BRCA1 is an essential mediator of vinorelbine-induced apoptosis in mesothelioma

The rapeutic options for malignant pleural mesothelioma (MPM) are limited despite the increasing incidence globally. The vinca alkaloid vinorelbine exhibits clinical activity; however, to date, treatment optimization has not been achieved using biomarkers. BRCA1 regulates sensitivity to microtubule poisons; however, its role in regulating vinorelbine-induced apoptosis in mesothelioma is unknown. Here we demonstrate that BRCA1 plays an essential role in mediating vinorelbine-induced apoptosis, as evidenced by (1) the strong correlation between vinorelbine sensitivity and BRCA1 expression level; (2) induction of resistance to vinorelbine by BRCA1 using siRNA oligonucleotides; (3) dramatic down-regulation of BRCA1 following selection for vinorelbine resistance; and (4) the re-activation of vinorelbine-induced apoptosis following re-expression of BRCA1 in resistant cells. To determine whether loss of BRCA1 expression in mesothelioma was potentially relevant in vivo, BRCA1 immunohistochemistry was subsequently performed on 144 primary mesothelioma specimens. Loss of BRCA1 protein expression was identified in 38.9% of samples. Together, these data suggest that BRCA1 plays a critical role in mediating apoptosis by vinorelbine in mesothelioma, warranting its clinical evaluation as a predictive biomarker. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

INT6/EIF3E interacts with ATM and is required for proper execution of the DNA damage response in human cells

Altered expression of the INT6 gene, encoding the e subunit of the translational initiation factor eIF3, occurs in human breast cancers, but how INT6 relates to carcinogenesis remains unestablished. Here, we show that INT6 is involved in the DNA damage response. INT6 was required for cell survival following γ-irradiation and G(2)-M checkpoint control. RNA interference-mediated silencing of INT6 reduced phosphorylation of the checkpoint kinases CHK1 and CHK2 after DNA damage. In addition, INT6 silencing prevented sustained accumulation of ataxia telangiectasia mutated (ATM) at DNA damage sites in cells treated with γ-radiation or the radiomimetic drug neocarzinostatin. Mechanistically, this result could be explained by interaction of INT6 with ATM, which together with INT6 was recruited to the sites of DNA damage. Finally, INT6 silencing also reduced ubiquitylation events that promote retention of repair proteins at DNA lesions. Accordingly, accumulation of the repair factor BRCA1 was defective in the absence of INT6. Our findings reveal unexpected and striking connections of INT6 with ATM and BRCA1 and suggest that the protective action of INT6 in the onset of breast cancers relies on its involvement in the DNA damage response.

Lineage-specific biomarkers predict response to FGFR inhibition

In this issue of Cancer Discovery, Guagnano and colleagues use a large and diverse annotated collection of cancer cell lines, the Cancer Cell Line Encyclopedia, to correlate whole-genome expression and genomic alteration datasets with cell line sensitivity data to the novel pan-fibroblast growth factor receptor (FGFR) inhibitor NVP-BGJ398. Their findings underscore not only the preclinical use of such cell line panels in identifying predictive biomarkers, but also the emergence of the FGFRs as valid therapeutic targets, across an increasingly broad range of malignancies.

Contribution of DEAF1 structural domains to the interaction with the breast cancer oncogene LMO4

The proteins LMO4 and DEAF1 contribute to the proliferation of mammary epithelial cells. During breast cancer LMO4 is upregulated, affecting its interaction with other protein partners. This may set cells on a path to tumour formation. LMO4 and DEAF1 interact, but it is unknown how they cooperate to regulate cell proliferation. In this study, we identify a specific LMO4-binding domain in DEAF1. This domain contains an unstructured region that directly contacts LMO4, and a coiled coil that contains the DEAF1 nuclear export signal (NES). The coiled coil region can form tetramers and has the typical properties of a coiled coil domain. Using a simple cell-based assay, we show that LMO4 modulates the activity of the DEAF NES, causing nuclear accumulation of a construct containing the LMO4-interaction region of DEAF1.

