Treatment with the vascular disruptive agent OXi4503 induces an immediate and widespread epithelial to mesenchymal transition in the surviving tumor

Epithelial to mesenchymal transition (EMT) is considered an important mechanism in tumor resistance to drug treatments; however, in vivo observation of this process has been limited. In this study we demonstrated an immediate and widespread EMT involving all surviving tumor cells following treatment of a mouse model of colorectal liver metastases with the vascular disruptive agent OXi4503. EMT was characterized by significant downregulation of E-cadherin, relocation and nuclear accumulation of b-catenin as well as significant upregulation of ZEB1 and vimentin. Concomitantly, significant temporal upregulation in hypoxia and the pro-angiogenic growth factors hypoxia-inducible factor 1-alpha, hepatocyte growth factor, vascular endothelial growth factor and transforming growth factor-beta were seen within the surviving tumor. The process of EMT was transient and by 5 days after treatment tumor cell reversion to epithelial morphology was evident. This reversal, termed mesenchymal to epithelial transition (MET) is a process implicated in the development of new metastases but has not been observed in vivo histologically. Similar EMT changes were observed in response to other antitumor treatments including chemotherapy, thermal ablation, and antiangiogenic treatments in our mouse colorectal metastasis model and in a murine orthotopic breast cancer model after OXi4503 treatment. These results suggest that EMT may be an early mechanism adopted by tumors in response to injury and hypoxic stress, such that inhibition of EMT in combination with other therapies could play a significant role in future cancer therapy.

Modeling of protein signaling networks in clinical proteomics

Molecular interactions that underlie pathophysiological states are being elucidated using techniques that profile proteomicend points in cellular systems. Within the field of cancer research, protein interaction networks play pivotal roles in the establishment and maintenance of the hallmarks of malignancy, including cell division, invasion, and migration. Multiple complementary tools enable a multifaceted view of how signal protein pathway alterations contribute to pathophysiological states.One pivotal technique is signal pathway profiling of patient tissue specimens. This microanalysis technology provides a proteomic snapshot at one point in time of cells directly procured from the native context of a tumor micro environment. To study the adaptive patterns of signal pathway events over time, before and after experimental therapy, it is necessary to obtain biopsies from patients before, during, and after therapy. A complementary approach is the profiling of cultured cell lines with and without treatment. Cultured cell models provide the opportunity to study short-term signal changes occurring over minutes to hours. Through this type of system, the effects of particular pharmacological agents may be used to test the effects of signal pathway inhibition or activation on multiple endpoints within a pathway. The complexity of the data generated has necessitated the development of mathematical models for optimal interpretation of interrelated signaling pathways. In combination,clinical proteomic biopsy profiling, tissue culture proteomic profiling, and mathematical modeling synergistically enable a deeper understanding of how protein associations lead to disease states and present new insights into the design of therapeutic regimens.

Mathematical Models of Tumor-Immune System Dynamics

"This collection of papers offers a broad synopsis of state-of-the-art mathematical methods used in modeling the interaction between tumors and the immune system. These papers were presented at the four-day workshop on Mathematical Models of Tumor-Immune System Dynamics held in Sydney, Australia from January 7th to January 10th, 2013. The workshop brought together applied mathematicians, biologists, and clinicians actively working in the field of cancer immunology to share their current research and to increase awareness of the innovative mathematical tools that are applicable to the growing field of cancer immunology. Recent progress in cancer immunology and advances in immunotherapy suggest that the immune system plays a fundamental role in host defense against tumors and could be utilized to prevent or cure cancer. Although theoretical and experimental studies of tumor-immune system dynamics have a long history, there are still many unanswered questions about the mechanisms that govern the interaction between the immune system and a growing tumor. The multidimensional nature of these complex interactions requires a cross-disciplinary approach to capture more realistic dynamics of the essential biology. The papers presented in this volume explore these issues and the results will be of interest to graduate students and researchers in a variety of fields within mathematical and biological sciences."--Publisher website

Speckle-type POZ protein mutations interrupt tumor suppressor function of speckle-type POZ protein in prostate cancer by affecting androgen receptor degradation

Large scale exome sequencing studies have revealed regions of the genome, which contribute to the castrate resistant prostate cancer (CRPC) phenotype. [1],[2],[3] Such studies have identified mutations in genes, which may have diagnostic/prognostic potential, or which may be targeted therapeutically. Two of these genes include the androgen receptor (AR) and speckle-type POZ protein (SPOP) genes. However, the findings from these exome sequencing studies can only be translated therapeutically once the functional consequences of these mutations have been determined. Here, we highlight the recent study by An et al. [4] which investigated the functional effects of mutations in the SPOP gene that were identified in the aforementioned exome sequencing studies, particularly in the context of SPOP-mediated degradation of the AR.

