising in women carrying mutations in the BRCA1 gene, encoding the tumor suppressor protein BRCA1, are thought to develop from the mammary stem cell. To explore early cellular changes that occur in BRCA1 mutation carriers, we have prospectively isolated distinct epithelial subpopulations from normal mammary tissue and preneoplastic specimens from individuals heterozygous for a BRCA1 mutation. We describe three epithelial subsets including basal stem/progenitor, luminal progenitor and mature luminal cells. Unexpectedly, we found that breast tissue from BRCA1 mutation carriers harbors an expanded luminal progenitor population that shows factor-independent growth in vitro. Moreover, gene expression profiling revealed that breast tissue heterozygous for a BRCA1 mutation and basal breast tumors were more similar to normal luminal progenitor cells than any other subset, including the stem cell-enriched population. The c-KIT tyrosine kinase receptor (encoded by KIT) emerged as a key marker of luminal progenitor cells and was more highly expressed in BRCA1-associated preneoplastic tissue and tumors. Our findings suggest that an aberrant luminal progenitor population is a target for transformation in BRCA1-associated basal tumors .

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.

Aberrant de novo methylation of the p16INK4A CpG island is initiated post gene silencing in association with chromatin remodelling and mimics nucleosome positioning.

Changes in the epigenetic landscape are widespread in neoplasia, with de novo methylation and histone repressive marks commonly enriched in CpG island associated promoter regions. DNA hypermethylation and histone repression correlate with gene silencing, however, the dynamics of this process are still largely unclear. The tumour suppressor gene p16(INK4A) is inactivated in association with CpG island methylation during neoplastic progression in a variety of cancers, including breast cancer. Here, we investigated the temporal progression of DNA methylation and histone remodelling in the p16(INK4A) CpG island in primary human mammary epithelial cell (HMEC) strains during selection, as a model for early breast cancer. Silencing of p16(INK4A) has been previously shown to be necessary before HMECs can escape from selection. Here, we demonstrate that gene silencing occurs prior to de novo methylation and histone remodelling. An increase in DNA methylation was associated with a rapid loss of both histone H3K27 trimethylation and H3K9 acetylation and a gradual gain of H3K9 dimethylation. Interestingly, we found that regional-specific 'seeding' methylation occurs early after post-selection and that the de novo methylation pattern observed in HMECs correlates with the apparent footprint of nucleosomes across the p16(INK4A) CpG island. Our results demonstrate for the first time that p16(INK4A) gene silencing is a precursor to epigenetic suppression and that subsequent de novo methylation initially occurs in nucleosome-free regions across the p16(INK4A) CpG island and this is associated with a dynamic change in histone modifications.

DNA replication licensing and progenitor numbers are increased by progesterone in normal human breast.

Proliferation in the nonpregnant human breast is highest in the luteal phase of the menstrual cycle when serum progesterone levels are high, and exposure to progesterone analogues in hormone replacement therapy is known to elevate breast cancer risk, yet the proliferative effects of progesterone in the human breast are poorly understood. In a model of normal human breast, we have shown that progesterone increased incorporation of 5-bromo-2'-deoxyuridine and increased cell numbers by activation of pathways involved in DNA replication licensing, including E2F transcription factors, chromatin licensing and DNA replication factor 1 (Cdt1), and the minichromosome maintenance proteins and by increased expression of proteins involved in kinetochore formation including Ras-related nuclear protein (Ran) and regulation of chromosome condensation 1 (RCC1). Progenitor cells competent to give rise to both myoepithelial and luminal epithelial cells were increased by progesterone, showing that progesterone influences epithelial cell lineage differentiation. Therefore, we have demonstrated that progesterone augments proliferation of normal human breast cells by both activating DNA replication licensing and kinetochore formation and increasing bipotent progenitor numbers.

Amplification of telomerase reverse transcriptase gene in human mammary epithelial cells with limiting telomerase RNA expression levels.

Activation of telomerase is a crucial step during cellular immortalization, and in some tumors this results from amplification of the human telomerase reverse transcriptase (hTERT) gene. Immortalization of normal human cells has been achieved by transduction with hTERT cDNA under the control of a strong heterologous enhancer/promoter, but this is sometimes an inefficient process, with periods of poor growth or even crisis occurring before immortalization. Here, we showed that normal human mammary epithelial cells expressing exogenous hTERT amplified the transgene extensively and expressed high levels of hTERT mRNA and protein. Paradoxically, the cells had low levels of telomerase activity and very short telomeres, indicating that telomerase activity did not correlate with hTERT expression. These cells contained only approximately 20 human telomerase RNA (hTR) molecules/cell (compared with approximately 120 hTR molecules per 293 cell). Expression of exogenous hTR caused increased telomerase activity and telomere lengthening. These data indicate that some hTERT-transduced normal cells may express high levels of the transgene but fail to up-regulate endogenous hTR expression sufficiently to enable expression of robust levels of telomerase activity.

Concordant epigenetic silencing of transforming growth factor-beta signaling pathway genes occurs early in breast carcinogenesis.

Human mammary epithelial cells (HMEC) grown under standard cell culture conditions enter a growth phase referred to as selection, but a subpopulation is able to escape from arrest and continue to proliferate. These cells, called post-selection or variant HMECs, may be derived from progenitor cells found in normal mammary epithelium that subsequently acquire premalignant lesions, including p16(INK4A) promoter hypermethylation. Epigenetic silencing of tumor suppressor genes through DNA methylation and histone modification is an early event in tumorigenesis. A major challenge is to find genes or gene pathways that are commonly silenced to provide early epigenetic diagnostic and therapeutic cancer targets. To identify very early epigenetic events that occur in breast cancer, we used microarrays to screen for gene pathways that were suppressed in post-selection HMECs but reactivated after treatment with the demethylation agent 5-aza-2'-deoxycytidine. We found that several members of the transforming growth factor beta (TGF-beta) signaling pathway were consistently down-regulated in the post-selection HMEC populations, and this was associated with a marked decrease in Smad4 nuclear staining. Gene suppression was not associated with DNA methylation but with chromatin remodeling, involving a decrease in histone H3 lysine 27 trimethylation and an increase in histone H3 lysine 9 dimethylation and deacetylation. These results show for the first time that TGF-beta2, its receptors TGF-beta R1 and TGF-beta R2, and activator thrombospondin-1 are concordantly suppressed early in breast carcinogenesis by histone modifications and indicate that the TGF-beta signaling pathway is a novel target for gene activation by epigenetic therapy.