Progression elevated gene-3, PEG-3, induces genomic instability in rodent and human tumor cells

Genomic instability is a fundamental component of cancer progression. Subtraction hybridization identified a novel rodent gene, progression elevated gene-3 (PEG-3) whose expression directly correlates with cancer aggressiveness and progression. Moreover, ectopic expression of PEG-3 in rodent or human tumor cells produces an aggressive transformed phenotype. We demonstrate that PEG-3 expression in rodent tumor cells correlates directly with genomic instability as characterized by alterations in chromosome composition and structure. Additionally, elevated endogenous or ectopic expression of PEG-3 in rodent and human tumor cells, respectively, enhances gene amplification, as monitored by resistance to methothrexate (MTX) and amplification of the dihydrofolate reductase (dhfr) gene. Stable expression of PEG-3 in normal cloned rat embryo fibroblast (CREF) cells marginally elevates MTX resistance, but morphology remains unaltered and anchorage independence is not induced, suggesting that these phenotypes are separable in immortal cells and gene amplification may precede the acquisition of morphological and oncogenic transformation. The present studies document that stable, inducible, and transient expression of PEG-3 in cancer cells augments genomic instability. In these contexts, one mechanism by which PEG-3 influences cancer progression may be by preferentially facilitating the development of genomic changes in evolving cancer cells.     

Unique Conditionally Replication Competent Bipartite Adenoviruses—Cancer Terminator Viruses (CTV): Efficacious Reagents for Cancer Gene Therapy

The frequent resistance of aggressive cancers to currently available therapies, such as radiotherapy and chemotherapy, mandates development of targeted, non-toxic and more efficacious treatment protocols. Conditionally replication competent adenoviruses (CRCAs) that induce oncolysis by cancer-specific replication are currently being evaluated in clinical trials. However, a single modality approach may not be sufficient to completely eradicate cancer in a patient, because most cancers arise from abnormalities in multiple genetic and signal transduction pathways. The promoter region of rodent progression elevated gene-3 (PEG-3), cloned and characterized in our laboratory, embodies the unique property of increased activity in a broad range of tumor cells, both rodent and human, when compared to normal counterparts. Bipartite adenoviruses were engineered to express the E1A gene, necessary for viral replication, under control of the PEG-3 promoter (PEG-Prom) and simultaneously express a second transgene in the E3 region that encodes an apoptosis-inducing and immunomodulatory cytokine, either immune interferon (IFN-?) or melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24). These conditionally replication competent bipartite adenoviruses, referred to as cancer terminator viruses (CTVs), facilitated cancer-selective adenovirus replication, robust transgene expression and apoptosis induction with complete eradication of both primary and distant (metastatic) human cancers xenotransplanted in athymic nude mice. These findings suggest that CTVs might prove efficacious for the therapy of primary and advanced neoplastic diseases.     

HMGA proteins and their genes as a potential neoplastic biomarkers

HMGA proteins and their genes are described in this article. HMGA proteins reveal ability to bind DNA in AT-rich regions, which are characteristic for gene promoter sequences. This interaction lead to gene silencing or their overexpression. In normal tissue HMGA proteins level is low or even undetectable. During embriogenesis their level is increasing. High HMGA proteins level is characteristic for tumor phenotype of spontaneous and experimental malignant neoplasms. High HMGA proteins expression correlate with bad prognostic factors and with metastases formation. HMGA genes expression can be used as a marker of tumor progression. Present studies connected with tumor gene therapy based on HMGA proteins sythesis inhibition by the use of viral vectors containing gene encoding these proteins in antisence orientation, as well as a new potential anticancer drugs acting as crosslinkers between DNA and HMGA proteins suggest their usefulness as a targets in cancer therapy.     

