Role played by BRCA1 in transcriptional regulation in response to therapy

BRCA1 (breast-cancer susceptibility gene 1) is a tumour suppressor, implicated in the hereditary predisposition to breast and ovarian cancer. BRCA1 has been implicated in a number of cellular processes including DNA repair and recombination, cell cycle checkpoint control, chromatin remodelling and ubiquitination. In addition, substantial data now exist to suggest a role for BRCA1 in transcriptional regulation; BRCA1 has been shown to interact with the Pol II holoenzyme complex and to interact with multiple transcription factors, such as p53 and c-Myc. We have previously identified a range of BRCA1 transcriptional targets and have linked these to specific cellular pathways, including cell cycle checkpoint activation and apoptosis. Current research is focused on the transcriptional mechanisms that underpin the association of BRCA1 deficiency with increased sensitivity to DNA damage-based chemotherapy and resistance to spindle poisons.

Uncovering functionally relevant signaling pathways using microarray-based expression profiling

The introduction of microarray technology to the scientific and medical communities has fundamentally altered the way in which we now address basic biomedical questions. Microarrays technology facilitates a more complete and inclusive experimental approach where alterations in the transcript level of entire genomes can be simultaneously assayed in response to a variety of stimuli. Conceptually different approaches to the development of microarray technology have resulted in the generation of two different array formats: oligonucleotide arrays and cDNA arrays. The application of microarray and related technologies to identify specific targets of defined genes that have clearly been implicated in cancer progression requires a specific experimental approach. The objective of tiffs approach is to define changes in transcriptional profile that occur in response to modulating the expression level of the gene to be studied. The resulting altered expression profile can then be viewed as a blueprint by which that gene effects its cellular function. We have used oligonucleotide array-based expression profiling in collaboration with Affymetrix to identify downstream transcriptional targets of the BRCA1 tumor-suppressor gene as a means of defining its function. BRCA1 has been implicated in at least three functional pathways, namely, mediating the cellular response to DNA damage, as a cell cycle checkpoint protein and in the regulation of transcription. The physiological significance of these properties and their implications for the function of BRCA1 as a tumor-suppressor gene remain to be defined.

Compromised Spindle Assembly Checkpoint due to Altered Expression of Ubch10 and Cdc20 in Human Papillomavirus Type 16 E6 and E7-Expressing Keratinocytes

Cells expressing human papillomavirus type 16 (HPV-16) E6 and E7 proteins exhibit deregulation of G(2)/M genes, allowing bypass of DNA damage arrest signals. Normally, cells with DNA damage that override the G(2) damage checkpoint would precociously enter mitosis and ultimately face mitotic catastrophe and apoptotic cell death. However, E6/E7-expressing cells (E6/E7 cells) have the ability to enter and exit mitosis in the presence of DNA damage and continue with the next round of the cell cycle. Little is known about the mechanism that allows these cells to gain entry into and exit from mitosis. Here, we show that in the presence of DNA damage, E6/E7 cells have elevated levels of cyclin B, which would allow entry into mitosis. Also, as required for exit from mitosis, cyclin B is degraded in these cells, permitting initiation of the next round of DNA synthesis and cell cycle progression. Proteasomal degradation of cyclin B by anaphase-promoting complex/cyclosome (APC/C) is, in part, due to elevated levels of the E2-conjugating enzyme, Ubch10, and the substrate recognition protein, Cdc20, of APC/C. Also, in E6/E7 cells with DNA damage, while Cdc20 is complexed with BubR1, indicating an active checkpoint, it is also present in complexes free of BubR1, presumably allowing APC/C activity and slippage through the checkpoint.

Cdk1 participates in BRCA1-dependent S phase checkpoint control in response to DNA damage.

Cdk2 and cdk1 are individually dispensable for cell-cycle progression in cancer cell lines because they are able to compensate for one another. However, shRNA-mediated depletion of cdk1 alone or small molecule cdk1 inhibition abrogated S phase cell-cycle arrest and the phosphorylation of a subset of ATR/ATM targets after DNA damage. Loss of DNA damage-induced checkpoint control was caused by a reduction in formation of BRCA1-containing foci. Mutation of BRCA1 at S1497 and S1189/S1191 resulted in loss of cdk1-mediated phosphorylation and also compromised formation of BRCA1-containing foci. Abrogation of checkpoint control after cdk1 depletion or inhibition in non-small-cell lung cancer cells sensitized them to DNA-damaging agents. Conversely, reduced cdk1 activity caused more potent G2/M arrest in nontransformed cells and antagonized the response to subsequent DNA damage. Cdk1 inhibition may therefore selectively sensitize BRCA1-proficient cancer cells to DNA-damaging treatments by disrupting BRCA1 function.

