Identification of tubulin as the molecular target of proapoptotic pyrrolo-1,5-benzoxazepines

We have demonstrated previously that certain members of a series of novel pyrrolo-1,5-benzoxazepine (PBOX) compounds potently induce apoptosis in a variety of human chemotherapy-resistant cancer cell lines and in primary ex vivo material derived from cancer patients. A better understanding of the molecular mechanisms underlying the apoptotic effects of these PBOX compounds is essential to their development as antineoplastic therapeutic agents. This study sought to test the hypothesis that proapoptotic PBOX compounds target the microtubules. We show that a representative proapoptotic PBOX compound, PBOX-6, induces apoptosis in both the MCF-7 and K562 cell lines. An accumulation of cells in G2/M precedes apoptosis in response to PBOX-6. PBOX-6 induces prometaphase arrest and causes an accumulation of cyclin B1 levels and activation of cyclin B1/CDK1 kinase in a manner similar to that of two representative antimicrotubule agents, nocodazole and paclitaxel. Indirect immunofluorescence demonstrates that both PBOX-6 and another pro-apoptotic PBOX compound, PBOX-15, cause microtubule depolymerization in MCF-7 cells. They also inhibit the assembly of purified tubulin in vitro, whereas a nonapoptotic PBOX compound (PBOX-21) has no effect on either the cellular microtubule network or on the assembly of purified tubulin. This suggests that the molecular target of the pro-apoptotic PBOX compounds is tubulin. PBOX-6 does not bind to either the vinblastine or the colchicine binding site on tubulin, suggesting that it binds to an as-yet-uncharacterised novel site on tubulin. The ability of PBOX-6 to bind tubulin and cause microtubule depolymerization confirms it as a novel candidate for antineoplastic therapy.

Molecular pathology of prostate cancer:the key to identifying new biomarkers of disease

Microarray technology has recently accelerated the study of the molecular events involved in prostate cancer, offering the prospect of more precise prognosis and new therapeutic strategies. This review summarises current knowledge of the molecular pathology of prostate cancer. The expression and function of numerous genes have been shown to be altered in prostate cancer. Many of these genes are involved in cell cycle regulation, steroid hormone metabolism or regulation of gene expression. The mechanisms by which androgen independence arises are discussed, including cross-activation, gene amplification and point mutations of the androgen receptor. Analysis of changes in the levels of expression of large numbers of genes during prostate cancer progression have provided a better understanding of the basis of the disease, yielding new molecular markers, such as hepsin, with potential use in diagnosis and prognosis.

Gene-based therapy in prostate cancer

Prostate cancer is one of the commonest causes of illness and death from cancer. Radical prostatectomy, radiotherapy, and hormonal therapy are the main conventional treatments. However, gene therapy is emerging as a promising adjuvant to conventional strategies, and several clinical trials are in progress. Here, we outline several approaches to gene therapy for prostate cancer that have been investigated. Methods of gene delivery are described, particularly those that have commonly been used in research on prostate cancer. We discuss efforts to achieve tissue-specific gene delivery, focusing on the use of tissue-specific gene promoters. Finally, the present use of gene therapy for prostate cancer is evaluated. The ability to deliver gene-therapy vectors directly to prostate tissue, and to regulate gene expression in a tissue-specific manner, offers promise for the use of gene therapy in prostate cancer.

Automated tumor analysis for molecular profiling in lung cancer

The discovery and clinical application of molecular biomarkers in solid tumors, increasingly relies on nucleic acid extraction from FFPE tissue sections and subsequent molecular profiling. This in turn requires the pathological review of haematoxylin & eosin (H&E) stained slides, to ensure sample quality, tumor DNA sufficiency by visually estimating the percentage tumor nuclei and tumor annotation for manual macrodissection. In this study on NSCLC, we demonstrate considerable variation in tumor nuclei percentage between pathologists, potentially undermining the precision of NSCLC molecular evaluation and emphasising the need for quantitative tumor evaluation. We subsequently describe the development and validation of a system called TissueMark for automated tumor annotation and percentage tumor nuclei measurement in NSCLC using computerized image analysis. Evaluation of 245 NSCLC slides showed precise automated tumor annotation of cases using Tissuemark, strong concordance with manually drawn boundaries and identical EGFR mutational status, following manual macrodissection from the image analysis generated tumor boundaries. Automated analysis of cell counts for % tumor measurements by Tissuemark showed reduced variability and significant correlation (p < 0.001) with benchmark tumor cell counts. This study demonstrates a robust image analysis technology that can facilitate the automated quantitative analysis of tissue samples for molecular profiling in discovery and diagnostics.

