Mitotic Arrest Deficiency Protein 2 (MAD2) and Histone Deacetylase 6 (HDAC6) Present a Complex Relationship in Their Regulation and Expression and Subsequent Impact on Chemoresponsiveness

Introduction:
Ovarian cancer patients presenting with advanced stage (III/IV)
canceraretreatedwithcarboplatinumincombinationwithpaclitaxel.Despitea
significant initial response rate, fewer than 20% of patients become long-term
survivors. We have published that low MAD2 expression levels associate with
reduced progression free survival (PFS) in patients with high-grade serous
epithelial ovarian cancer (EOC). Moreover, we have demonstrated that MAD2
expressionisdown-regulatedbythemicroRNAmiR-433(
Furlong et al., 2011
).
Interestingly, miR-433 also down-regulates HDAC6 (
Simon et al., 2010
), which
uniquely deacetylates
a
-tubulin prior to HDAC6s binding to
b
-tubulin.
In vitro
studies have shown that HDAC6 inhibition in combination with paclitaxel
treatment enhances chemoresistant cancer cell death. To date, an interaction
between MAD2 and HDAC6 has not been reported.
Experimental design:
MAD2 and HDAC6 immunohistochemistry (IHC) and
Western blot analyses were performed to investigate the role of HDAC6 and
MAD2 in chemoresistance to paclitaxel in high-grade serous EOC.
Results and Discussion:
In vitro
experiments demonstrated that overex-
pression of pre-miR-433, which targets MAD2, resulted in down-regulation
of HDAC6 in EOC cell lines. High levels of HDAC6 are co-expressed with
MAD2 in the paclitaxel resistant UPN251 and OVCAR7 cell lines. While, all
4 paclitaxel resistant EOC cell lines express higher levels of miR-433 than
the paclitaxel sensitive A2780 cells, only ovca432 and ovca433 demonstrated
down-regulation of both HDAC6 and MAD2. Paclitaxel binds to
b
-tubulin and
causesmicrotubulepolymerizationinpaclitaxelsensitivecellsasdemonstrated
by tubulin acetylation in A2780 cells. However, paclitaxel failed to cause a
significant acetylation of
a
-tubulin and microtubule stabilisation in the resistant
UPN251 cells. Therefore resistance in this cell line may be mediated by
aberrantly high HDAC6 activity. We have previously shown that MAD2 knock-
down cells are resistant to paclitaxel (
Furlong F., et al., 2011; Prencipe M.,
et al., 2009
). We measured HDAC6 protein expression in MAD2 knockdown
cells and showed that MAD2 knockdown is associated with concomitant
up-regulation of HDAC6. We hypothesise that the up-regulation of HDAC6
by MAD2 knockdown renders cancer cells more resistant to paclitaxel and
increases the invasive potential of these cells. On-going experiments will test
this hypothesis. Lastly we have observed differential MAD2 and HDAC6 IHC
staining intensity in formalin fixed paraffin embedded EOC samples.
In conclusion
, we have reported on a novel interaction between MAD2 and
HDAC6 which may have important consequences for paclitaxel resistant EOC.
Moreover, understanding chemo-responsiveness in ovarian tumours will lead
to improved patient management and treatment options for women diagnosed
with this disease

Recombinant Subgroup B Human Respiratory Syncytial Virus Expressing Enhanced Green Fluorescent Protein Efficiently Replicates in Primary Human Cells and Is Virulent in Cotton Rats

