PARP inhibitors and the treatment of breast cancer: beyond BRCA1/2?

Poly(ADP-ribose) polymerase (PARP) inhibitors have been explored as therapeutic agents for the treatment of hereditary breast and ovarian cancers harboring mutations in BRCA1 or BRCA2. In a new study, Inbar-Rozensal and colleagues show that phenanthridine-derived PARP inhibitors promote cell cycle arrest and cell death in breast cancer cells lacking BRCA1 and BRCA2 mutations and prevent the growth of tumors from xenografts of these cells in immunocompromised mice. These results suggest a potential broader utility of PARP-1 inhibitors in the treatment of breast cancer, although further mechanistic studies are needed

Clinical array-based karyotyping of breast cancer with equivocal HER2 status resolves gene copy number and reveals chromosome 17 complexity

<p>Abstract</p> <p>Background</p> <p><it>HER2 </it>gene copy status, and concomitant administration of trastuzumab (Herceptin), remains one of the best examples of targeted cancer therapy based on understanding the genomic etiology of disease. However, newly diagnosed breast cancer cases with equivocal HER2 results present a challenge for the oncologist who must make treatment decisions despite the patient's unresolved HER2 status. In some cases both immunohistochemistry (IHC) and fluorescence <it>in situ </it>hybridization (FISH) are reported as equivocal, whereas in other cases IHC results and FISH are discordant for positive versus negative results. The recent validation of array-based, molecular karyotyping for clinical oncology testing provides an alternative method for determination of HER2 gene copy number status in cases remaining unresolved by traditional methods.</p> <p>Methods</p> <p>In the current study, DNA extracted from 20 formalin fixed paraffin embedded (FFPE) tissue samples from newly diagnosed cases of invasive ductal carcinoma referred to our laboratory with unresolved HER2 status, were analyzed using a clinically validated genomic array containing 127 probes covering the HER2 amplicon, the pericentromeric regions, and both chromosome 17 arms.</p> <p>Results</p> <p>Array-based comparative genomic hybridization (array CGH) analysis of chromosome 17 resolved HER2 gene status in [20/20] (100%) of cases and revealed additional chromosome 17 copy number changes in [18/20] (90%) of cases. Array CGH analysis also revealed two false positives and one false negative by FISH due to "ratio skewing" caused by chromosomal gains and losses in the centromeric region. All cases with complex rearrangements of chromosome 17 showed genome-wide chromosomal instability.</p> <p>Conclusions</p> <p>These results illustrate the analytical power of array-based genomic analysis as a clinical laboratory technique for resolution of HER2 status in breast cancer cases with equivocal results. The frequency of complex chromosome 17 abnormalities in these cases suggests that the two probe FISH interphase analysis is inadequate and results interpreted using the HER2/CEP17 ratio should be reported "with caution" when the presence of centromeric amplification or monosomy is suspected by FISH signal gains or losses. The presence of these pericentromeric copy number changes may result in artificial skewing of the HER2/CEP17 ratio towards false negative or false positive results in breast cancer with chromosome 17 complexity. Full genomic analysis should be considered in all cases with complex chromosome 17 aneusomy as these cases are likely to have genome-wide instability, amplifications, and a poor prognosis.</p

Role of Endoplasmic Reticulum Stress in α-TEA Mediated TRAIL/DR5 Death Receptor Dependent Apoptosis