The multiple promoter methylation profile of PR gene and ERalpha gene in tumor cell lines

The changes of methylation status of various gene promoters are a common feature of malignant cells and these changes can occur early in the progression process. Therefore, abnormal methylation can be used as cancer marker. Such studies will first require the development of a panel of methylated markers that are methylated in cancer tissues but unmethylated in normal tissues or methylated status is different between cancer tissues and normal tissues. By using methylation-specific PCR (MSP) assay method, we observed alterations in DNA methylation at the double promoter regions of the progesterone receptor (PR) gene and estrogen receptor (ERalpha) gene in various tumor cell lines. Compared with normal white blood cell, the methylation status of PRA promoter in various cancer cell lines changed from unmethylation pattern to methylation pattern. That of PRB promoter changed from both unmethylated and methylated alleles to only methylated allele. The methylation status of ERalpha-A and ERalpha-B promoter in various cancer cell lines are cell -specific. This study indicates that PR promoter methylation may be a molecular marker in various cancer detections. And the methylation status of ERalpha-A and ERalpha-B is cell-specific.     

Tissue of origin determines cancer-associated CpG island promoter hypermethylation patterns

Background

Aberrant CpG island promoter DNA hypermethylation is frequently observed in cancer and is believed to contribute to tumor progression by silencing the expression of tumor suppressor genes. Previously, we observed that promoter hypermethylation in breast cancer reflects cell lineage rather than tumor progression and occurs at genes that are already repressed in a lineage-specific manner. To investigate the generality of our observation we analyzed the methylation profiles of 1,154 cancers from 7 different tissue types.

Results

We find that 1,009 genes are prone to hypermethylation in these 7 types of cancer. Nearly half of these genes varied in their susceptibility to hypermethylation between different cancer types. We show that the expression status of hypermethylation prone genes in the originator tissue determines their propensity to become hypermethylated in cancer; specifically, genes that are normally repressed in a tissue are prone to hypermethylation in cancers derived from that tissue. We also show that the promoter regions of hypermethylation-prone genes are depleted of repetitive elements and that DNA sequence around the same promoters is evolutionarily conserved. We propose that these two characteristics reflect tissue-specific gene promoter architecture regulating the expression of these hypermethylation prone genes in normal tissues.

Conclusions

As aberrantly hypermethylated genes are already repressed in pre-cancerous tissue, we suggest that their hypermethylation does not directly contribute to cancer development via silencing. Instead aberrant hypermethylation reflects developmental history and the perturbation of epigenetic mechanisms maintaining these repressed promoters in a hypomethylated state in normal cells.     

Novel Therapeutic Approaches for Various Cancer Types Using a Modified Sleeping Beauty-Based Gene Delivery System

Successful gene therapy largely depends on the selective introduction of therapeutic genes into the appropriate target cancer cells. One of the most effective and promising approaches for targeting tumor tissue during gene delivery is the use of viral vectors, which allow for high efficiency gene delivery. However, the use of viral vectors is not without risks and safety concerns, such as toxicities, a host immune response towards the viral antigens or potential viral recombination into the host''s chromosome; these risks limit the clinical application of viral vectors. The Sleeping Beauty (SB) transposon-based system is an attractive, non-viral alternative to viral delivery systems. SB may be less immunogenic than the viral vector system due to its lack of viral sequences. The SB-based gene delivery system can stably integrate into the host cell genome to produce the therapeutic gene product over the lifetime of a cell. However, when compared to viral vectors, the non-viral SB-based gene delivery system still has limited therapeutic efficacy due to the lack of long-lasting gene expression potential and tumor cell specific gene transfer ability. These limitations could be overcome by modifying the SB system through the introduction of the hTERT promoter and the SV40 enhancer. In this study, a modified SB delivery system, under control of the hTERT promoter in conjunction with the SV40 enhancer, was able to successfully transfer the suicide gene (HSV-TK) into multiple types of cancer cells. The modified SB transfected cancer cells exhibited a significantly increased cancer cell specific death rate. These data suggest that our modified SB-based gene delivery system can be used as a safe and efficient tool for cancer cell specific therapeutic gene transfer and stable long-term expression.     

