Tomokazu Ohishi

Targeting Tankyrase 1 as a therapeutic strategy for BRCA-associated cancer.

The BRCA1 and BRCA2 proteins are involved in the maintenance of genome stability and germ-line loss-of-function mutations in either BRCA1 or BRCA2 strongly predispose carriers to cancers of the breast and other organs. It has been demonstrated previously that inhibiting elements of the cellular DNA maintenance pathways represents a novel therapeutic approach to treating tumors in these individuals. Here, we show that inhibition of the telomere-associated protein, Tankyrase 1, is also selectively lethal with BRCA deficiency. We also demonstrate that the selectivity caused by inhibition of Tankyrase 1 is associated with an exacerbation of the centrosome amplification phenotype associated with BRCA deficiency. We propose that inhibition of Tankyrase 1 could be therapeutically exploited in BRCA-associated cancers.

Cross‐species difference in telomeric function of tankyrase 1

Telomeres protect chromosome ends from being recognized as DNA double‐strand breaks. Telomere shortening, which occurs due to incomplete replication of DNA termini, limits the proliferative capacity of human somatic cells and contributes as a barrier to carcinogenesis. In most human cancer cells, telomerase maintains telomere length whereas TRF1, a telomeric protein, represses telomere access to telomerase. Tankyrase 1 is a PARP that dissociates TRF1 from telomeres by poly(ADP‐ribosyl)ating TRF1. Thus, by reducing TRF1 loading on chromosome ends, tankyrase 1 enhances telomere access to telomerase and causes telomere elongation. Recent studies of knockout mice suggest that tankyrases may not regulate telomere length in mice (Mus musculus). Consistent with this idea is that mouse TRF1 has no canonical tankyrase‐binding motif. However, the presence of such a motif is not a prerequisite to bind tankyrase 1 in certain species. Here, we found that, in mice, tankyrase 1 does not bind or poly(ADP‐ribosyl)ate TRF1. Accordingly, mouse TRF1 was resistant to tankyrase 1‐mediated release from telomeres. These observations indicate that telomeric function of tankyrase 1 is not conserved in mice. We also found that the canonical tankyrase 1‐binding motif in TRF1 is conserved in several mammals but not in rats. Since mice and rats have much higher telomerase activity in their somatic tissues and much longer telomeres than those in other mammals, these rodent species might have evolved to resign the tankyrase 1‐mediated telomere maintenance system. Meanwhile, PARP inhibitors induced non‐telomeric tankyrase 1 foci in the nuclei, suggesting another function of tankyrase 1 at non‐telomeric loci. (Cancer Sci 2007; 98: 850–857)

TRF1 Mediates Mitotic Abnormalities Induced by Aurora-A Overexpression

Aurora-A, a conserved serine-threonine kinase, plays essential roles in mitosis. Aberrant upregulation of Aurora-A perturbs proper mitotic progression and results in a generation of multinucleated cells with centrosome amplification. The molecular mechanisms for these mitotic defects remain elusive. Here, we show that the overexpressed Aurora-A-induced mitotic defects depend on the telomeric protein TRF1. Live and fixed cell analyses revealed that Aurora-A overexpression in HeLa cells compromises chromosome biorientation, which leads to cytokinetic failure and tetraploidization with increased centrosome numbers. TRF1 depletion by small interfering RNAs or by tankyrase-1 overexpression suppresses Aurora-A-induced occurrence of unaligned chromosomes in metaphase, thus preventing the subsequent abnormalities. We found that Aurora-A binds and phosphorylates TRF1. When TRF1 knockdown cells are complemented with wild-type TRF1, Aurora-A-induced mitotic defects recur. By contrast, a TRF1 mutant that is not phosphorylatable by Aurora-A does not restore such Aurora-A-induced phenotype. We propose that TRF1 phosphorylation by excessive Aurora-A may provoke abnormal mitosis and chromosomal instability.

TRF1 Ensures the Centromeric Function of Aurora-B and Proper Chromosome Segregation

ABSTRACT A cancer is a robustly evolving cell population originating from a normal diploid cell. Improper chromosome segregation causes aneuploidy, a driving force of cancer development and malignant progression. Telomeric repeat binding factor 1 (TRF1) has been established as a telomeric protein that negatively regulates telomere elongation by telomerase and promotes efficient DNA replication at telomeres. Intriguingly, overexpression of a mitotic kinase, Aurora-A, compromises efficient microtubule-kinetochore attachment in a TRF1-dependent manner. However, the precise role of TRF1 in mitosis remains elusive. Here we demonstrate that TRF1 is required for the centromeric function of Aurora-B, which ensures proper chromosome segregation. TRF1 depletion abolishes centromeric recruitment of Aurora-B and loosens sister centromere cohesion, resulting in the induction of merotelic kinetochore attachments, lagging chromosomes, and micronuclei. Accordingly, an absence of TRF1 in human and mouse diploid cells induces aneuploidy. These phenomena seem to be telomere independent, because a telomere-unbound TRF1 mutant can suppress the TRF1 knockdown phenotype. These observations indicate that TRF1 regulates the rigidity of the microtubule-kinetochore attachment, contributing to proper chromosome segregation and the maintenance of genomic integrity.

