Antiproliferative small-molecule inhibitors of transcription factor LSF reveal oncogene addiction to LSF in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. Despite the prevalence of HCC, there is no effective, systemic treatment. The transcription factor LSF is a promising protein target for chemotherapy; it is highly expressed in HCC patient samples and cell lines, and promotes oncogenesis in rodent xenograft models of HCC. Here, we identify small molecules that effectively inhibit LSF cellular activity. The lead compound, factor quinolinone inhibitor 1 (FQI1), inhibits LSF DNA-binding activity both in vitro, as determined by electrophoretic mobility shift assays, and in cells, as determined by ChIP. Consistent with such inhibition, FQI1 eliminates transcriptional stimulation of LSF-dependent reporter constructs. FQI1 also exhibits antiproliferative activity in multiple cell lines. In LSF-overexpressing cells, including HCC cells, cell death is rapidly induced; however, primary or immortalized hepatocytes are unaffected by treatment with FQI1. The highly concordant structure-activity relationship of a panel of 23 quinolinones strongly suggests that the growth inhibitory activity is due to a single biological target or family. Coupled with the striking agreement between the concentrations required for antiproliferative activity (GI(50)s) and for inhibition of LSF transactivation (IC(50)s), we conclude that LSF is the specific biological target of FQIs. Based on these in vitro results, we tested the efficacy of FQI1 in inhibiting HCC tumor growth in a mouse xenograft model. As a single agent, tumor growth was dramatically inhibited with no observable general tissue cytotoxicity. These findings support the further development of LSF inhibitors for cancer chemotherapy.

A natural product telomerase activator as part of a health maintenance program.

Most human cells lack sufficient telomerase to maintain telomeres, hence these genetic elements shorten with time and stress, contributing to aging and disease. In January, 2007, a commercial health maintenance program, PattonProtocol-1, was launched that included a natural product-derived telomerase activator (TA-65®, 10-50 mg daily), a comprehensive dietary supplement pack, and physician counseling/laboratory tests at baseline and every 3-6 months thereafter. We report here analysis of the first year of data focusing on the immune system. Low nanomolar levels of TA-65® moderately activated telomerase in human keratinocytes, fibroblasts, and immune cells in culture; similar plasma levels of TA-65® were achieved in pilot human pharmacokinetic studies with single 10- to 50-mg doses. The most striking in vivo effects were declines in the percent senescent cytotoxic (CD8(+)/CD28(-)) T cells (1.5, 4.4, 8.6, and 7.5% at 3, 6, 9, and 12 months, respectively; p = not significant [N.S.], 0.018, 0.0024, 0.0062) and natural killer cells at 6 and 12 months (p = 0.028 and 0.00013, respectively). Most of these decreases were seen in cytomegalovirus (CMV) seropositive subjects. In a subset of subjects, the distribution of telomere lengths in leukocytes at baseline and 12 months was measured. Although mean telomere length did not increase, there was a significant reduction in the percent short (<4 kbp) telomeres (p = 0.037). No adverse events were attributed to PattonProtocol-1. We conclude that the protocol lengthens critically short telomeres and remodels the relative proportions of circulating leukocytes of CMV(+) subjects toward the more "youthful" profile of CMV(-) subjects. Controlled randomized trials are planned to assess TA-65®-specific effects in humans.

RNAi screen for telomerase reverse transcriptase transcriptional regulators identifies HIF1alpha as critical for telomerase function in murine embryonic stem cells.

In various types of stem cells, including embryonic stem (ES) cells and hematopoietic stem cells, telomerase functions to ensure long-term self-renewal capacity via maintenance of telomere reserve. Expression of the catalytic component of telomerase, telomerase reverse transcriptase (Tert), which is essential for telomerase activity, is limiting in many types of cells and therefore plays an important role in establishing telomerase activity levels. However, the mechanisms regulating expression of Tert in cells, including stem cells, are presently poorly understood. In the present study, we sought to identify genes involved in the regulation of Tert expression in stem cells by performing a screen in murine ES (mES) cells using a shRNA expression library targeting murine transcriptional regulators. Of 18 candidate transcriptional regulators of Tert expression identified in this screen, only one candidate, hypoxia inducible factor 1 alpha (Hif1alpha), did not have a significant effect on mES cell morphology, survival, or growth rate. Direct shRNA-mediated knockdown of Hif1alpha expression confirmed that suppression of Hif1alpha levels was accompanied by a reduction in both Tert mRNA and telomerase activity levels. Furthermore, gradual telomere attrition was observed during extensive proliferation of Hif1alpha-targeted mES cells. Switching Hif1alpha-targeted mES cells to a hypoxic environment largely restored Hif1alpha levels, as well as Tert expression, telomerase activity levels, and telomere length. Together, these findings suggest a direct effect of Hif1alpha on telomerase regulation in mES cells, and imply that Hif1alpha may have a physiologically relevant role in maintenance of functional levels of telomerase in stem cells.