Expression of the RNA helicase DDX3 and the hypoxia response in breast cancer

<p>Aims: DDX3 is an RNA helicase that has antiapoptotic properties, and promotes proliferation and transformation. In addition, DDX3 was shown to be a direct downstream target of HIF-1α (the master regulatory of the hypoxia response) in breast cancer cell lines. However, the relation between DDX3 and hypoxia has not been addressed in human tumors. In this paper, we studied the relation between DDX3 and the hypoxic responsive proteins in human breast cancer.</p> <p>Methods and Results: DDX3 expression was investigated by immunohistochemistry in breast cancer in comparison with hypoxia related proteins HIF-1α, GLUT1, CAIX, EGFR, HER2, Akt1, FOXO4, p53, ERα, COMMD1, FER kinase, PIN1, E-cadherin, p21, p27, Transferrin receptor, FOXO3A, c-Met and Notch1. DDX3 was overexpressed in 127 of 366 breast cancer patients, and was correlated with overexpression of HIF-1α and its downstream genes CAIX and GLUT1. Moreover, DDX3 expression correlated with hypoxia-related proteins EGFR, HER2, FOXO4, ERα and c-Met in a HIF-1α dependent fashion, and with COMMD1, FER kinase, Akt1, E-cadherin, TfR and FOXO3A independent of HIF-1α.</p> <p>Conclusions: In invasive breast cancer, expression of DDX3 was correlated with overexpression of HIF-1α and many other hypoxia related proteins, pointing to a distinct role for DDX3 under hypoxic conditions and supporting the oncogenic role of DDX3 which could have clinical implication for current development of DDX3 inhibitors.</p&gt

Hypoxia-driven cell motility reflects the interplay between JMY and HIF-1α.

Junction-mediating and regulatory protein (JMY) is a novel p53 cofactor that regulates p53 activity during stress. JMY interacts with p300/CBP, which are ubiquitous transcriptional co-activators that interact with a variety of sequence-specific transcription factors, including hypoxia-inducible factor-1α (HIF-1α). In addition, JMY is an actin-nucleating protein, which, through its WH2 domains, stimulates cell motility. In this study, we show that JMY is upregulated during hypoxia in a HIF-1α-dependent manner. The JMY gene contains HIF-responsive elements in its promoter region and HIF-1α is recruited to its promoter during hypoxia. HIF-1α drives transcription of JMY, which accounts for its induction under hypoxia. Moreover, the enhanced cell motility and invasion that occurs during hypoxia requires JMY, as depleting JMY under hypoxic conditions causes decreased cell motility. Our results establish the interplay between JMY and HIF-1α as a new mechanism that controls cell motility under hypoxic stress

The influence of invasive growth pattern and microvessel density on prognosis in colorectal cancer and colorectal liver metastases

The nature of the invasive growth pattern and microvessel density (MVD) have been suggested to be predictors of prognosis in primary colorectal cancer (CRC) and colorectal liver metastases. The purpose of the present study was to determine whether these two histological features were interrelated and to assess their relative influence on disease recurrence and survival following surgical resection. Archival tissue was retrieved from 55 patients who had undergone surgical resection for primary CRC and matching liver metastases. The nature of the invasive margin was determined by haematoxylin and eosin (H&E) histochemistry. Microvessel density was visualised using immunohistochemical detection of CD31 antigen and quantified using image capture computer software. Clinical details and outcome data were retrieved by case note review and collated with invasive margin and MVD data in a statistical database. Primary CRCs with a pushing margin tended to form capsulated liver metastases (P<0.001) and had a significantly better disease-free survival than the infiltrative margin tumours (log rank P=0.01). Primary cancers with a high MVD tended to form high MVD liver metastases (P=0.007). Microvessel density was a significant predictor of disease recurrence in primary CRCs (P=0.006), but not liver metastases. These results suggest that primary CRCs and their liver metastases show common histological features. This may reflect common mechanisms underlying the tumour–host interaction