Background -- α-TEA (RRR-α-tocopherol ether-linked acetic acid analog), a derivative of RRR-α-tocopherol (vitamin E) exhibits anticancer actions in vitro and in vivo in variety of cancer types. The objective of this study was to obtain additional insights into the mechanisms involved in α-TEA induced apoptosis in human breast cancer cells. Methodology/Principal Findings -- α-TEA induces endoplasmic reticulum (ER) stress as indicated by increased expression of CCAAT/enhancer binding protein homologous protein (CHOP) as well as by enhanced expression or activation of specific markers of ER stress such as glucose regulated protein (GRP78), phosphorylated alpha subunit of eukaryotic initiation factor 2 (peIF-2α), and spliced XBP-1 mRNA. Knockdown studies using siRNAs to TRAIL, DR5, JNK and CHOP as well as chemical inhibitors of ER stress and caspase-8 showed that: i) α-TEA activation of DR5/caspase-8 induces an ER stress mediated JNK/CHOP/DR5 positive amplification loop; ii) α-TEA downregulation of c-FLIP (L) protein levels is mediated by JNK/CHOP/DR5 loop via a JNK dependent Itch E3 ligase ubiquitination that further serves to enhance the JNK/CHOP/DR5 amplification loop by preventing c-FLIP's inhibition of caspase-8; and (iii) α-TEA downregulation of Bcl-2 is mediated by the ER stress dependent JNK/CHOP/DR5 signaling. Conclusion -- Taken together, ER stress plays an important role in α-TEA induced apoptosis by enhancing DR5/caspase-8 pro-apoptotic signaling and suppressing anti-apoptotic factors c-FLIP and Bcl-2 via ER stress mediated JNK/CHOP/DR5/caspase-8 signaling.The Clayton Foundation for Research, the National Institute of Environmental Health Sciences Center Grant ES007784, the Center for Molecular and Cellular Toxicology at the University of Texas at Austin and a NIEHS/NIH Toxicology Training Grant T32 ES07247. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Biological Sciences, School o

TgICMAP1 Is a Novel Microtubule Binding Protein in Toxoplasma gondii

The microtubule cytoskeleton provides essential structural support for all eukaryotic cells and can be assembled into various higher order structures that perform drastically different functions. Understanding how microtubule-containing assemblies are built in a spatially and temporally controlled manner is therefore fundamental to understanding cell physiology. Toxoplasma gondii, a protozoan parasite, contains at least five distinct tubulin-containing structures, the spindle pole, centrioles, cortical microtubules, the conoid, and the intra-conoid microtubules. How these five structurally and functionally distinct sets of tubulin containing structures are constructed and maintained in the same cell is an intriguing problem. Previously, we performed a proteomic analysis of the T. gondii apical complex, a cytoskeletal complex located at the apical end of the parasite that is composed of the conoid, three ring-like structures, and the two short intra-conoid microtubules. Here we report the characterization of one of the proteins identified in that analysis, TgICMAP1. We show that TgICMAP1 is a novel microtubule binding protein that can directly bind to microtubules in vitro and stabilizes microtubules when ectopically expressed in mammalian cells. Interestingly, in T. gondii, TgICMAP1 preferentially binds to the intra-conoid microtubules, providing us the first molecular tool to investigate the intra-conoid microtubule assembly process during daughter construction

Identification of germline alterations of the mad homology 2 domain of SMAD3 and SMAD4 from the Ontario site of the breast cancer family registry (CFR)

