Robert C. Bertheau

Telomerase inhibitor GRN163L inhibits myeloma cell growth in vitro and in vivo

Human telomerase, the reverse transcriptase which extends the life span of a cell by adding telomeric repeats to chromosome ends, is expressed in most cancer cells but not in the majority of normal somatic cells. Inhibition of telomerase therefore holds great promise as anticancer therapy. We have synthesized a novel telomerase inhibitor GRN163L, a lipid—attached phosphoramidate oligonucleotide complementary to template region of the RNA subunit of telomerase. Here, we report that GRN163L is efficiently taken up by human myeloma cells without any need of transfection and is resistant to nucleolytic degradation. The exposure of myeloma cells to GRN163L led to an effective inhibition of telomerase activity, reduction of telomere length and apoptotic cell death after a lag period of 2–3 weeks. Mismatch control oligonucleotides had no effect on growth of myeloma cells. The in vivo efficacy of GRN163L was confirmed in two murine models of human multiple myeloma. In three independent experiments, significant reduction in tumor cell growth and better survival than control mice was observed. Furthermore, GRN163L-induced myeloma cell death could be significantly enhanced by Hsp90 inhibitor 17AAG. These data provide the preclinical rationale for clinical evaluation of GRN163L in myeloma and in combination with 17AAG.

Telomerase inhibition by siRNA causes senescence and apoptosis in Barrett's adenocarcinoma cells: mechanism and therapeutic potential

BackgroundIn cancer cells, telomerase induction helps maintain telomere length and thereby bypasses senescence and provides enhanced replicative potential. Chemical inhibitors of telomerase have been shown to reactivate telomere shortening and cause replicative senescence and apoptotic cell death of tumor cells while having little or no effect on normal diploid cells.ResultsWe designed siRNAs against two different regions of telomerase gene and evaluated their effect on telomere length, proliferative potential, and gene expression in Barretts adenocarcinoma SEG-1 cells. The mixture of siRNAs in nanomolar concentrations caused a loss of telomerase activity that appeared as early as day 1 and was essentially complete at day 3. Inhibition of telomerase activity was associated with marked reduction in median telomere length and complete loss of detectable telomeres in more than 50% of the treated cells. Telomere loss caused senescence in 40% and apoptosis in 86% of the treated cells. These responses appeared to be associated with activation of DNA sensor HR23B and subsequent activation of p53 homolog p73 and p63 and E2F1. Changes in these gene regulators were probably the source of observed up-regulation of cell cycle inhibitors, p16 and GADD45. Elevated transcript levels of FasL, Fas and caspase 8 that activate death receptors and CARD 9 that interacts with Bcl10 and NFKB to enhance mitochondrial translocation and activation of caspase 9 were also observed.ConclusionThese studies show that telomerase siRNAs can cause effective suppression of telomerase and telomere shortening leading to both cell cycle arrest and apoptosis via mechanisms that include up-regulation of several genes involved in cell cycle arrest and apoptosis. Telomerase siRNAs may therefore be strong candidates for highly selective therapy for chemoprevention and treatment of Barretts adenocarcinoma.

Phenotypic and functional effects of heat shock protein 90 inhibition on dendritic cell

The 90-kDa heat shock protein (Hsp90) plays an important role in conformational regulation of cellular proteins and thereby cellular signaling and function. As Hsp90 is considered a key component of immune function and its inhibition has become an important target for cancer therapy, we here evaluated the role of Hsp90 in human dendritic cell (DC) phenotype and function. Hsp90 inhibition significantly decreased cell surface expression of costimulatory (CD40, CD80, CD86), maturation (CD83), and MHC (HLA-A, B, C and HLA-DP, DQ, DR) markers in immature DC and mature DC and was associated with down-regulation of both RNA and intracellular protein expression. Importantly, Hsp90 inhibition significantly inhibited DC function. It decreased Ag uptake, processing, and presentation by immature DC, leading to reduced T cell proliferation in response to tetanus toxoid as a recall Ag. It also decreased the ability of mature DC to present Ag to T cells and secrete IL-12 as well as induce IFN-γ secretion by allogeneic T cells. These data therefore demonstrate that Hsp90-mediated protein folding is required for DC function and, conversely, Hsp90 inhibition disrupts the DC function of significant relevance in the setting of clinical trials evaluating novel Hsp90 inhibitor therapy in cancer.

Telomere maintenance in laser capture microdissection-purified Barrett's adenocarcinoma cells and effect of telomerase inhibition in vivo.