Potent interaction of flavopiridol with MRP1

The multidrug resistance protein 1 (MRP1) is an ATP-dependent transport protein for organic anions, as well as neutral or positively charged anticancer agents. In this study we show that flavopiridol, a synthetic flavonoid currently studied in phase 1 trials for its anti-proliferative characteristics, interacts with MRP1 in a potent way. Flavopiridol, as well as other (iso)flavonoids stimulate the ATPase activity of MRP1 in a dose-dependent way at low micromolar concentrations. A new specific monoclonal antibody against MRP1 (MIB6) inhibits the (iso)flavonoid-induced ATPase activity of plasma membrane vesicles prepared from the MRP1 overexpressing cell line GLC4/ADR. The accumulation of daunorubicin in GLC4/ADR cells is increased by flavopiridol and by other non-glycosylated (iso)flavonoids that interact with MRP1 ATPase activity. However, flavopiridol is the only tested compound that affects the daunorubicin accumulation when present at concentrations below 1 μM. Glycosylated (iso)flavonoids do not affect MRP1-mediated transport or ATPase activity. Finally, MRP1 overexpressing and transfected cells are resistant to flavopiridol, but not to other (iso)flavonoids tested. These findings may be of relevance for the development of anticancer therapies with flavopiridol. © 1999 Cancer Research Campaig

A genistein derivative, ITB-301, induces microtubule depolymerization and mitotic arrest in multidrug-resistant ovarian cancer

PURPOSE: To investigate the mechanistic basis of the anti-tumor effect of the compound ITB-301. METHODS: Chemical modifications of genistein have been introduced to improve its solubility and efficacy. The anti-tumor effects were tested in ovarian cancer cells using proliferation assays, cell cycle analysis, immunofluorescence, and microscopy. RESULTS: In this work, we show that a unique glycoside of genistein, ITB-301, inhibits the proliferation of SKOv3 ovarian cancer cells. We found that the 50% growth inhibitory concentration of ITB-301 in SKOv3 cells was 0.5 μM. Similar results were obtained in breast cancer, ovarian cancer, and acute myelogenous leukemia cell lines. ITB-301 induced significant time- and dose-dependent microtubule depolymerization. This depolymerization resulted in mitotic arrest and inhibited proliferation in all ovarian cancer cell lines examined including SKOv3, ES2, HeyA8, and HeyA8-MDR cells. The cytotoxic effect of ITB-301 was dependent on its induction of mitotic arrest as siRNA-mediated depletion of BUBR1 significantly reduced the cytotoxic effects of ITB-301, even at a concentration of 10 μM. Importantly, efflux-mediated drug resistance did not alter the cytotoxic effect of ITB-301 in two independent cancer cell models of drug resistance. CONCLUSION: These results identify ITB-301 as a novel anti-tubulin agent that could be used in cancers that are multidrug resistant. We propose a structural model for the binding of ITB-301 to α- and β-tubulin dimers on the basis of molecular docking simulations. This model provides a rationale for future work aimed at designing of more potent analogs

Hypoxia Sensitive Metal β-Ketoiminate Complexes Showing Induced Single Strand DNA Breaks and Cancer Cell Death by Apoptosis

A series of ruthenium and iridium complexes have been synthesised and characterised with 20 novel crystal structures discussed. The library of β-ketoiminate complexes has been shown to be active against MCF-7 (human breast carcino-ma), HT-29 (human colon carcinoma), A2780 (human ovarian carcinoma) and A2780cis (cisplatin resistant human ovarian carcinoma) cell lines, with selected complexes being more than three times as active as cisplatin against the A2780cis cell line. Complexes have also been shown to be highly active under hypoxic conditions, with the activities of some complexes increasing with a decrease in O2 concentration. The enzyme thioredoxin reductase is over-expressed in cancer cells and complexes reported herein have the advantage of inhibiting this enzyme, with IC50 values measured in the nanomolar range. The anti-cancer activity of these complexes was further investigated to determine whether activity is due to effects on cellular growth or cell survival. The complexes were found to induce significant cancer cell death by apoptosis with levels induced correlating closely with activity in chemosensitivity studies. As a possible cause of cell death, the ability of the complexes to induce damage to cellular DNA was also assessed. The complexes failed to induce double strand DNA break or DNA crosslinking but induced significant levels of single DNA strand breaks indi-cating a different mechanism of action to cisplatin

P-glycoprotein and breast cancer resistance protein in acute myeloid leukaemia cells treated with the Aurora-B Kinase Inhibitor barasertib-hQPA