Circulating tumour cells, their role in metastasis and their clinical utility in lung cancer

Circulating tumour cells (CTCs) have attracted much recent interest in cancer research as a potential biomarker and as a means of studying the process of metastasis. It has long been understood that metastasis is a hallmark of malignancy, and conceptual theories on the basis of metastasis from the nineteenth century foretold the existence of a tumour "seed" which is capable of establishing discrete tumours in the "soil" of distant organs. This prescient "seed and soil" hypothesis accurately predicted the existence of CTCs; microscopic tumour fragments in the blood, at least some of which are capable of forming metastases. However, it is only in recent years that reliable, reproducible methods of CTC detection and analysis have been developed. To date, the majority of studies have employed the CellSearch™ system (Veridex LLC), which is an immunomagnetic purification method. Other promising techniques include microfluidic filters, isolation of tumour cells by size using microporous polycarbonate filters and flow cytometry-based approaches. While many challenges still exist, the detection of CTCs in blood is becoming increasingly feasible, giving rise to some tantalizing questions about the use of CTCs as a potential biomarker. CTC enumeration has been used to guide prognosis in patients with metastatic disease, and to act as a surrogate marker for disease response during therapy. Other possible uses for CTC detection include prognostication in early stage patients, identifying patients requiring adjuvant therapy, or in surveillance, for the detection of relapsing disease. Another exciting possible use for CTC detection assays is the molecular and genetic characterization of CTCs to act as a "liquid biopsy" representative of the primary tumour. Indeed it has already been demonstrated that it is possible to detect HER2, KRAS and EGFR mutation status in breast, colon and lung cancer CTCs respectively. In the course of this review, we shall discuss the biology of CTCs and their role in metastagenesis, the most commonly used techniques for their detection and the evidence to date of their clinical utility, with particular reference to lung cancer.

Generation and characterisation of Cisplatin-resistant non-small cell lung cancer cell lines displaying a stem-like signature

Introduction: Inherent and acquired cisplatin resistance reduces the effectiveness of this agent in the management of non-small cell lung cancer (NSCLC). Understanding the molecular mechanisms underlying this process may result in the development of novel agents to enhance the sensitivity of cisplatin. Methods: An isogenic model of cisplatin resistance was generated in a panel of NSCLC cell lines (A549, SKMES-1, MOR, H460). Over a period of twelve months, cisplatin resistant (CisR) cell lines were derived from original, age-matched parent cells (PT) and subsequently characterized. Proliferation (MTT) and clonogenic survival assays (crystal violet) were carried out between PT and CisR cells. Cellular response to cisplatin-induced apoptosis and cell cycle distribution were examined by FACS analysis. A panel of cancer stem cell and pluripotent markers was examined in addition to the EMT proteins, c-Met and β-catenin. Cisplatin-DNA adduct formation, DNA damage (γH2AX) and cellular platinum uptake (ICP-MS) was also assessed. Results: Characterisation studies demonstrated a decreased proliferative capacity of lung tumour cells in response to cisplatin, increased resistance to cisplatin-induced cell death, accumulation of resistant cells in the G0/G1 phase of the cell cycle and enhanced clonogenic survival ability. Moreover, resistant cells displayed a putative stem-like signature with increased expression of CD133+/CD44+cells and increased ALDH activity relative to their corresponding parental cells. The stem cell markers, Nanog, Oct-4 and SOX-2, were significantly upregulated as were the EMT markers, c-Met and β-catenin. While resistant sublines demonstrated decreased uptake of cisplatin in response to treatment, reduced cisplatin-GpG DNA adduct formation and significantly decreased γH2AX foci were observed compared to parental cell lines. Conclusion: Our results identified cisplatin resistant subpopulations of NSCLC cells with a putative stem-like signature, providing a further understanding of the cellular events associated with the cisplatin resistance phenotype in lung cancer. © 2013 Barr et al.