Association of CYP1B1 codon 432 mutant allele in head and neck squamous cell cancer is reflected by somatic mutations of p53 in tumor tissue

Tobacco use is causally associated with head and neck squamous cell cancer (HNSCC). Here, we present the results of a case-control study that investigated the effects that the genetic variants of the cytochrome (CYP)1A1, CYP1B1, glutathione-S-transferase (GST)M1, GSTT1, and GSTP1 genes have on modifying the risk of smoking-related HNSCC. Allelisms of the CYP1A1, GSTT1, GSTM1, and GSTT1 genes alone were not associated with an increased risk. CYP1B1 codon 432 polymorphism was found to be a putative susceptibility factor in smoking-related HNSCC. The frequency of CYP1B1 polymorphism was significantly higher (P < 0.001) in the group of smoking cases when compared with smoking controls. Additionally, an odds ratio (OR) of 4.53 (2.62-7.98) was discovered when investigating smoking and nonsmoking cases for the susceptible genotype CYP1B1*2/*2, when compared with the presence of the genotype wild type. In combination with polymorphic variants of the GST genes, a synergistic-effect OR was observed. The calculated OR for the combined genotype CYP1B1*2/*2 and GSTM1*2/*2 was 12.8 (4.09-49.7). The calculated OR for the combined genotype was 13.4 (2.92-97.7) for CYP1B1*2/*2 and GSTT1*2/*2, and 24.1 (9.36-70.5) for the combination of CYP1B1*2/*2 and GSTT1-expressors. The impact of the polymorphic variants of the CYP1B1 gene on HNSCC risk is reflected by the strong association with the frequency of somatic mutations of the p53 gene. Smokers with susceptible genotype CYP1B1*2/*2 were 20 times more likely to show evidence of p53 mutations than were those with CYP1B1 wild type. Combined genotype analysis of CYP1B1 and GSTM1 or GSTT1 revealed interactive effects on the occurrence of p53 gene mutations. The results of the present study indicate that polymorphic variants of CYP1B1 relate significantly to the individual susceptibility of smokers to HNSCC.

Pharmacokinetics and tumor disposition of PEGylated, methotrexate conjugated poly-l-lysine dendrimers

Dendrimers have potential for delivering chemotherapeutic drugs to solid tumours via the enhanced permeation and retention (EPR) effect. The impact of conjugation of hydrophobic anticancer drugs to hydrophilic PEGylated dendrimer surfaces, however, has not been fully investigated. The current study has therefore characterised the effect on dendrimer disposition of conjugating α-carboxyl protected methotrexate (MTX) to a series of PEGylated 3H-labelled poly-L-lysine dendrimers ranging in size from generation 3 (G3) to 5 (G5) in rats. Dendrimers contained 50% surface PEG and 50% surface MTX. Conjugation of MTX generally increased plasma clearance when compared to conjugation with PEG alone. Conversely, increasing generation reduced clearance, increased metabolic stability and reduced renal elimination of the administered radiolabel. For constructs with molecular weights >20 kDa increasing the molecular weight of conjugated PEG also reduced clearance and enhanced metabolic stability but had only a minimal effect on renal elimination. Tissue distribution studies revealed retention of MTX conjugated smaller (G3-G4) PEG570 dendrimers (or their metabolic products) in the kidneys. In contrast, the larger G5 dendrimer was concentrated more in the liver and spleen. The G5 PEG1100 dendrimer was also shown to accumulate in solid Walker 256 and HT1080 tumours and comparative disposition data in both rats (1 to 2% dose/g in tumour) and mice (11% dose/g in tumour) are presented. The results of this study further illustrate the potential utility of biodegradable PEGylated poly-L-lysine dendrimers as long circulating vectors for the delivery and tumour-targeting of hydrophobic drugs.