Myc-dependent purine biosynthesis affects nucleolar stress and therapy response in prostate cancer

The androgen receptor is a key transcription factor contributing to the development of all stages of prostate cancer (PCa). In addition, other transcription factors have been associated with poor prognosis in PCa, amongst which c-Myc (MYC) is a well-established oncogene in many other cancers. We have previously reported that the AR promotes glycolysis and anabolic metabolism; many of these metabolic pathways are also MYC-regulated in other cancers. In this study, we report that in PCa cells de novo purine biosynthesis and the subsequent conversion to XMP is tightly regulated by MYC and independent of AR activity. We characterized two enzymes, PAICS and IMPDH2, within the pathway as PCa biomarkers in tissue samples and report increased efficacy of established anti-androgens in combination with a clinically approved IMPDH inhibitor, mycophenolic acid (MPA). Treatment with MPA led to a significant reduction in cellular guanosine triphosphate (GTP) levels accompanied by nucleolar stress and p53 stabilization. In conclusion, targeting purine biosynthesis provides an opportunity to perturb PCa metabolism and enhance tumour suppressive stress responses.

The molecular signature of the stroma response in prostate cancer-induced osteoblastic bone metastasis highlights expansion of hematopoietic and prostate epithelial stem cell niches

The reciprocal interaction between cancer cells and the tissue-specific stroma is critical for primary and metastatic tumor growth progression. Prostate cancer cells colonize preferentially bone (osteotropism), where they alter the physiological balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, and elicit prevalently an osteoblastic response (osteoinduction). The molecular cues provided by osteoblasts for the survival and growth of bone metastatic prostate cancer cells are largely unknown. We exploited the sufficient divergence between human and mouse RNA sequences together with redefinition of highly species-specific gene arrays by computer-aided and experimental exclusion of cross-hybridizing oligonucleotide probes. This strategy allowed the dissection of the stroma (mouse) from the cancer cell (human) transcriptome in bone metastasis xenograft models of human osteoinductive prostate cancer cells (VCaP and C4-2B). As a result, we generated the osteoblastic bone metastasis-associated stroma transcriptome (OB-BMST). Subtraction of genes shared by inflammation, wound healing and desmoplastic responses, and by the tissue type-independent stroma responses to a variety of non-osteotropic and osteotropic primary cancers generated a curated gene signature ("Core" OB-BMST) putatively representing the bone marrow/bone-specific stroma response to prostate cancer-induced, osteoblastic bone metastasis. The expression pattern of three representative Core OB-BMST genes (PTN, EPHA3 and FSCN1) seems to confirm the bone specificity of this response. A robust induction of genes involved in osteogenesis and angiogenesis dominates both the OB-BMST and Core OB-BMST. This translates in an amplification of hematopoietic and, remarkably, prostate epithelial stem cell niche components that may function as a self-reinforcing bone metastatic niche providing a growth support specific for osteoinductive prostate cancer cells. The induction of this combinatorial stem cell niche is a novel mechanism that may also explain cancer cell osteotropism and local interference with hematopoiesis (myelophthisis). Accordingly, these stem cell niche components may represent innovative therapeutic targets and/or serum biomarkers in osteoblastic bone metastasis.