The cancer stem-cell hypothesis : its emerging role in lung cancer biology and its relevance for future therapy

The cancer stem-cell (CSC) hypothesis suggests that there is a small subset of cancer cells that are responsible for tumor initiation and growth, possessing properties such as indefinite self-renewal, slow replication, intrinsic resistance to chemotherapy and radiotherapy, and an ability to give rise to differentiated progeny. Through the use of xenotransplantation assays, putative CSCs have been identified in many cancers, often identified by markers usually expressed in normal stem cells. This is also the case in lung cancer, and the accumulated data on side population cells, CD133, CD166, CD44 and ALDH1 are beginning to clarify the true phenotype of the lung cancer stem cell. Furthermore, it is now clear that many of the pathways of normal stem cells, which guide cellular proliferation, differentiation, and apoptosis are also prominent in CSCs; the Hedgehog (Hh), Notch, and Wnt signaling pathways being notable examples. The CSC hypothesis suggests that there is a small reservoir of cells within the tumor, which are resistant to many standard therapies, and can give rise to new tumors in the form of metastases or relapses after apparent tumor regression. Therapeutic interventions that target CSC pathways are still in their infancy and clinical data of their efficacy remain limited. However Smoothened inhibitors, gamma-secretase inhibitors, anti-DLL4 antagonists, Wnt antagonists, and CBP/β-catenin inhibitors have all shown promising anticancer effects in early studies. The evidence to support the emerging picture of a lung cancer CSC phenotype and the development of novel therapeutic strategies to target CSCs are described in this review.

"Fair" government contracts for community service provision : time to curb unfettered executive freedom?

All Australian governments are significantly increasing the use of contracted community service provision through not for profit (NFP) organisations. These transactions occur through grant arrangements which take the form of standard contracts or deeds rather than drawing on statutory authority. Government inquiries bodies have consistently reported and raised concerns about the fairness of such standard grant contract terms, but failed to provide any mechanism whereby fairness can be assured. The Productivity Commission has suggested that the resulting poor relationship results in inappropriate risk transfer, micro-management, disincentives to innovate and poor service provision. This paper develops and tests a fairness measure based on the principles of the Australian Consumer Law which legislates fairness protections for standard consumer contracts.  

The role of Ureaplasma urealyticum in adverse pregnancy outcome

We investigated Ureaplasma urealyticum genital tract colonisation rates in an Australian population to determine whether colonisation was associated with adverse pregnancy outcome. Women attending an antenatal clinic were evaluated for lower genital tract colonisation at their first antenatal visit (162 women) and at 28 weeks gestation (120 women). Placentas from 92 women were cultured. U. urealyticum was the predominant isolate from the lower (57.4%) and upper (17.4%) genital tract in this population of pregnant women. U. urealyticum was a persistent coloniser during mid-trimester of pregnancy (in 88% of women colonised) whereas M. hominis, G. vaginalis, and Group B streptococcus were present as transient flora of the lower genital tract. Lower genital tract colonisation during pregnancy was not directly associated with adverse pregnancy outcome. However preterm delivery in afebrile, asymptomatic women, could possibly be associated with chorioamnionitis (four of 16 preterm births). Screening of women with a history of preterm birth may prevent upper genital tract infections and preterm delivery.

Comparison of PCR, nested PCR, and random amplified polymorphic DNA PCR for detection and typing of Ureaplasma urealyticum in specimens from pregnant women