Human respiratory syncytial virus (HRSV) is the most important viral cause of severe respiratory tract disease in infants. Two subgroups (A and B) have been identified, which cocirculate during, or alternate between, yearly epidemics and cause indistinguishable disease. Existing in vitro and in vivo models of HRSV focus almost exclusively on subgroup A viruses. Here, a recombinant (r) subgroup B virus (rHRSV(B05)) was generated based on a consensus genome sequence obtained directly from an unpassaged clinical specimen from a hospitalized infant. An additional transcription unit containing the gene encoding enhanced green fluorescent protein (EGFP) was introduced between the phosphoprotein and matrix genes (position 5) of the genome to generate rHRSV(B05)EGFP(5). The recombinant viruses replicated efficiently in both HEp-2 cells and in well-differentiated normal human bronchial cells grown at air-liquid interface. Intranasal infection of cotton rats (Sigmodon hispidus) resulted in high numbers of EGFP(+) cells in epithelia of the nasal septum and conchae. When administered in a relatively large inoculum volume, the virus also replicated efficiently in bronchiolar epithelial cells and spread extensively in both the upper and lower respiratory tracts. Virus replication was not observed in ciliated epithelial cells of the trachea. This is the first virulent rHRSV strain with the genetic composition of a currently circulating wild-type virus. In vivo tracking of infected cells by means of EGFP fluorescence in the absence of cytopathic changes increases the sensitivity of virus detection in HRSV pathogenesis studies.

IMPORTANCE

Virology as a discipline has depended on monitoring cytopathic effects following virus culture in vitro. However, wild-type viruses isolated from patients often do not cause significant changes to infected cells, necessitating blind passage. This can lead to genetic and phenotypic changes and the generation of high-titer, laboratory-adapted viruses with diminished virulence in animal models of disease. To address this, we determined the genome sequence of an unpassaged human respiratory syncytial virus from a sample obtained directly from an infected infant, assembled a molecular clone, and recovered a wild-type recombinant virus. Addition of a gene encoding enhanced green fluorescent protein allowed this wild-type virus to be tracked in primary human cells and living animals in the absence of significant cytopathic effects. Imaging of fluorescent cells proved to be a highly valuable tool for monitoring the spread of virus and may help improve assays for evaluating novel intervention strategies.

Prokineticin ligands and receptors are expressed in the human fetal ovary and regulate germ cell expression of COX2

CONTEXT: Fetal ovarian development and primordial follicle formation underpin future female fertility. Prokineticin (PROK) ligands regulate cell survival, proliferation and angiogenesis in adult reproductive tissues including the ovary. However, their expression and function during fetal ovarian development remains unclear.

OBJECTIVE: To investigate expression and localization of the PROK ligands, receptors and their downstream transcriptional targets in the human fetal ovary.

SETTING: This study was conducted at the University of Edinburgh.

PARTICIPANTS: Ovaries were collected from 37 morphologically normal human fetuses.

DESIGN AND MAIN OUTCOME MEASURES: mRNA and protein expression of PROK ligands and receptors was determined in human fetal ovaries using qRT-PCR, immunoblotting and immunohistochemistry. Functional studies were performed using a human germ tumour cell line (TCam-2) stably transfected with PROKR1.

RESULTS: Expression of PROK1 and PROKR1 was significantly higher in mid-gestation ovaries (17-20 weeks) than at earlier gestations (8-11 and 14-16 weeks). PROK2 significantly increased across the gestations examined. PROKR2 expression remained unchanged. PROK ligand and receptor proteins were predominantly localised to germ cells (including oocytes within primordial follicles) and endothelial cells, indicating these cell types to be the targets of PROK signalling in the human fetal ovary. PROK1 treatment of a germ cell line stably-expressing PROKR1 resulted in ERK phosphorylation, and elevated COX2 expression.

CONCLUSIONS: Developmental changes in expression and regulation of COX2 and pERK by PROK1 suggest that PROK ligands may be novel regulators of germ cell development in the human fetal ovary, interacting within a network of growth and survival factors prior to primordial follicle formation.

