Marxa L. Figueiredo

Comprehensive evaluation of the role of EZH2 in the growth, invasion, and aggression of a panel of prostate cancer cell lines.

Although most prostate cancers respond well to initial treatments, a fraction of prostate cancers are more aggressive and will recur and metastasize. At that point, there are few treatment options available. Significant efforts have been made to identify biomarkers that will identify these more aggressive cancers to tailor a more vigorous treatment in order to improve outcome. Polycomb Group protein enhancer of zeste 2 (EZH2) was found to be overexpressed in metastatic prostate tumors, and is considered an excellent candidate for such a biomarker. Scattered studies have found that EZH2 overexpression causes neoplastic transformation, invasion, and growth of prostate cells. However, these studies utilized different systems and cell lines, and so are difficult to correlate with one another.

Polycomb group protein enhancer of zeste 2 is an oncogene that promotes the neoplastic transformation of a benign prostatic epithelial cell line

Polycomb group protein enhancer of zeste 2 (EZH2) is a master regulatory protein that plays a critical role in development as part of the polycomb repressive complex 2. Polycomb repressive complex 2 controls numerous cell cycle and regulatory genes through trimethylation of histone 3, which results in chromatin condensation and transcriptional silencing. EZH2 overexpression has been correlated with high incidence of more aggressive, metastatic prostate cancers. Although this correlation means EZH2 could prove valuable as a biomarker in clinical settings, the question remains whether EZH2 is actually responsible for the initiation of these more aggressive tumor types. In this study, EZH2-mediated neoplastic transformation of the normal prostate epithelial cell line benign prostate hyperplasia 1 (BPH1) was confirmed by in vivo tumor growth and in vitro colony formation. Furthermore, EZH2 transformation resulted in increased invasive behavior of BPH1 cells, indicating that EZH2 may be responsible for aggressive behavior in prostate cancers. BPH1 was also transformed with the classic oncogenes myristoylated Akt and activated Ras(V12) to allow phenotype comparisons with the EZH2-transformed cells. This study marks the first demonstration of neoplastic transformation in prostate cells mediated by EZH2 and establishes that EZH2 possesses stronger transforming activity than Akt but weaker activity than activated Ras. (Mol Cancer Res 2009;7(9):1456–65)

Transient receptor potential melastatin type 7 channel is critical for the survival of bone marrow derived mesenchymal stem cells

The transient receptor potential melastatin type 7 channel (TRPM7) is a member of the TRP family of ion channels that is essential for cell proliferation and viability. Mesenchymal stem cells (MSCs) from bone marrow are a potential source for tissue repair due to their ability to differentiate into specialized cells. However, the role of TRPM7 in stem cells is unknown. In this study, we characterized TRPM7 in mouse MSCs using molecular biology, immunocytochemistry, and patch clamp. We also investigated TRPM7 function using a lentiviral vector and specific shRNA to knockdown gene expression. By RT-PCR and immunocytochemistry, we identified TRPM7, but not TRPM6, a close family member with similar function. Electrophysiological recordings during depletion of intracellular Mg(2+) or Mg(2+)-ATP resulted in the development of currents typical for the channel. Furthermore, 2-aminoethoxydiphenyl borate (1 pM-100 microM) inhibited TRPM7 in a concentration-dependent manner. The molecular suppression of TRPM7 significantly decreased MSC proliferation and viability as determined by MTT assay. In addition, TRPM7 gene expression was up-regulated during osteogenesis. These findings demonstrate that TRPM7 is required for MSC survival and perhaps involved in the differentiation process.

PLGA Nanoparticles for Ultrasound-Mediated Gene Delivery to Solid Tumors

This paper focuses on novel approaches in the field of nanotechnology-based carriers utilizing ultrasound stimuli as a means to spatially target gene delivery in vivo, using nanoparticles made with either poly(lactic-co-glycolic acid) (PLGA) or other polymers. We specifically discuss the potential for gene delivery by particles that are echogenic (amenable to destruction by ultrasound) composed either of polymers (PLGA, polystyrene) or other contrast agent materials (Optison, SonoVue microbubbles). The use of ultrasound is an efficient tool to further enhance gene delivery by PLGA or other echogenic particles in vivo. Echogenic PLGA nanoparticles are an attractive strategy for ultrasound-mediated gene delivery since this polymer is currently approved by the US Food and Drug Administration for drug delivery and diagnostics in cancer, cardiovascular disease, and also other applications such as vaccines and tissue engineering. This paper will review recent successes and the potential of applying PLGA nanoparticles for gene delivery, which include (a) echogenic PLGA used with ultrasound to enhance local gene delivery in tumors or muscle and (b) PLGA nanoparticles currently under development, which could benefit in the future from ultrasound-enhanced tumor targeted gene delivery.