The ADAMTS1 protease gene is required for mammary tumor growth and metastasis

A disintegrin and metalloprotease with thrombospondin motifs protein 1 (ADAMTS1) is a protease commonly up-regulated in metastatic carcinoma. Its overexpression in cancer cells promotes experimental metastasis, but whether ADAMTS1 is essential for metastatic progression is unknown. To address this question, we investigated mammary cancer progression and spontaneous metastasis in the MMTV-PyMT mouse mammary tumor model in Adamts1 knockout mice. Adamts1−/−/PyMT mice displayed significantly reduced mammary tumor and lung metastatic tumor burden and increased survival, compared with their wild-type and heterozygous littermates. Histological examination revealed an increased proportion of tumors with ductal carcinoma in situ and a lower proportion of high-grade invasive tumors in Adamts1−/−/PyMT mice, compared with Adamts1+/+/PyMT mice. Increased apoptosis with unaltered proliferation and vascular density in the Adamts1−/−/PyMT tumors suggested that reduced cell survival accounts for the lower tumor burden in ADAMTS1-deficient mice. Furthermore, Adamts1−/− tumor stroma had significantly lesser amounts of proteolytically cleaved versican and increased numbers of CD45+ leukocytes. Characterization of immune cell gene expression indicated that cytotoxic cell activation was increased in Adamts1−/− tumors, compared with Adamts1+/+ tumors. This finding is supported by significantly elevated IL-12+ cell numbers in Adamts1−/− tumors. Thus, in vivo ADAMTS1 may promote mammary tumor growth and progression to metastasis in the PyMT model and is a potential therapeutic target to prevent metastatic breast cancer.

NADPH oxidases as regulators of tumor angiogenesis : current and emerging concepts

Significance Reactive oxygen species (ROS) such as superoxide, hydrogen peroxide, and peroxynitrite are generated ubiquitously by all mammalian cells and have been understood for many decades as inflicting cell damage and as causing cancer by oxidation and nitration of macromolecules, including DNA, RNA, proteins, and lipids. Recent Advances A current concept suggests that ROS can also promote cell signaling pathways triggered by growth factors and transcription factors that ultimately regulate cell proliferation, differentiation, and apoptosis, all of which are important hallmarks of tumor cell proliferation and angiogenesis. Moreover, an emerging concept indicates that ROS regulate the functions of immune cells that infiltrate the tumor environment and stimulate angiogenesis, such as macrophages and specific regulatory T cells. Critical Issues In this article, we highlight that the NADPH oxidase family of ROS-generating enzymes are the key sources of ROS and, thus, play an important role in redox signaling within tumor, endothelial, and immune cells thereby promoting tumor angiogenesis. Future Directions Knowledge of these intricate ROS signaling pathways and identification of the culprit NADPH oxidases is likely to reveal novel therapeutic opportunities to prevent angiogenesis that occurs during cancer and which is responsible for the revascularization after current antiangiogenic treatment.

Tumor-suppressor gene promoter hypermethylation in saliva of head and neck cancer patients

Head and neck squamous cell carcinoma (HNSCC) accounts for a bulk of the oral and laryngeal cancers, the majority (70%) of which are associated with smoking and excessive drinking, major known risk factors for the development of HNSCC. In contrast to reports that suggest an inverse relationship between smoking and global DNA CpG methylation, hypermethylation of promoters of a number of genes was detected in saliva collected from patients with HNSCC. Using a sensitive methylation-specific polymerase chain reaction (MSP) assay to determine specific methylation events in the promoters of RASSF1A, DAPK1, and p16 genes, we demonstrate that we can detect tumor presence with an overall accuracy of 81% in the DNA isolated from saliva of patients with HNSCC (n = 143) when compared with the DNA isolated from the saliva of healthy nonsmoker controls (n = 31). The specificity for this MSP panel was 87% and the sensitivity was 80%(with a Fisher exact test P < .0001). In addition, the test panel performed extremely well in the detection of the early stages of HNSCCs, with a sensitivity of 94% and a specificity of 87%, and a high. concordance value of 0.8, indicating an excellent overall agreement between the presence of HNSCC and a positive MSP panel result. In conclusion, we demonstrate that the promoter methylation of RASSF1A, DAPK1, and p16 MSP panel is useful in detecting hypermethylation events in a noninvasive manner in patients with HNSCC.

Tissue-engineered 3D tumor angiogenesis models : potential technologies for anti-cancer drug discovery

Angiogenesis is indispensable for solid tumor expansion, and thus it has become a major target of cancer research and anti-cancer therapies. Deciphering the arcane actions of various cell populations during tumor angiogenesis requires sophisticated research models, which could capture the dynamics and complexity of the process. There is a continuous need for improvement of existing research models, which engages interdisciplinary approaches of tissue engineering with life sciences. Tireless efforts to develop a new model to study tumor angiogenesis result in innovative solutions, which bring us one step closer to decipher the dubious nature of cancer. This review aims to overview the recent developments, current limitations and future challenges in three-dimensional tissue-engineered models for the study of tumor angiogenesis and for the purpose of elucidating novel targets aimed at anti-cancer drug discovery.