Mutant p53 expression in fallopian tube epithelium drives cell migration

Ovarian cancer is the fifth leading cause of cancer death among US women. Evidence supports the hypothesis that high-grade serous ovarian cancers (HGSC) may originate in the distal end of the fallopian tube. Although a heterogeneous disease, 96% of HGSC contain mutations in p53. In addition, the "p53 signature," or overexpression of p53 protein (usually associated with mutation), is a potential precursor lesion of fallopian tube derived HGSC suggesting an essential role for p53 mutation in early serous tumorigenesis. To further clarify p53-mutation dependent effects on cells, murine oviductal epithelial cells (MOE) were stably transfected with a construct encoding for the R273H DNA binding domain mutation in p53, the most common mutation in HGSC. Mutation in p53 was not sufficient to transform MOE cells but did significantly increase cell migration. A similar p53 mutation in murine ovarian surface epithelium (MOSE), another potential progenitor cell for serous cancer, was not sufficient to transform the cells nor change migration suggesting tissue specific effects of p53 mutation. Microarray data confirmed expression changes of pro-migratory genes in p53(R273H) MOE compared to parental cells, which could be reversed by suppressing Slug expression. Combining p53(R273H) with KRAS(G12V) activation caused transformation of MOE into high-grade sarcomatoid carcinoma when xenografted into nude mice. Elucidating the specific role of p53(R273H) in the fallopian tube will improve understanding of changes at the earliest stage of transformation. This information can help develop chemopreventative strategies to prevent the accumulation of additional mutations and reverse progression of the "p53 signature" thereby, improving survival rates.

Tumorigenesis and peritoneal colonization from fallopian tube epithelium

Ovarian cancer is the most lethal gynecological malignancy, primarily because its origin and initiation factors are unknown. A secretory murine oviductal epithelial (MOE) model was generated to address the hypothesis that the fallopian tube is an origin for high-grade serous cancer. MOE cells were stably altered to express mutation in p53, silence PTEN, activate AKT, and amplify KRAS alone and in combination, to define if this cell type gives rise to tumors and what genetic alterations are required to drive malignancy. Cell lines were characterized in vitro and allografted into mice. Silencing PTEN formed high-grade carcinoma with wide spread tumor explants including metastasis into the ovary. Addition of p53 mutation to PTEN silencing did not enhance this phenotype, whereas addition of KRAS mutation reduced survival. Interestingly, PTEN silencing and KRAS mutation originating from ovarian surface epithelium generated endometrioid carcinoma, suggesting that different cellular origins with identical genetic manipulations can give rise to distinct cancer histotypes. Defining the roles of specific signaling modifications in tumorigenesis from the fallopian tube/oviduct is essential for early detection and development of targeted therapeutics. Further, syngeneic MOE allografts provide an ideal model for pre-clinical testing in an in vivo environment with an intact immune system.

Three-dimensional modeling of the human fallopian tube fimbriae

OBJECTIVE: Ovarian cancer is the most lethal gynecological malignancy that affects women. Recent data suggests that the disease may originate in the fallopian fimbriae; however, the anatomical origin of ovarian carcinogenesis remains unclear. This is largely driven by our lack of knowledge regarding the structure and function of normal fimbriae and the relative paucity of models that accurately recapitulate the in vivo fallopian tube. Therefore, a human three-dimensional (3D) culture system was developed to examine the role of the fallopian fimbriae in serous tumorigenesis.

METHODS: Alginate matrix was utilized to support human fallopian fimbriae ex vivo. Fimbriae were cultured with factors hypothesized to contribute to carcinogenesis, namely; H2O2 (1mM) a mimetic of oxidative stress, insulin (5μg/ml) to stimulate glycolysis, and estradiol (E2, 10nM) which peaks before ovulation. Cultures were evaluated for changes in proliferation and p53 expression, criteria utilized to identify potential precursor lesions. Further, secretory factors were assessed after treatment with E2 to identify if steroid signaling induces a pro-tumorigenic microenvironment.

RESULTS: 3D fimbriae cultures maintained normal tissue architecture up to 7days, retaining both epithelial subtypes. Treatment of cultures with H2O2 or insulin significantly induced proliferation. However, p53 stabilization was unaffected by any particular treatment, although it was induced by ex vivo culturing. Moreover, E2-alone treatment significantly induced its canonical target PR and expression of IL8, a factor linked to poor outcome.