HES6 drives a critical AR transcriptional programme to induce castration-resistant prostate cancer through activation of an E2F1-mediated cell cycle network

Castrate-resistant prostate cancer (CRPC) is poorly characterized and heterogeneous and while the androgen receptor (AR) is of singular importance, other factors such as c-Myc and the E2F family also play a role in later stage disease. HES6 is a transcription co-factor associated with stem cell characteristics in neural tissue. Here we show that HES6 is up-regulated in aggressive human prostate cancer and drives castration-resistant tumour growth in the absence of ligand binding by enhancing the transcriptional activity of the AR, which is preferentially directed to a regulatory network enriched for transcription factors such as E2F1. In the clinical setting, we have uncovered a HES6-associated signature that predicts poor outcome in prostate cancer, which can be pharmacologically targeted by inhibition of PLK1 with restoration of sensitivity to castration. We have therefore shown for the first time the critical role of HES6 in the development of CRPC and identified its potential in patient-specific therapeutic strategies.

Molecular subtyping of primary prostate cancer reveals specific and shared target genes of different ETS rearrangements

This work aimed to evaluate whether ETS transcription factors frequently involved in rearrangements in prostate carcinomas (PCa), namely ERG and ETV1, regulate specific or shared target genes. We performed differential expression analysis on nine normal prostate tissues and 50 PCa enriched for different ETS rearrangements using exon-level expression microarrays, followed by in vitro validation using cell line models. We found specific deregulation of 57 genes in ERG-positive PCa and 15 genes in ETV1-positive PCa, whereas deregulation of 27 genes was shared in both tumor subtypes. We further showed that the expression of seven tumor-associated ERG target genes (PLA1A, CACNA1D, ATP8A2, HLA-DMB, PDE3B, TDRD1, and TMBIM1) and two tumor-associated ETV1 target genes (FKBP10 and GLYATL2) was significantly affected by specific ETS silencing in VCaP and LNCaP cell line models, respectively, whereas the expression of three candidate ERG and ETV1 shared targets (GRPR, KCNH8, and TMEM45B) was significantly affected by silencing of either ETS. Interestingly, we demonstrate that the expression of TDRD1, the topmost overexpressed gene of our list of ERG-specific candidate targets, is inversely correlated with the methylation levels of a CpG island found at -66 bp of the transcription start site in PCa and that TDRD1 expression is regulated by direct binding of ERG to the CpG island in VCaP cells. We conclude that ETS transcription factors regulate specific and shared target genes and that TDRD1, FKBP10, and GRPR are promising therapeutic targets and can serve as diagnostic markers for molecular subtypes of PCa harboring specific fusion gene rearrangements.

Androgen receptor driven transcription in molecular apocrine breast cancer is mediated by FoxA1

Breast cancer is a heterogeneous disease and several distinct subtypes exist based on differential gene expression patterns. Molecular apocrine tumours were recently identified as an additional subgroup, characterised as oestrogen receptor negative and androgen receptor positive (ER- AR+), but with an expression profile resembling ER+ luminal breast cancer. One possible explanation for the apparent incongruity is that ER gene expression programmes could be recapitulated by AR. Using a cell line model of ER- AR+ molecular apocrine tumours (termed MDA-MB-453 cells), we map global AR binding events and find a binding profile that is similar to ER binding in breast cancer cells. We find that AR binding is a near-perfect subset of FoxA1 binding regions, a level of concordance never previously seen with a nuclear receptor. AR functionality is dependent on FoxA1, since silencing of FoxA1 inhibits AR binding, expression of the majority of the molecular apocrine gene signature and growth cell growth. These findings show that AR binds and regulates ER cis-regulatory elements in molecular apocrine tumours, resulting in a transcriptional programme reminiscent of ER-mediated transcription in luminal breast cancers.

LYRIC/AEG-1 is targeted to different subcellular compartments by ubiquitinylation and intrinsic nuclear localization signals

PURPOSE: LYRIC/AEG-1 has been reported to influence breast cancer survival and metastases, and its altered expression has been found in a number of cancers. The cellular function of LYRIC/AEG-1 has previously been related to its subcellular distribution in cell lines. LYRIC/AEG-1 contains three uncharacterized nuclear localization signals (NLS), which may regulate its distribution and, ultimately, function in cells.