AURKA overexpression accompanies dysregulation of DNA-damage response genes in invasive urothelial cell carcinoma

Invasive urothelial cell carcinoma (UCC) is characterized by increased chromosomal instability and follows an aggressive clinical course in contrast to non-invasive disease. To identify molecular processes that confer and maintain an aggressive malignant phenotype, we used a high-throughput genome-wide approach to interrogate a cohort of high and low clinical risk UCC tumors. Differential expression analyses highlighted cohesive dysregulation of critical genes involved in the G(2)/M checkpoint in aggressive UCC. Hierarchical clustering based on DNA Damage Response (DDR) genes separated tumors according to a pre-defined clinical risk phenotype. Using array-comparative genomic hybridization, we confirmed that the DDR was disrupted in tumors displaying high genomic instability. We identified DNA copy number gains at 20q13.2-q13.3 (AURKA locus) and determined that overexpression of AURKA accompanied dysregulation of DDR genes in high risk tumors. We postulated that DDR-deficient UCC tumors are advantaged by a selective pressure for AURKA associated override of M phase barriers and confirmed this in an independent tissue microarray series. This mechanism that enables cancer cells to maintain an aggressive phenotype forms a rationale for targeting AURKA as a therapeutic strategy in advanced stage UCC.

Characterization of a thymidylate synthase (TS)-inducible cell line: a model system for studying sensitivity to TS- and non-TS-targeted chemotherapies

Thymidylate synthase (TS) is responsible for the de novo synthesis of thymidylate, which is required for DNA synthesis and repair and which is an important target for fluoropyrimidines such as 5-fluorouracil (5-FU), and antifolates such as Tomudex (TDX), ZD9331, and multitargeted antifolate (MTA). To study the importance of TS expression in determining resistance to these agents, we have developed an MDA435 breast cancer-derived cell line with tetracycline-regulated expression of TS termed MTS-5. We have demonstrated that inducible expression of TS increased the IC(50) dose of the TS-targeted therapeutic agents 5-FU, TDX, and ZD9331 by 2-, 9- and 24-fold respectively. An IC(50) dose for MTA was unobtainable when TS was overexpressed in these cells, which indicated that MTA toxicity is highly sensitive to increased TS expression levels. The growth inhibitory effects of the chemotherapeutic agents CPT-11, cisplatin, oxaliplatin, and Taxol were unaffected by TS up-regulation. Cell cycle analyses revealed that IC(50) doses of 5-FU, TDX and MTA caused an S-phase arrest in cells that did not overexpress TS, and this arrest was overcome when TS was up-regulated. Furthermore, the S-phase arrest was accompanied by 2- to 4-fold increased expression of the cell cycle regulatory genes cyclin E, cyclin A, and cyclin dependent kinase 2 (cdk2). These results indicate that acute increases in TS expression levels play a key role in determining cellular sensitivity to TS-directed chemotherapeutic drugs by modulating the degree of S-phase arrest caused by these agents. Moreover, CPT-11, cisplatin, oxaliplatin, and Taxol remain highly cytotoxic in cells that overexpress TS.

Effects of the aurora kinase inhibitors AZD1152-HQPA and ZM447439 on growth arrest and polyploidy in acute myeloid leukemia cell lines and primary blasts.