<p>Abstract</p> <p>Background</p> <p>Aurora kinases play an essential role in orchestrating chromosome alignment, segregation and cytokinesis during mitotic progression, with both aurora-A and B frequently over-expressed in a variety of human malignancies. Over-expression of the ABC drug transporter proteins P-glycoprotein (Pgp) and Breast cancer resistance protein (BCRP) is a major obstacle for chemotherapy in many tumour types with Pgp conferring particularly poor prognosis in acute myeloid leukaemia (AML). Barasertib-hQPA is a highly selective inhibitor of aurora-B kinase that has shown tumouricidal activity against a range tumour cell lines including those of leukaemic AML origin.</p> <p>Methods</p> <p>Effect of barasertib-hQPA on the pHH3 biomarker and cell viability was measured in a panel of leukaemic cell lines and 37 primary AML samples by flow cytometry. Pgp status was determined by flow cytometry and BCRP status by flow cytometry and real-time PCR.</p> <p>Results</p> <p>In this study we report the creation of the cell line OCI-AML3DNR, which over-expresses Pgp but not BCRP or multidrug resistance-associated protein (MRP), through prolonged treatment of OCI-AML3 cells with daunorubicin. We demonstrate that Pgp (OCI-AML3DNR and KG-1a) and BCRP (OCI-AML6.2) expressing AML cell lines are less sensitive to barasertib-hQPA induced pHH3 inhibition and subsequent loss of viability compared to transporter negative cell lines. We also show that barasertib-hQPA resistance in these cell lines can be reversed using known Pgp and BCRP inhibitors. We report that barasertib-hQPA is not an inhibitor of Pgp or BCRP, but by using ¹⁴[C]-barasertib-hQPA that it is effluxed by these transporters. Using phosphoHistone H3 (pHH3) as a biomarker of barasertib-hQPA responsiveness in primary AML blasts we determined that Pgp and BCRP positive primary samples were less sensitive to barasertib-hQPA induced pHH3 inhibition (p = <0.001) than samples without these transporters. However, we demonstrate that IC₅₀inhibition of pHH3 by barasertib-hQPA was achieved in 94.6% of these samples after 1 hour drug treatment, in contrast to the resistance of the cell lines.</p> <p>Conclusion</p> <p>We conclude that Pgp and BCRP status and pHH3 down-regulation in patients treated with barasertib should be monitored in order to establish whether transporter-mediated efflux is sufficient to adversely impact on the efficacy of the agent.</p

Inhibition of Hypoxia-Inducible Factor-1α (HIF-1α) Protein Synthesis by DNA damage inducing agents

10.1371/journal.pone.0010522PLoS ONE55

Genetic Ablation of Bcl-x Attenuates Invasiveness without Affecting Apoptosis or Tumor Growth in a Mouse Model of Pancreatic Neuroendocrine Cancer

Tumor cell death is modulated by an intrinsic cell death pathway controlled by the pro- and anti-apoptotic members of the Bcl-2 family. Up-regulation of anti-apoptotic Bcl-2 family members has been shown to suppress cell death in pre-clinical models of human cancer and is implicated in human tumor progression. Previous gain-of-function studies in the RIP1-Tag2 model of pancreatic islet carcinogenesis, involving uniform or focal/temporal over-expression of Bcl-xL, demonstrated accelerated tumor formation and growth. To specifically assess the role of endogenous Bcl-x in regulating apoptosis and tumor progression in this model, we engineered a pancreatic β-cell-specific knockout of both alleles of Bcl-x using the Cre-LoxP system of homologous recombination. Surprisingly, there was no appreciable effect on tumor cell apoptosis rates or on tumor growth in the Bcl-x knockout mice. Other anti-apoptotic Bcl-2 family members were expressed but not substantively altered at the mRNA level in the Bcl-x-null tumors, suggestive of redundancy without compensatory transcriptional up-regulation. Interestingly, the incidence of invasive carcinomas was reduced, and tumor cells lacking Bcl-x were impaired in invasion in a two-chamber trans-well assay under conditions mimicking hypoxia. Thus, while the function of Bcl-x in suppressing apoptosis and thereby promoting tumor growth is evidently redundant, genetic ablation implicates Bcl-x in selectively facilitating invasion, consistent with a recent report documenting a pro-invasive capability of Bcl-xL upon exogenous over-expression