Purpose: The aims of this study were to investigate telomere function in normal and Barretts esophageal adenocarcinoma (BEAC) cells purified by laser capture microdissection and to evaluate the effect of telomerase inhibition in cancer cells in vitro and in vivo. Experimental Design: Epithelial cells were purified from surgically resected esophagi. Telomerase activity was measured by modified telomeric repeat amplification protocol and telomere length was determined by real-time PCR assay. To evaluate the effect of telomerase inhibition, adenocarcinoma cell lines were continuously treated with a specific telomerase inhibitor (GRN163L) and live cell number was determined weekly. Apoptosis was evaluated by Annexin labeling and senescence by β-galactosidase staining. For in vivo studies, severe combined immunodeficient mice were s.c. inoculated with adenocarcinoma cells and following appearance of palpable tumors, injected i.p. with saline or GRN163L. Results: Telomerase activity was significantly elevated whereas telomeres were shorter in BEAC cells relative to normal esophageal epithelial cells. The treatment of adenocarcinoma cells with telomerase inhibitor, GRN163L, led to loss of telomerase activity, reduction in telomere length, and growth arrest through induction of both the senescence and apoptosis. GRN163L-induced cell death could also be expedited by addition of the chemotherapeutic agents doxorubicin and ritonavir. Finally, the treatment with GRN163L led to a significant reduction in tumor volume in a subcutaneous tumor model. Conclusions: We show that telomerase activity is significantly elevated whereas telomeres are shorter in BEAC and suppression of telomerase inhibits proliferation of adenocarcinoma cells both in vitro and in vivo.

Genomic evolution in Barrett's adenocarcinoma cells: Critical roles of elevated hsRAD51, homologous recombination and Alu sequences in the genome

A prominent feature of most cancers including Barretts adenocarcinoma (BAC) is genetic instability, which is associated with development and progression of disease. In this study, we investigated the role of recombinase (hsRAD51), a key component of homologous recombination (HR)/repair, in evolving genomic changes and growth of BAC cells. We show that the expression of RAD51 is elevated in BAC cell lines and tissue specimens, relative to normal cells. HR activity is also elevated and significantly correlates with RAD51 expression in BAC cells. The suppression of RAD51 expression, by short hairpin RNA (shRNA) specifically targeting this gene, significantly prevented BAC cells from acquiring genomic changes to either copy number or heterozygosity (P<0.02) in several independent experiments employing single-nucleotide polymorphism arrays. The reduction in copy-number changes, following shRNA treatment, was confirmed by Comparative Genome Hybridization analyses of the same DNA samples. Moreover, the chromosomal distributions of mutations correlated strongly with frequencies and locations of Alu interspersed repetitive elements on individual chromosomes. We conclude that the hsRAD51 protein level is systematically elevated in BAC, contributes significantly to genomic evolution during serial propagation of these cells and correlates with disease progression. Alu sequences may serve as substrates for elevated HR during cell proliferation in vitro, as they have been reported to do during the evolution of species, and thus may provide additional targets for prevention or treatment of this disease.

T1176 Role of Recombinase (RAD51) in Ongoing Genomic Instability and Proliferation in Barrett's Adenocarcinoma Cells

activity. Exposure to 15mmHg increased extracellular pressure stimulated serine phosphorylation of FAK (p-FAK) in Caco-2 and primary human colon cancer cells isolated from surgical specimens. This pressure-induced increase in serine p-FAK was blocked by Akt inhibitor and by siRNA silencing of Akt1 but not by silencing Akt2. Co-precipitation demonstrated that Akt associates directly with FAK. Akt-FAK association was increased by pressure and this increased association was blocked by inhibiting FAK or silencing Akt1 but not Akt2. Scanning the FAK sequence with Scansite software revealed three serine-containing consensus sequences for AKT phosphorylation in the FAK sequence. We therefore constructed a FAK non-phosphorylatable mutant with point mutations (S®A) at these three putative serine phosphorylation sites (S517/601/695) of FAK by Akt to investigate their relevance to pressurestimulated cell adhesion and tyrosine p-FAK. Indeed, overexpression of the triple mutant of FAK (S517/601/695®A) in Caco-2 cells, in contrast to wild type FAK, prevented the increase in p-FAK at Y397 and cancer cell adhesion induced by extracellular pressure. These results suggest that Akt regulates pressure-induced cancer cell adhesion by binding directly to and phosphorylating FAK at S509/601/695. This serine phosphorylation, in turn, permits the pressure-dependent tyrosine autophosphorylation of p-FAK at Y397, the conventional initiator of FAK activation. Although Akt is therefore required for FAK activation in response to pressure, further studies demonstrated that FAK also potentiates Akt activation. Blocking or silencing FAK by three different FAK-specific siRNA sequences prevented the increases in serine p-Akt (S473) and tyrosine p-FAK(Y397) induced by increased pressure. Thus, FAK and Akt bind directly and potentiate each others activation. This novel mechanism of FAKAkt interaction suggests that FAK and Akt1 may be important dual therapeutic targets for preventing cancer cell adhesion, and eventually cancer metastasis.