BACKGROUND:
Aurora kinases play an essential role in the orchestration of chromosome separation and cytokinesis during mitosis. Small-molecule inhibition of the aurora kinases has been shown to result in inhibition of cell division, phosphorylation of histone H3 and the induction of apoptosis in a number of cell systems. These characteristics have led aurora kinase inhibitors to be considered as potential therapeutic agents.
DESIGN AND METHODS:
Aurora kinase gene expression profiles were assessed in 101 samples from patients with acute myeloid leukemia. Subsequently, aurora kinase inhibitors were investigated for their in vitro effects on cell viability, histone H3 phosphorylation, cell cycle and morphology in acute myeloid leukemia cell lines and primary acute myeloid leukemia samples.
RESULTS:
The aurora kinase inhibitors AZD1152-HQPA and ZM447439 induced growth arrest and the accumulation of hyperploid cells in acute myeloid leukemia cell lines and primary acute myeloid leukemia cultures. Furthermore, both agents inhibited histone H3 phosphorylation and this preceded perturbations in cell cycle and the induction of apoptosis. Single cell cloning assays were performed on diploid and polyploid cells to investigate their colony-forming capacities. Although the polyploid cells showed a reduced capacity for colony formation when compared with their diploid counterparts, they were consistently able to form colonies.
CONCLUSIONS:
AZD1152-HQPA- and ZM447439 are effective apoptosis-inducing agents in acute myeloid leukemia cell lines and primary acute myeloid leukemia cultures. However, their propensity to induce polyploidy does not inevitably result in apoptosis.

Hoxa6 potentiates short-term hemopoietic cell proliferation and extended self-renewal.

Hemopoietic progenitor cells express clustered homeobox (Hox) genes in a pattern characteristic of their lineage and stage of differentiation. In general, HOX expression tends to be higher in more primitive and lower in lineage-committed cells. These trends have led to the hypothesis that self-renewal of hemopoietic stem/progenitor cells is HOX-dependent and that dysregulated HOX expression underlies maintenance of the leukemia-initiating cell. Gene expression profile studies support this hypothesis and specifically highlight the importance of the HOXA cluster in hemopoiesis and leukemogenesis. Within this cluster HOXA6 and HOXA9 are highly expressed in patients with acute myeloid leukemia and form part of the "Hox code" identified in murine models of this disease. We have examined endogenous expression of Hoxa6 and Hoxa9 in purified primary progenitors as well as four growth factor-dependent cell lines FDCP-Mix, EML, 32Dcl3, and Ba/F3, representative of early multipotential and later committed precursor cells respectively. Hoxa6 was consistently higher expressed than Hoxa9, preferentially expressed in primitive cells and was both growth-factor and cell-cycle regulated. Enforced overexpression of HOXA6 or HOXA9 in FDCP-Mix resulted in increased proliferation and colony formation but had negligible effect on differentiation. In both FDCP-Mix and the more committed Ba/F3 precursor cells overexpression of HOXA6 potentiated factor-independent proliferation. These findings demonstrate that Hoxa6 is directly involved in fundamental processes of hemopoietic progenitor cell development.

Ribosomal 18S RNA processing by the IGF-I-responsive WDR3 protein is integrated with p53 function in cancer cell proliferation.

Insulin-like growth factor-I (IGF-I) signaling is strongly associated with cell growth and regulates the rate of synthesis of the rRNA precursor, the first and the key stage of ribosome biogenesis. In a screen for mediators of IGF-I signaling in cancer, we recently identified several ribosome-related proteins, including NEP1 (nucleolar essential protein 1) and WDR3 (WD repeat 3), whose homologues in yeast function in ribosome processing. The WDR3 gene and its locus on chromosome 1p12-13 have previously been linked with malignancy. Here we show that IGF-I induces expression of WDR3 in transformed cells. WDR3 depletion causes defects in ribosome biogenesis by affecting 18 S rRNA processing and also causes a transient down-regulation of precursor rRNA levels with moderate repression of RNA polymerase I activity. Suppression of WDR3 in cells expressing functional p53 reduced proliferation and arrested cells in the G1 phase of the cell cycle. This was associated with activation of p53 and sequestration of MDM2 by ribosomal protein L11. Cells lacking functional p53 did not undergo cell cycle arrest upon suppression of WDR3. Overall, the data indicate that WDR3 has an essential function in 40 S ribosomal subunit synthesis and in ribosomal stress signaling to p53-mediated regulation of cell cycle progression in cancer cells.

