Therapeutic Target In Cancer Cells Research Articles

Microsomal prostaglandin E synthase 1 determines tumor growth in vivo of prostate and lung cancer cells

There is strong evidence for a role of prostaglandin E(2) (PGE(2)) in cancer cell proliferation and tumor development. In PGE(2) biosynthesis, cyclooxygenases (COX-1/COX-2) convert arachidonic acid to PGH(2), which can be isomerized to PGE(2) by microsomal PGE-synthase-1 (MPGES-1). The human prostate cancer cell line DU145 expressed high amounts of MPGES-1 in a constitutive manner. MPGES-1 expression also was detectable in human prostate cancer tissues, where it appeared more abundant compared with benign hyperplasia. By using shRNA, we established stable and practically complete knockdown of MPGES-1, both in DU145 cells with high constitutive expression and in the non-small cell lung cancer cell line A549, where MPGES-1 is inducible. For microsomes prepared from knockdown clones, conversion of PGH(2) to PGE(2) was reduced by 85-90%. This resulted in clear phenotypic changes: MPGES-1 knockdown conferred decreased clonogenic capacity and slower growth of xenograft tumors (with disintegrated tissue structure) in nude mice. For DU145 cells, MPGES-1 knockdown gave increased apoptosis in response to genotoxic stress (adriamycin), which could be rescued by exogenous PGE(2). The results suggest that MPGES-1 is an alternative therapeutic target in cancer cells expressing this enzyme.

Melatonin modulates microfilament phenotypes in epithelial cells: implications for adhesion and inhibition of cancer cell migration.

Cell migration and adhesion are cytoskeleton- dependent functions that play a key role in epithelial physiology. Specialized epithelial cells in water transport have specific microfilament rearrangements that make these cells adopt a polyhedral shape, forming a sealed monolayer which functions as permeability barrier. Also, specific polarized microfilament phenotypes are formed at the front and the rear of migratory epithelial cells. In pathological processes such as cancer, increased migration occurs in invasive cells driven by the formation of polarized and differential microfilament phenotypes. Melatonin, the main product secreted by the pineal gland during dark phase of the photoperiod, acts as a cytoskeletal modulator in normal and cancer cells. In this paper we will summarize evidence supporting that melatonin acts as a microfilament modulator in epithelial MDCK cells, and we will describe its effects on cytoskeleton organization involved in the mechanism by which melatonin synchronizes water transport. In addition, we will review recent data that indicate that melatonin is able to switch microfilament phenotypes in MCF-7 human mammary cancer cells, from invasive migratory cells to dormant microfilament phenotypes that occur in non- migratory cells. Moreover, we will discuss the implications of the cytoskeleton as therapeutic target for cancer cells.

Histone Deacetylase Inhibition with LBH589 Inhibits the Rapamycin Insensitive Rictor-mTOR (mTORC2) Complex and Translation Initiation Factor eIF4E Activation in Diffuse Large B-Cell Lymphoma