Abstract Introduction A common feature of neoplastic cells is that mutations in SMADs can contribute to the loss of sensitivity to the anti-tumor effects of transforming growth factor-β (TGF-β). However, germline mutation analysis of SMAD3 and SMAD4, the principle substrates of the TGF-β signaling pathway, has not yet been conducted in breast cancer. Thus, it is currently unknown whether germline SMAD3 and SMAD4 mutations are involved in breast cancer predisposition. Methods We performed mutation analysis of the highly conserved mad-homology 2 (MH2) domains for both genes in genomic DNA from 408 non-BRCA1/BRCA2 breast cancer cases and 710 population controls recruited by the Ontario site of the breast cancer family registry (CFR) using denaturing high-performance liquid chromatography (DHPLC) and direct DNA sequencing. The results were interpreted in several ways. First, we adapted nucleotide diversity analysis to quantitatively assess whether the frequency of alterations differ between the two genes. Next, in silico tools were used to predict variants' effect on domain function and mRNA splicing. Finally, 37 cases or controls harboring alterations were tested for aberrant splicing using reverse-transcription polymerase chain reaction (PCR) and real-time PCR statistical comparison of germline expressions by non-parametric Mann-Whitney test of independent samples. Results We identified 27 variants including 2 novel SMAD4 coding variants c.1350G > A (p.Gln450Gln), and c.1701A > G (p.Ile525Val). There were no inactivating mutations even though c.1350G > A was predicted to affect exonic splicing enhancers. However, several additional findings were of note: 1) nucleotide diversity estimate for SMAD3 but not SMAD4 indicated that coding variants of the MH2 domain were more infrequent than expected; 2) in breast cancer cases SMAD3 was significantly over-expressed relative to controls (P A was associated with elevated germline expression (> 5-fold); 3) separate analysis using tissue expression data showed statistically significant over-expression of SMAD3 and SMAD4 in breast carcinomas. Conclusions This study shows that inactivating germline alterations in SMAD3 and SMAD4 are rare, suggesting a limited role in driving tumorigenesis. Nevertheless, aberrant germline expressions of SMAD3 and SMAD4 may be more common in breast cancer than previously suspected and offer novel insight into their roles in predisposition and/or progression of breast cancer

Relationship of imatinib-free plasma levels and target genotype with efficacy and tolerability

Imatinib has revolutionised the treatment of chronic myeloid leukaemia (CML) and gastrointestinal stromal tumours (GIST). Using a nonlinear mixed effects population model, individual estimates of pharmacokinetic parameters were derived and used to estimate imatinib exposure (area under the curve, AUC) in 58 patients. Plasma-free concentration was deduced from a model incorporating plasma levels of alpha1-acid glycoprotein. Associations between AUC (or clearance) and response or incidence of side effects were explored by logistic regression analysis. Influence of KIT genotype was also assessed in GIST patients. Both total (in GIST) and free drug exposure (in CML and GIST) correlated with the occurrence and number of side effects (e.g. odds ratio 2.7±0.6 for a two-fold free AUC increase in GIST; P<0.001). Higher free AUC also predicted a higher probability of therapeutic response in GIST (odds ratio 2.6±1.1; P=0.026) when taking into account tumour KIT genotype (strongest association in patients harbouring exon 9 mutation or wild-type KIT, known to decrease tumour sensitivity towards imatinib). In CML, no straightforward concentration–response relationships were obtained. Our findings represent additional arguments to further evaluate the usefulness of individualising imatinib prescription based on a therapeutic drug monitoring programme, possibly associated with target genotype profiling of patients

EZH2 Depletion Blocks the Proliferation of Colon Cancer Cells

The Enhancer of Zeste 2 (EZH2) protein has been reported to stimulate cell growth in some cancers and is therefore considered to represent an interesting new target for therapeutic intervention. Here, we investigated a possible role of EZH2 for the growth control of colon cancer cells. RNA interference (RNAi)-mediated intracellular EZH2 depletion led to cell cycle arrest of colon carcinoma cells at the G1/S transition. This was associated with a reduction of cell numbers upon transient transfection of synthetic EZH2-targeting siRNAs and with inhibition of their colony formation capacity upon stable expression of vector-borne siRNAs. We furthermore tested whether EZH2 may repress the growth-inhibitory p27 gene, as reported for pancreatic cancer. However, expression analyses of colon cancer cell lines and colon cancer biopsies did not reveal a consistent correlation between EZH2 and p27 levels. Moreover, EZH2 depletion did not re-induce p27 expression in colon cancer cells, indicating that p27 repression by EZH2 may be cell- or tissue-specific. Whole genome transcriptome analyses identified cellular genes affected by EZH2 depletion in colon cancer cell lines. They included several cancer-associated genes linked to cellular proliferation or invasion, such as Dag1, MageD1, SDC1, Timp2, and Tob1. In conclusion, our results demonstrate that EZH2 depletion blocks the growth of colon cancer cells. These findings might provide benefits for the treatment of colon cancer