Activated oncogenic pathways and therapeutic targets in extranodal nasal-type NK/T cell lymphoma revealed by gene expression profiling

We performed comprehensive genome-wide gene expression profiling (GEP) of extranodal nasal-type natural killer/T-cell lymphoma (NKTL) using formalin-fixed, paraffin-embedded tissue (n = 9) and NK cell lines (n = 5) in comparison with normal NK cells, with the objective of understanding the oncogenic pathways involved in the pathogenesis of NKTL and to identify potential therapeutic targets. Pathway and network analysis of genes differentially expressed between NKTL and normal NK cells revealed significant enrichment for cell cycle-related genes and pathways, such as PLK1, CDK1, and Aurora-A. Furthermore, our results demonstrated a pro-proliferative and anti-apoptotic phenotype in NKTL characterized by activation of Myc and nuclear factor kappa B (NF-kappa B), and deregulation of p53. In corroboration with GEP findings, a significant percentage of NKTLs (n = 33) overexpressed c-Myc (45.4%), p53 (87.9%), and NF-kappa B p50 (67.7%) on immunohistochemistry using a tissue microarray containing 33 NKTL samples. Notably, overexpression of survivin was observed in 97% of cases. Based on our findings, we propose a model of NKTL pathogenesis where deregulation of p53 together with activation of Myc and NF-kappa B, possibly driven by EBV LMP-1, results in the cumulative up-regulation of survivin. Down-regulation of survivin with Terameprocol (EM-1421, a survivin inhibitor) results in reduced cell viability and increased apoptosis in tumour cells, suggesting that targeting survivin may be a potential novel therapeutic strategy in NKTL. Copyright (C) 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Runx2, p53, and pRB Status as Diagnostic Parameters for Deregulation of Osteoblast Growth and Differentiation in a New Pre-Chemotherapeutic Osteosarcoma Cell Line (OS1)

Osteosarcomas are the most prevalent primary bone tumors found in pediatric patients. To understand their molecular etiology, cell culture models are used to define disease mechanisms under controlled conditions. Many osteosarcoma cell lines (e.g., SAOS-2, U2OS, MG63) are derived from Caucasian patients. However, patients exhibit individual and ethnic differences in their responsiveness to irradiation and chemotherapy. This motivated the establishment of osteosarcoma cell lines (OS1, OS2, OS3) from three ethnically Chinese patients. OS1 cells, derived from a pre-chemotherapeutic tumor in the femur of a 6-year-old female, were examined for molecular markers characteristic for osteoblasts, stem cells, and cell cycle control by immunohistochemistry, reverse transcriptase-PCR, Western blotting and flow cytometry. OS I have aberrant G-banded karyotypes, possibly reflecting chromosomal abnormalities related to p53 deficiency. OS I had ossification profiles similar to human fetal osteoblasts rather than SAOS-2 which ossifies ab initio, (P

Cell cycles and endocycles in the model brown seaweed, Ectocarpus siliculosus

The recent announcement of the first genome sequence of a brown macroalga, the filamentous Ectocarpus, has been accompanied by a number of companion papers in New Phytologist. In a paper which contributes to this special issue, we classified the core cell cycle components of Ectocarpus, comparing them to the previously studied cell cycle components of diatoms. We then carried out fluorescence microscopy experiments to show that the Ectocarpus cell cycle could be deregulated during early development to give endopolyploid adults. We discuss here how our findings complement recent studies on endopolyploidy in plant and algal systems.

Acetylation of Rb by PCAF is required for nuclear localization and keratinocyte differentiation

Although the retinoblastoma protein (Rb) functions as a checkpoint in the cell cycle, it also regulates differentiation. It has recently been shown that Rb is acetylated during differentiation; however, the role of this modification has not been identified. Depletion of Rb levels with short hairpin RNA resulted in inhibition of human keratinocyte differentiation, delayed cell cycle exit and allowed cell cycle re-entry. Restoration of Rb levels rescued defects in differentiation and cell cycle exit and re-entry; however, re-expression of Rb with the major acetylation sites mutated did not. During keratinocyte differentiation, acetylation of Rb is mediated by PCAF and it is further shown that PCAF acetyltransferase activity is also required for normal differentiation. The major acetylation sites in Rb are located within the nuclear localization sequence and, although mutation did not alter Rb localization in cycling cells, the mutant is mislocalized to the cytoplasm during differentiation. Studies indicate that acetylation is a mechanism for controlling Rb localization in human keratinocytes, with either reduction of the PCAF or exogenous expression of the deacetylase SIRT1, resulting in mislocalization of Rb. These findings identify PCAF-mediated acetylation of Rb as an event required to retain Rb within the nucleus during keratinocyte differentiation.