Diffuse large B cell lymphoma (DLBCL) is an aggressive form of non-Hodgkin lymphoma and the treatment of these patients has improved with rituximab-based chemo-immunotherapy. However, approximately 40% of patients die of their disease and new agents with novel mechanisms of action are needed for this disease. The mammalian target of rapamycin (mTOR) has emerged as an important therapeutic target in cancer cells and rapamycin and its derivatives, which specifically inhibit mTOR, are now being actively evaluated. Recent clinical trials of mTOR inhibitors have demonstrated that a fraction of patients (35%) with relapsed DLBCL respond to single-agent temsirolimus (Smith S et al J Clin Oncol 26; May 20 Supplement abstract 8514) or everolimus (Reeder et al 2007 ASH Annual Meeting Abstracts 110 (11) abstract 121) in relapsed DLBCL, however most patients are resistant to this therapy. The mechanism of this resistance remains a subject of major therapeutic significance. Herein, we report that cells from DLBCL lines (DHL-6, Ly7 and Ly3) and primary tissues from DLBCL patients (n=10) display persistent activation of the mTOR pathway as determined by phosphorylation of mTOR targets S6 ribosomal protein (S6rp) and 4E-binding protein 1 (4E-BP1). Treatment of human DLBCL cells with various doses of rapamycin predictably demonstrated a decrease in proliferation but less than 10% reduction in overall cell survival. However, rapamycin suppressed the phosphorylation of S6rp and 4E-BP1, indicating an inhibition of raptor-mTOR (mTORC1) signaling. Paradoxically, rapamycin also concurrently increased, through a negative feedback mechanism, the phosphorylation of Akt that may contribute to drug resistance. Interestingly we found that rapamycin treatment also increased the phosphorylation of eIF4E, a survival protein downstream of mTOR, which may also be responsible for resistance of rapamycin along with Akt. To determine whether the observations found in vitro are clinically relevant, we obtained peripheral blood samples from patients with aggressive lymphoma treated with the rapamycin derivative everolimus. The levels of phosphorylated Akt and eIF4E were increased in 3 of 3 patient samples at 48 hrs and after 1 cycle compared to untreated control. Our observations therefore suggest that rapamycin derivatives potently activate Akt and eIF4E activity via activation of the mTORC2 assembly, in addition to its well-characterized ability to suppress the mTORC1 pathway. Treatment of DLBCL cells with histone deacetylase (HDAC) inhibitor LBH589 induced growth inhibition of DLBCL cells at nanomolar concentrations in a dose dependent manner in association with hyperacetylation of histones H3 and H4. LBH alone was able to inhibit the phosphorylation of S6rp and 4E-BP1, while combined treatment with rapamycin inhibited the phosphorylation of S6rp and 4EBP1 to a greater extent than either agent alone. Surprisingly LBH inhibited constitutive as well as rapamycin-induced activation of Akt and eIF4E in a time and dose dependent manner. Our co-immunoprecipitation data suggest that LBH alone was able to alter the level of intact mTORC2 by reducing the amounts of rictor bound to mTOR, which is further decreased when combined with rapamycin. In support of this model, rapamycin combined with LBH exhibited enhanced synergistic inhibitory effects on survival and proliferation of DLBCL cells. The mTOR pathway is also considered to control the translation of specific mRNA species, some of which are involved in cell cycle control and angiogenesis (e.g. cyclin D1, c-Myc and HIF-1a). LBH alone down-regulated expression of c-Myc and HIF-1a, while have no effect on cyclin D1 expression. Combination of LBH with rapamycin further decreased expression of c-Myc and HIF-1a. Overall these results indicate that pharmacological inhibition of the mTOR pathway by rapamycin and LBH interferes with essential survival and proliferating pathways in DLBCL cells. We propose that the rapamycin-induced functional blockade of Akt and eIF4E is inhibited by LBH, and the combination increased the anti tumor activity of rapamycin. In summary, our data provide a mechanistic basis for enhancing mTORtargeted cancer therapy by combining an mTOR inhibitor with a HDAC inhibitor such as LBH. A phase I/II trial of this combination is planned.

The Transforming Acidic Coiled Coil 3 Protein Is Essential for Spindle-dependent Chromosome Alignment and Mitotic Survival

Cancer-associated centrosomal transforming acidic coiled coil (TACC) proteins are involved in mitotic spindle function. By employing gene targeting, we have recently described a nonredundant and essential role of TACC3 in regulating cell proliferation. In this study, we used an inducible RNA interference approach to characterize the molecular function of TACC3 and its role in mitotic progression and cell survival. Our data demonstrate that a TACC3 knockdown arrests G(1) checkpoint-compromised HeLa cells prior to anaphase with aberrant spindle morphology and severely misaligned chromosomes. Interestingly, TACC3-depleted cells fail to accumulate the mitotic kinase Aurora B and the checkpoint protein BubR1 to normal levels at kinetochores. Moreover, localization of the structural protein Ndc80 at outer kinetochores is reduced, indicating a defective kinetochore-microtubule attachment in TACC3-deficient cells. As a consequence of prolonged TACC3 depletion, cells undergo caspase-dependent cell death that relies on a spindle checkpoint-dependent mitotic arrest. TACC3 knockdown cells that escape from this arrest by mitotic slippage become highly polyploid and accumulate supernumerary centrosomes. Similarly, deficiency of the post-mitotic cell cycle inhibitor p21(WAF) exacerbates the effects of TACC3 depletion. Our findings therefore point to an essential role of TACC3 in spindle assembly and cellular survival and identify TACC3 as a potential therapeutic target in cancer cells.

