Maintenance of telomeres is implicated in chromosome stabilization and cell immortalization. Telomerase, which catalyzes de novo synthesis of telomeres, is activated in germ cells and most cancers. Telomerase activity is regulated by gene expression for its catalytic subunit, TERT, whereas several lines of evidence have suggested a post‐translational regulation of telomerase activity. Here we identify the 14‐3‐3 signaling proteins as human TERT (hTERT)‐binding partners. A dominant‐negative 14‐3‐3 redistributed hTERT, which was normally predominant in the nucleus, into the cytoplasm. Consistent with this observation, hTERT‐3A, a mutant that could not bind 14‐3‐3, was localized into the cytoplasm. Leptomycin B, an inhibitor of CRM1/exportin 1‐mediated nuclear export, or disruption of a nuclear export signal (NES)‐like motif located just upstream of the 14‐3‐3 binding site in hTERT impaired the cytoplasmic localization of hTERT. Compared with wild‐type hTERT, hTERT‐3A increased its association with CRM1. 14‐3‐3 binding was not required for telomerase activity either in vitro or in cell extracts. These observations suggest that 14‐3‐3 enhances nuclear localization of TERT by inhibiting the CRM1 binding to the TERT NES‐like motif.
Telomere elongation by telomerase is repressed in cis by the telomeric protein TRF1. Tankyrase 1 poly(ADP-ribosyl)ates TRF1 and releases it from telomeres, allowing access of telomerase to telomeres. Here we demonstrate that tankyrase 1 inhibition in human cancer cells enhances telomere shortening by a telomerase inhibitor and hastens cell death. Conversely, either tankyrase 1 upregulation or telomere shortening, each of which decreased TRF1 loading on a chromosome end, attenuated the impact of telomerase inhibition. These results are consistent with the idea that telomeres having fewer TRF1s increase the efficiency of their elongation by telomerase. This study implies that both enzyme activity and accessibility to telomeres can be targets for telomerase inhibition.
In human cells, telomere elongation by telomerase is repressed in cis by the telomeric protein TRF1. Tankyrase
In most colorectal cancers, Wnt/β-catenin signaling is activated by loss-of-function mutations in the () gene and plays a critical role in tumorigenesis. Tankyrases poly(ADP-ribosyl)ate and destabilize Axins, a negative regulator of β-catenin, and upregulate β-catenin signaling. Tankyrase inhibitors downregulate β-catenin and are expected to be promising therapeutics for colorectal cancer. However, colorectal cancer cells are not always sensitive to tankyrase inhibitors, and predictive biomarkers for the drug sensitivity remain elusive. Here we demonstrate that the short-form mutations predict the sensitivity of colorectal cancer cells to tankyrase inhibitors. By using well-established colorectal cancer cell lines, we found that tankyrase inhibitors downregulated β-catenin in the drug-sensitive, but not resistant, colorectal cancer cells. The drug-sensitive cells showed higher Tcf/LEF transcriptional activity than the resistant cells and possessed "short" truncated APCs lacking all seven β-catenin-binding 20-amino acid repeats (20-AARs). In contrast, the drug-resistant cells possessed "long" APC retaining two or more 20-AARs. Knockdown of the long APCs with two 20-AARs increased β-catenin, Tcf/LEF transcriptional activity and its target gene expression. Under these conditions, tankyrase inhibitors were able to downregulate β-catenin in the resistant cells. These results indicate that the long APCs are hypomorphic mutants, whereas they exert a dominant-negative effect on Axin-dependent β-catenin degradation caused by tankyrase inhibitors. Finally, we established 16 patient-derived colorectal cancer cells and confirmed that the tankyrase inhibitor-responsive cells harbor the short-form APC mutations. These observations exemplify the predictive importance of mutations, the most common genetic alteration in colorectal cancers, for molecular targeted therapeutics..
Poly(4,4‘-dialkyl-2,2‘-bithiazole-5,5‘-diyl)s (PRBTz, alkyl = methyl (PMeBTz), butyl (PBuBTz), and heptyl (PHepBTz)) and their analogues comprised of 33−150 thiazole rings have been prepared by organometallic polycondensation, and their chemical and physical properties are compared with those of π-conjugated poly(thiophene-2,5-diyl) (PTh) and poly(pyridine-2,5-diyl) (PPy). Electrochemical n-doping of PRBTz takes place at E° = −1.77 to −2.30 V vs Ag/Ag+ and is accompanied by the appearance of a new absorption band in the near-infrared. PMeBTz assumes a relatively stiff structure in solution and shows a large refractive index increment of 0.55 cm3 g-1; powder X-ray diffraction analysis of PMeBTz supports a face-to-face type stacking of the polymer chains in the solid state. All of the polymers show photoluminescence in solutions and in the solid, and an electroluminescence device using PMeBTz as the emitting layer gives emission of light at λmax = 680 nm with 100 cd m-2 at 8 V. A thin film of PMeBTz gives an optical third-order nonlinear susceptibility χ(3) of 2.5 × 10-11 esu, which is larger than observed with PTh and PPy films, and comparison of the χ(3) value with that (0.3 × 10-11 esu) of nonregioregular poly(4-methylthiazole-2,5-diyl) (PMeTz) reveals the importance of the regioregular structure of PMeBTz to give the larger χ(3) value.