Potentiation of Inflammatory CXCL8 Signalling Sustains Cell Survival in PTEN-deficient Prostate Carcinoma

Background: Inflammation and genetic instability are enabling characteristics of prostate carcinoma (PCa). Inactivation of the tumour suppressor gene phosphatase and tensin homolog (PTEN) is prevalent in early PCa. The relationship of PTEN deficiency to inflammatory signalling remains to be characterised.

Objective: To determine how loss of PTEN functionality modulates expression and efficacy of clinically relevant, proinflammatory chemokines in PCa.

Design, setting and participants: Experiments were performed in established cell-based PCa models, supported by pathologic analysis of chemokine expression in prostate tissue harvested from PTEN heterozygous (Pten(+/-)) mice harbouring inactivation of one PTEN allele.

Interventions: Small interfering RNA (siRNA)- or small hairpin RNA (shRNA)-directed strategies were used to repress PTEN expression and resultant interleukin-8 (CXCL8) signalling, determined under normal and hypoxic culture conditions.

Outcome measurements and statistical analysis: Changes in chemokine expression in PCa cells and tissue were analysed by real-time polymerase chain reaction (PCR), immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry; effects of chemokine signalling on cell function were assessed by cell cycle analysis, apoptosis, and survival assays.

Results and limitations: Transient (siRNA) or prolonged (shRNA) PTEN repression increased expression of CXCL8 and its receptors, chemokine (C-X-C motif) receptor (CXCR) 1 and CXCR2, in PCa cells. Hypoxia-induced increases in CXCL8, CXCR1, and CXCR2 expression were greater in magnitude and duration in PTEN-depleted cells. Autocrine CXCL8 signalling was more efficacious in PTEN-depleted cells, inducing hypoxia-inducible factor-1 (HIF-1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-?B) transcription and regulating genes involved in survival and angiogenesis. Increased expression of the orthologous chemokine KC was observed in regions displaying atypical cytologic features in Pten(+/-) murine prostate tissue relative to normal epithelium in wild-type PTEN (Pten(WT)) glands. Attenuation of CXCL8 signalling decreased viability of PCa cells harbouring partial or complete PTEN loss through promotion of G1 cell cycle arrest and apoptosis. The current absence of clinical validation is a limitation of the study.

Conclusions: PTEN loss induces a selective upregulation of CXCL8 signalling that sustains the growth and survival of PTEN-deficient prostate epithelium.

Cellular senescence induced by aberrant MAD2 levels impacts on paclitaxel responsiveness in vitro

BACKGROUND: The mitotic arrest deficiency protein 2 (MAD2) is a key component of the mitotic spindle assembly checkpoint, monitoring accurate chromosomal alignment at the metaphase plate before mitosis. MAD2 also has a function in cellular senescence and in a cell’s response to microtubule inhibitory (MI) chemotherapy exemplified by paclitaxel.
METHODS: Using an siRNA approach, the impact of MAD2 down-regulation on cellular senescence and paclitaxel responsiveness was investigated. The endpoints of senescence, cell viability, migration, cytokine expression, cell cycle analysis and anaphase bridge scoring were carried out using standard approaches.
RESULTS: We show that MAD2 down-regulation induces premature senescence in the MCF7 breast epithelial cancer cell line. These MAD2-depleted (MAD2k) cells are also significantly replicative incompetent but retain viability. Moreover, they show significantly higher levels of anaphase bridges and polyploidy compared to controls. In addition, these cells secrete higher levels of IL-6 and IL-8
representing key components of the senescence-associated secretory phenotype (SASP) with the ability to impact on neighbouring cells. In support of this, MAD2kcells show enhanced migratory ability. At 72 h after paclitaxel, MAD2kcells show a significant further induction of senescence compared with paclitaxel naive controls. In addition, there are significantly more viable cells in the MAD2k MCF7 cell line after paclitaxel reflecting the observed increase in senescence.
CONCLUSION: Considering that paclitaxel targets actively dividing cells, these senescent cells will evade cytotoxic kill. In conclusion, compromised MAD2 levels induce a population of senescent cells resistant to paclitaxel.