Isolation of Human Prostatic Epithelial Plasma Membranes for Proteomics Using Mirror Image Tissue Banking of Radical Prostatectomy Specimens

To isolate human prostatic epithelial plasma membranes for the identification of cell surface proteins in the therapeutic targeting of cancer cells while permitting the retrieval of banked samples for clinical purposes. Radical prostatectomies from 84 patients (median, 61 years; prostate-specific antigen, 5.9; 66% nonpalpable) were processed with alternate, mirror image slices submitted for histology and tissue banking. Benign and malignant foci were macrodissected from the banked sections using the pathologically mapped, mirror image histology sections as a guide. Epithelial plasma membranes were isolated using novel immunomagnetic purification and their purity was assessed. Tissue homogenates were probed by Western blot for malignant (AMACR) and benign (p63) markers to test the accuracy of this protocol. Selected banked tissue slices were retrieved, thawed, and compared pathologically to their corresponding routinely processed alternate slices. Plasma membrane preparations showed the enrichment of epithelial plasma membrane markers (prostate-specific membrane antigen and epithelial-specific antigen) with minimal marker expression from nonepithelial cells or intracellular organelles. Cancer homogenates showed up-regulated AMACR and down-regulated p63, whereas benign homogenates showed up-regulated p63 and down-regulated AMACR. There was 30% benign (p63+) contamination in cancer slices and <6% cancer (AMACR+) contamination in benign slices. Retrieved tissues showed the retention of immunoreactivity while their histology was always adequate for diagnosis. We have successfully isolated purified epithelial plasma membranes from benign and malignant human prostates and provided validation data for the accuracy of our protocol in a prostate-specific antigen-screened cohort. Our method also enabled the retrieval of banked tissues for clinical purposes with the retention of good histologic and immunohistochemical quality.

Induction of Nitric Oxide Synthase-Dependent Telomere Shortening after Functional Inhibition of Hsp90 in Human Tumor Cells

In most cancer cells, the lengths of telomeres, the functional DNA-protein complexes located at chromosome ends, are maintained by the ribonucleoprotein telomerase. Hsp90 facilitates the assembly of telomerase and remains associated with the functional complex, implying a direct involvement of Hsp90 in telomere length regulation. In an effort to elucidate the effects of Hsp90 inhibition on function and viability of human prostate cancer cells, both pharmacological (radicicol) and genetic (small interfering RNA) approaches were utilized to target Hsp90. Depletion of functional Hsp90 caused dramatic telomere shortening followed by apoptosis. Of particular significance, these cells exhibit a high level of nitric oxide synthase (NOS)-dependent free radical production, and simultaneous treatment of cells with the NOS inhibitor L-NAME resulted in telomere elongation and prevention of apoptosis. In addition, we observe significant DNA damage assessed by telomere dysfunction, although in the absence of a classical DNA damage response. Overall, our data suggest a novel mechanism whereby inhibition of Hsp90 disrupts free radical homeostasis and contributes directly to telomere erosion, further implicating Hsp90 as a potential therapeutic target for cancer cells.

Evolving Molecular-Based Targeted Therapy for Cancer: An Exciting Field

The development of molecular biology tools has led to the identification of numerous therapeutic targets in cancer cells. In this article, we discuss the rational and clinical data supporting the use of agents that selectively target epidermal and vascular endothelial growth factors in selected cancers.

Mitogen-activated protein kinase antisense oligonucleotide inhibits the growth of human lung cancer cells.

Mitogen-activated protein kinase (MAPK) pathway is proposed to be a therapeutic target for cancer cells. In order to find the potential therapeutic usefulness of MAPK for cancer cells, the effect of EAS1, an antisense oligonucleotide for an MAPK, on cancer-cell-growth were investigated in vitro. EAS1 effectively inhibited the growth of several human lung cancer cell lines such as PC-14 cells upon exposure to 10-0-10-1 microM of EAS1 determined dye-formation (MTT) assay. The ED50 values were comparable to those obtained for the inhibition of MAPK activity, DNA synthesis. EAS1 arrested the PC-14 cells at the G2/M phase of cell cycle followed by apoptosis in a dose-dependent manner. In order to determine the factors which influence the cellular sensitivity against MAPK inhibition, the effect of EAS1 on H-ras-transformed murine fibroblast cells were compared with that on parental cells. The NIH3T3 cells transformed by the H-ras gene (PT22-3) showed higher sensitivity against the effects of EAS1. Because MAPK activity was activated by H-ras gene transfection in PT22-3, the status of the MAPK cascade in cells was the determining factor for the efficacy of EAS1. In addition, cell permeabilization by digitonin enhanced the growth inhibitory effect of EAS1. Penetration of the cell membrane by EAS1 is also crucial for the growth inhibitory effect of EAS1. In conclusion, MAPK is an important target for cancer treatment and MAPK antisense oligonucleotide is a potentially significant antitumor oligonucleotide.