Pigment Epithelial-Derived Factor and Melanoma Differentiation Associated Gene-7 Cytokine Gene Therapies Delivered by Adipose-Derived Stromal/Mesenchymal Stem Cells are Effective in Reducing Prostate Cancer Cell Growth

Adipose-derived stromal/mesenchymal stem cells (ASC) have gained interest as promising tools for delivering cancer therapy. Adipose tissue can be obtained readily in amounts sufficient for ASC isolation, which can be expanded rapidly, allowing its use at low passage numbers, and can be transduced by viral and nonviral means. Our goal was to examine the potential of ASC to deliver cytokine gene therapies melanoma differentiation associated gene-7 (MDA-7) or pigment epithelial-derived factor (PEDF) to cancer cells. These novel cytokines are a potent proapoptotic and an antiangiogenesis mediator, respectively, with potential as antitumor agents. Expression of cytokine therapies did not adversely affect ASC biology, and these cells were still able to differentiate and retain normal viability. The ASC cytokine therapies were efficient in reducing tumor cell growth in coculture and also in suppressing in vitro angiogenesis phenotypes. We also observed that ASC retained their innate ability to migrate toward tumor cells in coculture, and this ability could be blocked by inhibition of CXCR4 signaling. The ASC were found to be nontumorigenic in vitro using a soft agar assay, as well as in vivo, utilizing 2 prostate cancer xenograft models. The ASC-MDA7 only reduced tumor growth in the TRAMP-C2-Ras (TC2Ras) prostate cancer model. The ASC-PEDF, however, reduced growth in both the TC2Ras and the PC3 highly aggressive prostate cancer models, and it was able to completely prevent prostate tumor establishment in vivo. In conclusion, ASC expressing PEDF and MDA7 could effectively reduce prostate tumor growth in vivo, suggesting ASC-cytokine therapies might have translational applications, especially the PEDF modality.

Regulation of Ca2+-entry in pancreatic α-cell line by transient receptor potential melastatin 4 plays a vital role in glucagon release

Elevation in the intracellular Ca(2+) concentration stimulates glucagon secretion from pancreatic α-cells. The Transient Receptor Potential Melastatin 4 channel (TRPM4) is critical for Ca(2+) signaling. However, its role in glucagon secreting α-cells has not been investigated. We identified TRPM4 gene expression and protein in the αTC1-6 cell line using RT-PCR and immunocytochemistry. Furthermore, we performed a detailed biophysical characterization of the channel using the patch-clamp technique to confirm that currents typical for TRPM4 were present in αTC1-6 cells. To investigate TRPM4 function, we generated a stable knockdown clone using shRNA and a lentiviral vector. Inhibition of TRPM4 significantly reduced the responses to different agonists during Ca(2+) imaging analysis with Fura-2AM. The reduction in the magnitude of Ca(2+) signals resulted in decreased glucagon secretion. These results suggested that depolarization by TRPM4 may play an important role in controlling glucagon secretion from α-cells and perhaps glucose homeostasis.

Cell cycle regulator cdk2ap1 inhibits prostate cancer cell growth and modifies androgen‐responsive pathway function

We evaluated the effect of expressing the cell cycle regulator cdk2ap1, downregulated in prostate cancer cell lines, in inhibiting prostate cancer cell growth.

Cell-cycle regulators cdk2ap1 and bicalutamide suppress malignant biological interactions between prostate cancer and bone cells.

We examined whether the novel cell‐cycle regulator cdk2‐associated protein 1 (p12cdk2ap1 or cdk2ap1), recently shown to regulate prostate cancer cell cycle and apoptosis, could have the capacity to reduce invasiveness and/or reduce malignant biological interactions between prostate cancer and bone cells. We also examined whether combining two cell‐cycle arrest stimuli, cdk2ap1 plus bicalutamide (or casodex, CDX), could help enhance inhibition of prostate cancer cell phenotypes.

Expression of cell cycle regulator cdk2ap1 suppresses tumor cell phenotype by non-cell-autonomous mechanisms

We evaluated the effect of expressing the cell cycle regulator cdk2ap1 in epithelial or stromal cell compartments to reduce SCC growth in vitro and in vivo. Cell-autonomous and/or non-cell-autonomous expression of cdk2ap1 reduced tumor growth and invasion and altered cell cycle, adhesion, invasion, angiogenesis, and apoptotic gene expression, as assessed by several in vitro phenotype assays, quantitative real-time PCR, and in vivo molecular imaging using a novel three-way xenograft animal model. Our findings suggest that the interactions between cancer cells and fibroblasts that promote abnormal growth can be minimized by expressing cdk2ap1, supporting a novel concept by which tumor/growth suppressor genes can impact tumorigenesis phenotypes from non-cell-autonomous interactions within the tumor microenvironment.

Polymer-peptide delivery platforms: effect of oligopeptide orientation on polymer-based DNA delivery.

The success of nonviral transfection using polymers hinges on efficient nuclear uptake of nucleic acid cargo and overcoming intra- and extracellular barriers. By incorporating PKKKRKV heptapeptide pendent groups as nuclear localization signals (NLS) on a polymer backbone, we demonstrate protein expression levels higher than those obtained from JetPEI and Lipofectamine 2000, the latter being notorious for coupling high transfection efficiency with cytotoxicity. The orientation of the NLS peptide grafts markedly affected transfection performance. Polymers with the sequence attached to the backbone from the valine residue achieved a level of nuclear translocation higher than the levels of those having the NLS groups attached in the opposite orientation. The differences in nuclear localization and DNA complexation strength between the two orientations correlated with a striking difference in protein expression, both in cell culture and in vivo. Polyplexes formed from these comb polymer structures exhibited transfection efficiencies superior to those of Lipofectamine 2000 but with greatly reduced toxicity. Moreover, these novel polymers, when administered by intramuscular ultrasound-mediated delivery, allowed a high level of reporter gene expression in mice, demonstrating their therapeutic promise in vivo.