CONCLUSIONS: 3D alginate cultures of human fallopian fimbriae provide an important microphysiological model, which can be further utilized to investigate serous tumorigenesis originating from the fallopian tube.

Developmentally regulated IL6-type cytokines signal to germ cells in the human fetal ovary

Fetal ovarian development and primordial follicle formation are imperative for adult fertility in the female. Data suggest the interleukin (IL)6-type cytokines, leukaemia inhibitory factor (LIF), IL6, oncostatin M (OSM) and ciliary neurotrophic factor (CNTF), are able to regulate the survival, proliferation and differentiation of fetal murine germ cells (GCs) in vivo and in vitro. We postulated that these factors may play a similar role during early human GC development and primordial follicle formation. To test this hypothesis, we have investigated the expression and regulation of IL6-type cytokines, using quantitative reverse transcription polymerase chain reaction and immunohistochemistry. Expression of transcripts encoding OSM increased significantly across the gestational range examined (8-20 weeks), while expression of IL6 increased specifically between the first (8-11 weeks) and early second (12-16 weeks) trimesters, co-incident with the initiation of meiosis. LIF and CNTF expression remained unchanged. Expression of the genes encoding the LIF and IL6 receptors, and their common signalling subunit gp130, was also found to be developmentally regulated, with expression increasing significantly with increasing gestation. LIF receptor and gp130 proteins localized exclusively to GCs, including oocytes in primordial follicles, indicating this cell type to be the sole target of IL6-type cytokine signalling in the human fetal ovary. These data establish that IL6-type cytokines and their receptors are expressed in the human fetal ovary and may directly influence GC development at multiple stages of maturation.

An original phylogenetic approach identified mitochondrial haplogroup T1a1 as inversely associated with breast cancer risk in BRCA2 mutation carriers

Introduction: Individuals carrying pathogenic mutations in the BRCA1 and BRCA2 genes have a high lifetime risk of breast cancer. BRCA1 and BRCA2 are involved in DNA double-strand break repair, DNA alterations that can be caused by exposure to reactive oxygen species, a main source of which are mitochondria. Mitochondrial genome variations affect electron transport chain efficiency and reactive oxygen species production. Individuals with different mitochondrial haplogroups differ in their metabolism and sensitivity to oxidative stress. Variability in mitochondrial genetic background can alter reactive oxygen species production, leading to cancer risk. In the present study, we tested the hypothesis that mitochondrial haplogroups modify breast cancer risk in BRCA1/2 mutation carriers.

Methods: We genotyped 22,214 (11,421 affected, 10,793 unaffected) mutation carriers belonging to the Consortium of Investigators of Modifiers of BRCA1/2 for 129 mitochondrial polymorphisms using the iCOGS array. Haplogroup inference and association detection were performed using a phylogenetic approach. ALTree was applied to explore the reference mitochondrial evolutionary tree and detect subclades enriched in affected or unaffected individuals.

Results: We discovered that subclade T1a1 was depleted in affected BRCA2 mutation carriers compared with the rest of clade T (hazard ratio (HR) = 0.55; 95% confidence interval (CI), 0.34 to 0.88; P = 0.01). Compared with the most frequent haplogroup in the general population (that is, H and T clades), the T1a1 haplogroup has a HR of 0.62 (95% CI, 0.40 to 0.95; P = 0.03). We also identified three potential susceptibility loci, including G13708A/rs28359178, which has demonstrated an inverse association with familial breast cancer risk.

Conclusions: This study illustrates how original approaches such as the phylogeny-based method we used can empower classical molecular epidemiological studies aimed at identifying association or risk modification effects.

Explaining variation in cancer survival between 11 jurisdictions in the International Cancer Benchmarking Partnership: a primary care vignette survey

OBJECTIVES: The International Cancer Benchmarking Partnership (ICBP) is a collaboration between 6 countries and 12 jurisdictions with similar primary care-led health services. This study investigates primary care physician (PCP) behaviour and systems that may contribute to the timeliness of investigating for cancer and subsequently, international survival differences.