A PCR assay, using three primer pairs, was developed for the detection of Ureaplasma urealyticum, parvo biovar, mba types 1, 3, and 6, in cultured clinical specimens. The primer pairs were designed by using the polymorphic base positions within a 310- to 311-bp fragment of the 5* end and upstream control region of the mba gene. The specificity of the assay was confirmed with reference serovars 1, 3, 6, and 14 and by the amplified-fragment sizes (81 bp for mba 1, 262 bp for mba 3, and 193 bp for mba 6). A more sensitive nested PCR was also developed. This involved a first-step PCR, using the primers UMS-125 and UMA226, followed by the nested mba-type PCR described above. This nested PCR enabled the detection and typing of small numbers of U. urealyticum cells, including mixtures, directly in original clinical specimens. By using random amplified polymorphic DNA (RAPD) PCR with seven arbitrary primers, we were also able to differentiate the two biovars of U. urealyticum and to identify 13 RAPD-PCR subtypes. By applying these subtyping techniques to clinical samples collected from pregnant women, we established that (i) U. urealyticum is often a persistent colonizer of the lower genital tract from early midtrimester until the third trimester of pregnancy, (ii) mba type 6 was isolated significantly more often (P 5 0.048) from women who delivered preterm than from women who delivered at term, (iii) no particular ureaplasma subtype(s) was associated with placental infections and/or adverse pregnancy outcomes, and (iv) the ureaplasma subtypes most frequently isolated from women were the same subtypes most often isolated from infected placentas.

The phylogeny of Ureaplasma urealyticum based on the mba gene fragment

Sequencing of mba gene fragments of reference strains of Ureaplasma urealyticum serovars 1, 3, 6, 14, in addition to 33 clinical U. urealyticum isolates is reported. A phylogenetic tree deduced from an alignment of these sequences clearly demonstrates two major clusters (confidence limit 100%), which equate to the parvo and T960 biovars, and five types which we have designated mba 1, 3, 6, 8 and X. These relationships are supported by bootstrap analysis. Polymorphisms within the mba fragment of types mba 1, 3, and 6 were used to define nine subtypes (mba 1a, 1b, 3a, 3b, 3c, 3d, 3e, 6a, and 6b) thus facilitating high resolution typing of U. urealyticum. Inclusion of the reference strains for serovars 1, 3, 6, and 8 in the mba typing scheme showed that the results of this analysis are broadly consistent with currently accepted serotyping. In addition a ure gene fragment from nine of the clinical isolates was amplified and sequenced. Comparisons of the sequences clearly distinguished the two biovars of U. urealyticum; however this fragment was invariant within the parvo biovar. This study has shown that the sequence of the mba can reveal the fine details of the relationships between U. urealyticum isolates and also supports the significant evolutionary gap between the two biovars.

Cytokine synthesis by intestinal intraepithelial lymphocytes. Both gamma/delta T cell receptor-positive and alpha/beta T cell receptor-positive T cells in the G1 phase of cell cycle produce IFN-gamma and IL-5

Murine intestinal intraepithelial lymphocytes (IEL) have been shown to contain subsets of alpha/beta TCR+ and gamma/delta TCR+ T cells that spontaneously produce cytokines such as IFN-gamma and IL-5. We have now determined the nature and cell cycle stage of these cytokine-producing T lymphocytes in EIL by using IFN-gamma- and IL-5-specific ELISPOT assay, cytokine-specific mRNA-cDNA dot-blot hybridization and polymerase chain reaction, and flow cytometry (FACS) for DNA analysis. When CD3+ T cells from IEL of normal C3H/HeN mice were separated into low and high density fractions by discontinuous Percoll gradients, IFN-gamma and IL-5 spot-forming cells were only found in the former population. Analysis of mRNA for these cytokines by both IFN-gamma- and IL-5-specific dot-blot hybridization and polymerase chain reaction revealed that higher levels of message for IFN-gamma and IL-5 were also seen in the low density fraction. However, cell cycle analysis of these two fractions by FACS using propidium iodide showed a similar pattern of cell cycle stages in both low and high density populations (G0 + G1 approximately 96 to 98% and S/G2 + M approximately 2 to 4%). Finally, mRNA from gamma/delta TCR+ and alpha/beta TCR+ T cells in both low and high density fractions of IEL were analyzed for IFN-gamma and IL-5 message by polymerase chain reaction. After 35 cycles of amplification, both gamma/delta TCR+ and alpha/beta TCR+ T cells in the low density fraction expressed higher levels of message for these two cytokines when compared with the high density population. These results have now shown that both gamma/delta and alpha/beta TCR+ IEL can be separated into low and high density subsets and both fractions possess a similar stage of cell cycle. However, only the low density cells (in G1 phase) of both gamma/delta and alpha/beta TCR types possess increased cytokine-specific mRNA and produce the cytokines IFN-gamma and IL-5. Our results suggest that alpha/beta TCR+ and gamma/delta TCR+ IEL can produce cytokines without cell proliferation.