EXPERIMENTAL DESIGN: Immunohistochemistry of a human prostate tissue microarray composed of 179 prostate cancer and 24 benign samples was used to assess LYRIC/AEG-1 distribution. Green fluorescent protein-NLS fusion proteins and deletion constructs were used to show the ability of LYRIC/AEG-1 NLS to target green fluorescent protein from the cytoplasm to the nucleus. Immunoprecipitation and Western blotting were used to show posttranslational modification of LYRIC/AEG-1 NLS regions.

RESULTS: Using a prostate tissue microarray, significant changes in the distribution of LYRIC/AEG-1 were observed in prostate cancer as an increased cytoplasmic distribution in tumors compared with benign tissue. These differences were most marked in high grade and aggressive prostate cancers and were associated with decreased survival. The COOH-terminal extended NLS-3 (amino acids 546-582) is the predominant regulator of nuclear localization, whereas extended NLS-1 (amino acids 78-130) regulates its nucleolar localization. Within the extended NLS-2 region (amino acids 415-486), LYRIC/AEG-1 can be modified by ubiquitin almost exclusively within the cytoplasm.

CONCLUSIONS: Changes in LYRIC/AEG-1 subcellular distribution can predict Gleason grade and survival. Two lysine-rich regions (NLS-1 and NLS-3) can target LYRIC/AEG-1 to subcellular compartments whereas NLS-2 is modified by ubiquitin in the cytoplasm.

Pathogenesis of prostate cancer and hormone refractory prostate cancer

Prostate cancer is the second most common malignancy in males and the leading cause of cancer death. Prostate cancer is initially androgen dependent and relies upon the androgen receptor (AR) to mediate the effects of androgens. The AR is also the target for therapy using antiandrogens and LHRH analogues. However, all cancers eventually become androgen independent, often referred to as hormone refractory prostate cancer. The processes involved in this transformation are yet to be fully understood but research in this area has discovered numerous potential mechanisms including AR amplification, over-expression or mutation and alterations in the AR signaling pathway. This review of the recent literature examines the current knowledge and developments in the understanding of the molecular biology of prostate cancer and hormone refractory prostate cancer, summarizing the well characterized pathways involved as well as introducing new concepts that may offer future solutions to this difficult problem.

Assessing the in vivo efficacy of biologic antiangiogenic therapies

PURPOSE: To review key clinical issues underlying the assessment of in vivo efficacy when using antiangiogenic therapies for cancer treatment.

METHODS: Literature relevant to use of antiangiogenic therapies in cancer was reviewed, with particular emphasis on the assessment of in vivo efficacy of these agents, as well as additional angiogenic factors that could play a role in escape from angiogenesis inhibition.

RESULTS: In order to grow and metastasize, tumors need to continually acquire new blood supplies; therefore, therapeutic inhibition of angiogenesis has become a component of anticancer treatment for many tumor types. Bevacizumab, a humanized monoclonal antibody directed at vascular endothelial growth factor A (VEGF-A), has shown activity in combination with chemotherapy in metastatic colorectal cancer. Nevertheless, the use of antiangiogenic therapies remains suboptimal; specifically, optimal dose, duration of therapy, and combination of agents remain unknown. Also, at present, it is not possible to determine which patients are most likely to respond to a given form of antiangiogenic therapy. There has been increased recognition of alternative pathways possibly associated with disease progression in patients undergoing antiangiogenic therapy targeted at VEGF-A. Multiligand-targeted antiangiogenic therapies, such as ziv-aflibercept (formerly known as aflibercept, VEGF Trap), are currently undergoing clinical evaluation. Ziv-aflibercept forms monomeric complexes with VEGF-A, VEGF-B, and PlGF, which have a long half-life, allowing optimization of ziv-aflibercept doses and angiogenic blockage.

CONCLUSIONS: Although antiangiogenic therapies have increased treatment options for cancer patients, their use is limited by a lack of established and standardized methodology to evaluate their efficacy in vivo. Circulating endothelial cells, hypertension, and several molecular and imaging-based markers have potential for use as biomarkers in these patients and may better define appropriate patient populations.