DESIGN: A validated survey administered to PCPs via the internet set out in two parts: direct questions on primary care structure and practice relating to cancer diagnosis, and clinical vignettes, assessing management of scenarios relating to the diagnosis of lung, colorectal or ovarian cancer.

PARTICIPANTS: 2795 PCPs in 11 jurisdictions: New South Wales and Victoria (Australia), British Columbia, Manitoba, Ontario (Canada), England, Northern Ireland, Wales (UK), Denmark, Norway and Sweden.

PRIMARY AND SECONDARY OUTCOME MEASURES: Analysis compared the cumulative proportion of PCPs in each jurisdiction opting to investigate or refer at each phase for each vignette with 1-year survival, and conditional 5-year survival rates for the relevant cancer and jurisdiction. Logistic regression was used to explore whether PCP characteristics or system differences in each jurisdiction affected the readiness to investigate.

RESULTS: 4 of 5 vignettes showed a statistically significant correlation (p<0.05 or better) between readiness to investigate or refer to secondary care at the first phase of each vignette and cancer survival rates for that jurisdiction. No consistent associations were found between readiness to investigate and selected PCP demographics, practice or health system variables.

CONCLUSIONS: We demonstrate a correlation between the readiness of PCPs to investigate symptoms indicative of cancer and cancer survival rates, one of the first possible explanations for the variation in cancer survival between ICBP countries. No specific health system features consistently explained these findings. Some jurisdictions may consider lowering thresholds for PCPs to investigate for cancer-either directly, or by specialist referral, to improve outcomes.

BRCA1, a 'complex' protein involved in the maintenance of genomic stability

BRCA1 is a major breast and ovarian cancer susceptibility gene, with mutations in this gene predisposing women to a very high risk of developing breast and ovarian tumours. BRCA1 primarily functions to maintain genomic stability via critical roles in DNA repair, cell cycle checkpoint control, transcriptional regulation, apoptosis and mRNA splicing. As a result, BRCA1 mutations often result in defective DNA repair, genomic instability and sensitivity to DNA damaging agents. BRCA1 carries out these different functions through its ability to interact, and form complexes with, a vast array of proteins involved in multiple cellular processes, all of which are considered to contribute to its function as a tumour suppressor. This review discusses and highlights recent research into the functions of BRCA1-related protein complexes and their roles in maintaining genomic stability and tumour suppression.

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 BRCA1-regulated signalling pathways by microarray-based expression profiling

The introduction of microarray technology to the scientific and medical communities has dramatically changed the way in which we now address basic biomedical questions. Expression profiling using microarrays facilitates an experimental approach where alterations in the transcript level of entire transcriptomes can be simultaneously assayed in response to defined stimuli. We have used microarray analysis to identify downstream transcriptional targets of the BRCA1 (Breast Cancer 1) tumour-suppressor gene as a means of defining its function. BRCA1 has been implicated in the predisposition to early onset breast and ovarian cancer and while its exact function remains to be defined, roles in DNA repair, cell-cycle control and transcriptional regulation have been implied. In the current study we have generated cell lines with tetracycline-regulated, inducible expression of BRCA1 as a tool to identify genes, which might represent important effectors of BRCA1 function. Oligonucleotide array-based expression profiling identified a number of genes that were upregulated at various times following inducible expression of BRCA1 including the DNA damage-responsive gene GADD45 (Growth Arrest after DNA Damage). Identified targets were confirmed by Northern blot analysis and their functional significance as BRCA1 targets examined.

Brca1 and brca2:Role in the DNA damage response, cancer formation and treatment

BRCA1 and BRCA2 are highly penetrant breast and ovarian cancer susceptibility genes that are mutated in a significant proportion of familial breast and ovarian cancer syndromes. Both of these genes are tumour suppressors, the products of which play vital roles in the cellular response to DNA damage. These proteins function in a number of cellular pathways in order to maintain genomic stability including DNA damage signaling, DNA repair, cell cycle regulation, protein ubiquitination, chromatin remodeling, transcriptional regulation and apoptosis. This chapter will discuss the functions of these proteins and how they relate to tumour development, and therapy. © 2009 Springer Science+Business Media B.V.

Novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines, induce apoptosis in multi-drug-resistant cancer cells

PURPOSE: The development of multi-drug resistance (MDR) due to the expression of members of the ATP binding cassette (ABC) transporter family is a major obstacle in cancer treatment. The broad range of substrate specificities associated with these transporters leads to the efflux of many anti-cancer drugs from tumour cells. Therefore, the development of new chemotherapeutic agents that are not substrates of these transporters is important. We have recently demonstrated that some members of a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds are microtubule-depolymerising agents that potently induce apoptosis in several cancer cell lines and impair growth of mouse breast tumours. The aim of this current study was to establish whether PBOXs were capable of inducing apoptosis in cancer cells expressing either P-glycoprotein or breast cancer resistance protein (BCRP), two of the main ABC transporters associated with MDR.

METHODS: We performed in vitro studies to assess the effects of PBOXs on cell proliferation, cell cycle and apoptosis in human cancer cell lines and their drug-resistant substrains expressing either P-glycoprotein or BCRP. In addition, we performed a preliminary molecular docking study to examine interactions between PBOXs and P-glycoprotein.

RESULTS: We established that three representative PBOXs, PBOX-6, -15 and -16 were capable of inducing apoptosis in drug-resistant HL60-MDR1 cells (expressing P-glycoprotein) and HL60-ABCG2 cells (expressing BCRP) with similar potencies as in parental human promyelocytic leukaemia HL60 cells. Likewise, resistance to PBOX-6 and -16 was not evident in P-glycoprotein-expressing A2780-ADR cells in comparison with parent human ovarian carcinoma A2780 cells. Finally, we deduced by molecular docking that PBOX-6 is not likely to form favourable interactions with the substrate binding site of P-glycoprotein.

CONCLUSION: Our results suggest that pro-apoptotic PBOX compounds may be potential candidates for the treatment of P-glycoprotein- or BCRP-associated MDR cancers.

Dual targeting of tumour cells and host endothelial cells by novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines

PURPOSE: Some members of a novel series of pyrrolo-1,5-benzoxazepines (PBOXs) are microtubule-targeting agents capable of inducing apoptosis in a variety of human cancerous cells, hence, they are currently being developed as potential anti-cancer agents. The purpose of this study was to first characterise the activities of a novel PBOX analogue, PBOX-16 and then investigate the anti-angiogenic potential of both PBOX-16 and its prototype PBOX-6.

METHODS: The effects of PBOX-6 and -16 on cancerous cells (chronic myeloid leukaemia K562 cells and ovarian carcinoma A2780 cells) and primary cultured human umbilical vein endothelial cells (HUVECs) were examined by assessing cell proliferation, microtubular organisation, DNA analysis of cell cycle progression and caspase-3/7 activity. Their anti-angiogenic properties were then investigated by examining their ability to interfere with HUVEC differentiation into capillary-like structures and vascular endothelial growth factor (VEGF)-stimulated HUVEC migration.

RESULTS: PBOX-6 and -16 inhibited proliferation of K562, A2780 and HUVEC cells in a concentration-dependent manner. PBOX-16, confirmed as a novel depolymerising agent, was approximately tenfold more potent than PBOX-6. Inhibition of cell proliferation was mediated by G(2)/M arrest followed by varying degrees of apoptosis depending on the cell type; endothelial cells underwent less apoptosis than either of the cancer cell lines. In addition to the antitumourigenic properties, we also describe a novel antiangiogenic function for PBOXs: treatment with PBOXs inhibited the spontaneous differentiation of HUVECs into capillary-like structures when grown on a basement membrane matrix preparation (Matrigel™) and also significantly reduced VEGF-stimulated HUVEC migration.

CONCLUSION: Dual targeting of both the tumour cells and the host endothelial cells by PBOX compounds might enhance the anti-cancer efficacy of these drugs.