The Ghrelin axis : does it have an appetite for cancer progression?

Ghrelin, the endogenous ligand for the GH secretagogue receptor (GHSR), is a peptide hormone with diverse physiological roles. Ghrelin regulates GH release, appetite and feeding, gut motility, and energy balance and also has roles in the cardiovascular, immune, and reproductive systems. Ghrelin and the GHSR are expressed in a wide range of normal and tumor tissues, and a fluorescein-labeled, truncated form of ghrelin is showing promise as a biomarker for prostate cancer. Plasma ghrelin levels are generally inversely related to body mass index and are unlikely to be useful as a biomarker for cancer, but may be useful as a marker for cancer cachexia. Some single nucleotide polymorphisms in the ghrelin and GHSR genes have shown associations with cancer risk; however, larger studies are required. Ghrelin regulates processes associated with cancer, including cell proliferation, apoptosis, cell migration, cell invasion, inflammation, and angiogenesis; however, the role of ghrelin in cancer is currently unclear. Ghrelin has predominantly antiinflammatory effects and may play a role in protecting against cancer-related inflammation. Ghrelin and its analogs show promise as treatments for cancer-related cachexia. Further studies using in vivo models are required to determine whether ghrelin has a role in cancer progression.

Signal transduction mechanisms underlying growth hormone receptor action

Our understanding of the mechanisms of action of GH and its receptor, the GHR, has advanced significantly in the last decade and has provided some important surprises. It is now clear that the GH-GHR axis activates a number of inter-related signalling pathways, not all of which are dependent on the intracellular tyrosine kinase, JAK2 as originally postulated. JAK2-independent pathways, mediated via the Src family kinases, together with a number of negative regulators of GH signalling and emerging cross-talk mechanisms with other growth factor receptors, provide a complex array of mechanisms that are capable of fine-tuning responses to GH in a cell context dependent manner. Additionally, it is also now clear that GH and the GHR can translocate to the nucleus of target cells and initiate, as yet not well defined, nuclear responses. Continued emphasis on elucidation of these complex mechanisms is critical to provide further insights into the diverse physiological and pathophysiological effects of GH.

IGF2 increases de novo steroidogenesis in prostate cancer cells

Androgen-dependent pathways regulate maintenance and growth of normal and malignant prostate tissues. Androgen deprivation therapy (ADT) exploits this dependence and is used to treat metastatic prostate cancer; however, regression initially seen with ADT gives way to development of incurable castration-resistant prostate cancer (CRPC). Although ADT generates a therapeutic response, it is also associated with a pattern of metabolic alterations consistent with metabolic syndrome including elevated circulating insulin. Because CRPC cells are capable of synthesizing androgens de novo, we hypothesized that insulin may also influence steroidogenesis in CRPC. In this study, we examined this hypothesis by evaluating the effect of insulin on steroid synthesis in prostate cancer cell lines. Treatment with 10 nmol/L insulin increased mRNA and protein expression of steroidogenesis enzymes and upregulated the insulin receptor substrate insulin receptor substrate 2 (IRS-2). Similarly, insulin treatment upregulated intracellular testosterone levels and secreted androgens, with the concentrations of steroids observed similar to the levels reported in prostate cancer patients. With similar potency to dihydrotestosterone, insulin treatment resulted in increased mRNA expression of prostate-specific antigen. CRPC progression also correlated with increased expression of IRS-2 and insulin receptor in vivo. Taken together, our findings support the hypothesis that the elevated insulin levels associated with therapeutic castration may exacerbate progression of prostate cancer to incurable CRPC in part by enhancing steroidogenesis.