The effect of HLA-DR on susceptibility to rheumatoid arthritis is influenced by the associated lymphotoxin α-tumor necrosis factor haplotype

Objective. HLA-DRB1, a major genetic determinant of susceptibility to rheumatoid arthritis (RA), is located within 1,000 kb of the gene encoding tumor necrosis factor (TNF). Because certain HLA-DRB1*04 subtypes increase susceptibility to RA, investigation of the role of the TNF gene is complicated by linkage disequilibrium (LD) between TNF and DRB1 alleles. By adequately controlling for this LD, we aimed to investigate the presence of additional major histocompatibility complex (MHC) susceptibility genes. Methods. We identified 274 HLA-DRB1*04-positive cases of RA and 271 HLA-DRB1*04-positive population controls. Each subject was typed for 6 single-nucleotide polymorphisms within a 4.5-kb region encompassing TNF and lymphotoxin a (LTA). LTA-TNF haplotypes in these unrelated individuals were determined using a combination of family data and the PHASE software program. Results. Significant differences in LTA-TNF haplotype frequencies were observed between different subtypes of HLA-DRB1*04. The LTA-TNF haplotypes observed were very restricted, with only 4 haplotypes constituting 81% of all haplotypes present. Among individuals carrying DRB1*0401, the LTA-TNF 2 haplotype was significantly underrepresented in cases compared with controls (odds ratio 0.5 [95% confidence interval 0.3-0.8], P = 0.007), while in those with DRB1*0404, the opposite effect was observed (P = 0.007). Conclusion. These findings suggest that the MHC contains genetic elements outside the LTA-TNF region that modify the effect of HLA-DRB1 on susceptibility to RA.

Brief report: Intestinal dysbiosis in ankylosing spondylitis

Objective Ankylosing spondylitis (AS) is a common, highly heritable immune-mediated arthropathy that occurs in genetically susceptible individuals exposed to an unknown but likely ubiquitous environmental trigger. There is a close relationship between the gut and spondyloarthritis, as exemplified in patients with reactive arthritis, in whom a typically self-limiting arthropathy follows either a gastrointestinal or urogenital infection. Microbial involvement in AS has been suggested; however, no definitive link has been established. The aim of this study was to determine whether the gut in patients with AS carries a distinct microbial signature compared with that in the gut of healthy control subjects. Methods Microbial profiles for terminal ileum biopsy specimens obtained from patients with recent-onset tumor necrosis factor antagonist-naive AS and from healthy control subjects were generated using culture-independent 16S ribosomal RNA gene sequencing and analysis techniques. Results Our results showed that the terminal ileum microbial communities in patients with AS differ significantly (P < 0.001) from those in healthy control subjects, driven by a higher abundance of 5 families of bacteria (Lachnospiraceae [P = 0.001], Ruminococcaceae [P = 0.012], Rikenellaceae [P = 0.004], Porphyromonadaceae [P = 0.001], and Bacteroidaceae [P = 0.001]) and a decrease in the abundance of 2 families of bacteria (Veillonellaceae [P = 0.01] and Prevotellaceae [P = 0.004]). Conclusion We show evidence for a discrete microbial signature in the terminal ileum of patients with AS compared with healthy control subjects. The microbial composition was demonstrated to correlate with disease status, and greater differences were observed between disease groups than within disease groups. These results are consistent with the hypothesis that genes associated with AS act, at least in part, through effects on the gut microbiome.

Utility of temporal artery biopsy samples for genome-wide analysis of giant cell arteritis

Giant Cell Arteritis (GCA) is the most common vasculitis affecting the elderly. Archived formalin-fixed paraffin-embedded (FFPE) temporal artery biopsy (TAB) specimens potentially represent a valuable resource for large-scale genetic analysis of this disease. FFPE TAB samples were obtained from 12 patients with GCA. Extracted TAB DNA was assessed by real time PCR before restoration using the Illumina HD FFPE Restore Kit. Paired FFPE-blood samples were genotyped on the Illumina OmniExpress FFPE microarray. The FFPE samples that passed stringent quality control measures had a mean genotyping success of >97%. When compared with their matching peripheral blood DNA, the mean discordant heterozygote and homozygote single nucleotide polymorphisms calls were 0.0028 and 0.0003, respectively, which is within the accepted tolerance of reproducibility. This work demonstrates that it is possible to successfully obtain high-quality microarray-based genotypes FFPE TAB samples and that this data is similar to that obtained from peripheral blood.