The dual EGFR/HER2 inhibitor lapatinib synergistically enhances the antitumor activity of the histone deacetylase inhibitor panobinostat in colorectal cancer models

As key molecules that drive progression and chemoresistance in gastrointestinal cancers, epidermal growth factor receptor (EGFR) and HER2 have become efficacious drug targets in this setting. Lapatinib is an EGFR/HER2 kinase inhibitor suppressing signaling through the RAS/RAF/MEK (MAP/ERK kinase)/MAPK (mitogen-activated protein kinase) and PI3K (phosphoinositide 3-kinase)/AKT pathways. Histone deacetylase inhibitors (HDACi) are a novel class of agents that induce cell cycle arrest and apoptosis following the acetylation of histone and nonhistone proteins modulating gene expression and disrupting HSP90 function inducing the degradation of EGFR-pathway client proteins. This study sought to evaluate the therapeutic potential of combining lapatinib with the HDACi panobinostat in colorectal cancer (CRC) cell lines with varying EGFR/HER2 expression and KRAS/BRAF/PIK3CA mutations. Lapatinib and panobinostat exerted concentration-dependent antiproliferative effects in vitro (panobinostat range 7.2-30 nmol/L; lapatinib range 7.6-25.8 μmol/L). Combined lapatinib and panobinostat treatment interacted synergistically to inhibit the proliferation and colony formation in all CRC cell lines tested. Combination treatment resulted in rapid induction of apoptosis that coincided with increased DNA double-strand breaks, caspase-8 activation, and PARP cleavage. This was paralleled by decreased signaling through both the PI3K and MAPK pathways and increased downregulation of transcriptional targets including NF-κB1, IRAK1, and CCND1. Panobinostat treatment induced downregulation of EGFR, HER2, and HER3 mRNA and protein through transcriptional and posttranslational mechanisms. In the LoVo KRAS mutant CRC xenograft model, the combination showed greater antitumor activity than either agent alone, with no apparent increase in toxicity. Our results offer preclinical rationale warranting further clinical investigation combining HDACi with EGFR and HER2-targeted therapies for CRC treatment.

Prognostic role of lemur tyrosine kinase-3 germline polymorphisms in adjuvant gastric cancer in Japan and the United States

Lemur tyrosine kinase-3 (LMTK3) was recently identified as an estrogen receptor (ER)-α modulator related to endocrine therapy resistance, and its polymorphisms rs9989661 (T>C) T/T genotype and rs8108419 (G>A) G/G or A/G genotype predicted improved outcomes in breast cancer. Because different predominant ER distributions link to breast and gastric cancer and little is known of the prognostic role of LMTK3 in gastric cancer, this study was carried out to clarify the prognostic role of these polymorphisms in gastric cancer. One-hundred and sixty-nine Japanese and 137 U.S. patients with localized gastric adenocarcinoma were enrolled. Genomic DNA was extracted from blood or tissue, and all samples were analyzed by PCR-based direct DNA sequencing. Overall, these polymorphisms were not associated with survival in both cohorts. When gender was considered, in multivariate analysis, harboring rs9989661 T/T genotype was associated with disease-free survival [HR, 4.37; 95% confidence interval (CI), 2.08-9.18; P < 0.0001] and overall survival (OS; HR, 3.69; 95% CI, 1.65-8.24; P = 0.0014) in the Japanese males and time to recurrence (HR, 7.29; 95% CI, 1.07-49.80; P = 0.043) in the U.S. females. Meanwhile, harboring rs8108419 G/G genotype was associated with OS in the Japanese females (HR, 3.04; 95% CI, 1.08-8.56; P = 0.035) and the U.S. males (HR, 3.39; 95% CI, 1.31-8.80; P = 0.012). The prognostic role of these polymorphisms may be negative in gastric cancer. These findings suggest that the estrogen pathway may play a prognostic role in patients with gastric cancer but this may be dependent on the regional differences both in physiology and genetic alterations of gastric cancer.