Heparanase enhances syndecan-1 shedding: a novel mechanism for stimulation of tumor growth and metastasis

When shed from the cell surface, the heparan sulfate proteoglycan syndecan-1 can facilitate the growth, angiogenesis, and metastasis of tumors. Here we report that tumor cell expression of heparanase, an enzyme known to be a potent promoter of tumor progression and metastasis, regulates both the level and location of syndecan-1 within the tumor microenvironment by enhancing its synthesis and subsequent shedding from the tumor cell surface. Heparanase regulation of syndecan-1 is detected in both human myeloma and breast cancer cell lines. This regulation requires the presence of active enzyme, because mutated forms of heparanase lacking heparan sulfate-degrading activity failed to influence syndecan-1 expression or shedding. Removal of heparan sulfate from the cell surface using bacterial heparitinase dramatically accelerated syndecan-1 shedding, suggesting that the effects of heparanase on syndecan-1 expression by tumor cells may be due, at least in part, to enzymatic removal or reduction in the size of heparan sulfate chains. Animals bearing tumors formed from cells expressing high levels of heparanase or animals transgenic for heparanase expression exhibited elevated levels of serum syndecan-1 as compared with controls, indicating that heparanase regulation of syndecan-1 expression and shedding can occur in vivo and impact cancer progression and perhaps other pathological states. These results reveal a new mechanism by which heparanase promotes an aggressive tumor phenotype and suggests that heparanase and syndecan-1 act synergistically to fine tune the tumor microenvironment and ensure robust tumor growth.     

The 3D tissue microenvironment modulates DNA methylation and E-cadherin expression in squamous cell carcinoma

The microenvironment plays a significant role in human cancer progression. However, the role of the tumor microenvironment in the epigenetic control of genes critical to cancer progression remains unclear. As transient E-cadherin expression is central to many stages of neoplasia and is sensitive to regulation by the microenvironment, we have studied if microenvironmental control of E-cadherin expression is linked to transient epigenetic regulation of its promoter, contributing to the unstable and reversible expression of E-cadherin seen during tumor progression. We used 3D, bioengineered human tissue constructs that mimic the complexity of their in vivo counterparts, to show that the tumor microenvironment can direct the re-expression of E-cadherin through the reversal of methylation-mediated silencing of its promoter. This loss of DNA methylation results from the induction of homotypic cell-cell interactions as cells undergo tissue organization. E-cadherin re-expression is associated with multiple epigenetic changes including altered methylation of a small number of CpGs, specific histone modifications, and control of miR-148a expression. These epigenetic changes may drive the plasticity of E-cadherin-mediated adhesion in different tissue microenvironments during tumor cell invasion and metastasis. Thus, we suggest that epigenetic regulation is a mechanism through which tumor cell colonization of metastatic sites occurs as E-cadherin-expressing cells arise from E-cadherin-deficient cells.     

Effect of magnetic fields on tumor growth and viability

Breast cancer is the most common nonskin cancer and is the second leading cause of cancer-related deaths in women. Most methods of intervention involve combinations of surgery, chemotherapy, and ionizing radiation. Both chemotherapy and ionizing radiation can be effective against many types of cancer, but they also harm normal tissues. The use of nonionizing, magnetic fields has shown early promise in a number of in vitro and animal studies. Our study tested the effect of varying durations of magnetic exposure on tumor growth and viability in mice injected with breast cancer cells. Cancer cells were labeled through stable expression of firefly luciferase for monitoring of tumor growth and progression by using an in vivo imaging system. We hypothesized that magnetic field exposure would influence tumor growth and progression. Our results showed that exposure of the mice to magnetic fields for 360 min daily for as long as 4 wk suppressed tumor growth. Our study is unique in that it uses an in vivo imaging system to monitor the growth and progression of tumors in real time in individual mice. Our findings support further exploration of the potential of magnetic fields in cancer therapeutics, either as adjunct or primary therapy.     

Assessing responses to cancer therapy using molecular imaging

Tumor responses to therapy in the clinic are still evaluated primarily from non-invasive imaging measurements of reductions in tumor size. This approach, however, lacks sensitivity and can only give a delayed indication of a positive response to treatment. Major advances in our understanding of the molecular mechanisms responsible for cancer, combined with new targeted clinical imaging technologies designed to detect the molecular correlates of disease progression and response to treatment, are set to revolutionize our approach to the detection and treatment of the disease. We describe here the imaging technologies available to image tumor cell proliferation and migration, metabolism, receptor and gene expression, apoptosis and tumor angiogenesis and vascular function, and show how measurements of these parameters can be used to give early indications of positive responses to treatment or to detect drug resistance and/or disease recurrence. Special emphasis has been placed on those applications that are already used in the clinic and those that are likely to translate into clinical application in the near future or whose use in preclinical studies is likely to facilitate translation of new treatments into the clinic.