Aberrant activation of Wnt/β‐catenin signaling causes tumorigenesis and promotes the proliferation of colorectal cancer cells. Porcupine inhibitors, which block secretion of Wnt ligands, may have only limited clinical impact for the treatment of colorectal cancer, because most colorectal cancer is caused by loss‐of‐function mutations of the tumor suppressor adenomatous polyposis coli (APC) downstream of Wnt ligands. Tankyrase poly(ADP‐ribosyl)ates (PARylates) Axin, a negative regulator of β‐catenin. This post‐translational modification causes ubiquitin‐dependent degradation of Axin, resulting in β‐catenin accumulation. Tankyrase inhibitors downregulate β‐catenin and suppress the growth of APC‐mutated colorectal cancer cells. Herein, we report a novel tankyrase‐specific inhibitor RK‐287107, which inhibits tankyrase‐1 and ‐2 four‐ and eight‐fold more potently, respectively, than G007‐LK, a tankyrase inhibitor that has been previously reported as effective in mouse xenograft models. RK‐287107 causes Axin2 accumulation and downregulates β‐catenin, T‐cell factor/lymphoid enhancer factor reporter activity and the target gene expression in colorectal cancer cells harboring the shortly truncated APC mutations. Consistently, RK‐287107 inhibits the growth of APC‐mutated (β‐catenin‐dependent) colorectal cancer COLO‐320DM and SW403 cells but not the APC‐wild (β‐catenin‐independent) colorectal cancer RKO cells. Intraperitoneal or oral administration of RK‐287107 suppresses COLO‐320DM tumor growth in NOD‐SCID mice. Rates of tumor growth inhibition showed good correlation with the behavior of pharmacodynamic biomarkers, such as Axin2 accumulation and MYC downregulation. These observations indicate that RK‐287107 exerts a proof‐of‐concept antitumor effect, and thus may have potential for tankyrase‐directed molecular cancer therapy.
dLimitless reproductive potential is one of the hallmarks of cancer cells. This ability is due to the maintenance of telomeres, erosion of which causes cellular senescence or death. While most cancer cells activate telomerase, a telomere-elongating enzyme, it remains elusive as to why cancer cells often maintain shorter telomeres than the cells in the surrounding normal tissues. Here, we show that forced telomere elongation in cancer cells promotes their differentiation in vivo. We elongated the telomeres of human prostate cancer cells that possess short telomeres by enhancing their telomerase activity. The resulting cells had long telomeres and retained the ability to form tumors in nude mice. Strikingly, these tumors exhibited many duct-like structures and reduced N-cadherin expression, reminiscent of well-differentiated adenocarcinoma. These changes were caused by telomere elongation and not by enhanced telomerase activity. Gene expression profiling revealed that tumor formation was accompanied by the expression of innate immune system-related genes, which have been implicated in maintaining tumor cells in an undifferentiated state and poor-prognosis cancers. In tumors derived from the telomere-elongated cells, upregulation of such gene sets is not observed. Our observations suggest a functional contribution of short telomeres to tumor malignancy by regulation of cancer cell differentiation.
The canonical WNT pathway plays an important role in cancer pathogenesis. Inhibition of poly(ADP-ribose) polymerase catalytic activity of the tankyrases (TNKS/TNKS2) has been reported to reduce the Wnt/β-catenin signal by preventing poly ADP-ribosylation-dependent degradation of AXIN, a negative regulator of Wnt/β-catenin signaling. With the goal of investigating the effects of tankyrase and Wnt pathway inhibition on tumor growth, we set out to find small-molecule inhibitors of TNKS/TNKS2 with suitable drug-like properties. Starting from 1a, a high-throughput screening hit, the spiroindoline derivative 40c (RK-287107) was discovered as a potent TNKS/TNKS2 inhibitor with >7000-fold selectivity against the PARP1 enzyme, which inhibits WNT-responsive TCF reporter activity and proliferation of human colorectal cancer cell line COLO-320DM. RK-287107 also demonstrated dose-dependent tumor growth inhibition in a mouse xenograft model. These observations suggest that RK-287107 is a promising lead compound for the development of novel tankyrase inhibitors as anticancer agents.
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