Bladder Cancer Stem-Like Cells: Their Origin and Therapeutic Perspectives

Bladder cancer (BC), the most common cancer arising from the human urinary tract, consists of two major clinicopathological phenotypes: muscle-invasive bladder cancer (MIBC) and non-muscle-invasive bladder cancer (NMIBC). MIBC frequently metastasizes and is associated with an unfavorable prognosis. A certain proportion of patients with metastatic BC can achieve a remission with systemic chemotherapy; however, the disease relapses in most cases. Evidence suggests that MIBC comprises a small population of cancer stem cells (CSCs), which may be resistant to these treatments and may be able to form new tumors in the bladder or other organs. Therefore, the unambiguous identification of bladder CSCs and the development of targeted therapies are urgently needed. Nevertheless, it remains unclear where bladder CSCs originate and how they are generated. We review recent studies on bladder CSCs, specifically focusing on their proposed origin and the possible therapeutic options based on the CSC theory.

Evaluation of Tankyrase Inhibition in Whole Cells

The telomeric poly(ADP-ribose) polymerase (PARP), tankyrase 1, modulates the impact of telomerase inhibition on human cancer cells. Thus, overexpression of tankyrase 1 in telomerase-positive cancer cells confers resistance to telomerase inhibitors, such as MST-312, whereas pharmacological inhibition of tankyrase 1 enhances telomere shortening by MST-312. These facts indicate that tankyrase 1 could be a target for telomere-directed molecular cancer therapy. Here, the authors describe a convenient method to monitor the telomeric function of tankyrase 1. This protocol takes much less time than the telomere Southern blot analysis and can be utilized as a rapid screening system for tankyrase 1 inhibitors that are effective in intact cells. For direct monitoring of tankyrase 1 PARP activity, a protocol for the in vitro enzyme assay is also described.

SF-1 deficiency causes lipid accumulation in Leydig cells via suppression of STAR and CYP11A1

Genetic mutations of steroidogenic factor 1 (also known as Ad4BP or Nr5a1) have increasingly been reported in patients with 46,XY disorders of sex development (46,XY disorders of sex development). However, because the phenotype of 46,XY disorders of sex development with a steroidogenic factor 1 mutation is wide-ranging, its precise diagnosis remains a clinical problem. We previously reported the frequent occurrence of lipid accumulation in Leydig cells among patients with 46,XY disorders of sex development with a steroidogenic factor 1 mutation, an observation also reported by other authors. To address the mechanism of lipid accumulation in this disease, we examined the effects of steroidogenic factor 1 deficiency on downstream targets of steroidogenic factor 1 in in vitro and in vivo. We found that lipid accumulation in Leydig cells was enhanced after puberty in heterozygous steroidogenic factor 1 knockout mice compared with wild-type mice, and was accompanied by a significant decrease in steroidogenic acute regulatory protein and CYP11A1 expression. In mouse Leydig cell lines, steroidogenic factor 1 knockdown induced a remarkable accumulation of neutral lipids and cholesterol with reduced androgen levels. Steroidogenic factor 1 knockdown reduced the expression of steroidogenic acute regulatory protein and CYP11A1, both of which are transcriptional targets of steroidogenic factor 1 and key molecules for steroidogenesis from cholesterol in the mitochondria. Knockdown of either steroidogenic acute regulatory protein or CYP11A1 also induced lipid accumulation, and knockdown of both had an additive effect. Our data suggested that lipid accumulation in the Leydig cells of the 46,XY disorders of sex development phenotype with a steroidogenic factor 1 mutation is due, at least in part, to the suppression of steroidogenic acute regulatory protein and CYP11A1, and a resulting increase in unmetabolized cholesterol.

Tankyrase-1 assembly to large protein complexes blocks its telomeric function.

MINT‐7987998: Tankyrase‐1 (uniprotkb:O95271) physically interacts (MI:0915) with Tankyrase‐1 (uniprotkb:O95271) by anti tag coimmunoprecipitation (MI:0007).

Tankyrase-binding protein TNKS1BP1 regulates actin cytoskeleton rearrangement and cancer cell invasion

Tankyrase, a PARP that promotes telomere elongation and Wnt/β-catenin signaling, has various binding partners, suggesting that it has as-yet unidentified functions. Here, we report that the tankyrase-binding protein TNKS1BP1 regulates actin cytoskeleton and cancer cell invasion, which is closely associated with cancer progression. TNKS1BP1 colocalized with actin filaments and negatively regulated cell invasion. In TNKS1BP1-depleted cells, actin filament dynamics, focal adhesion, and lamellipodia ruffling were increased with activation of the ROCK/LIMK/cofilin pathway. TNKS1BP1 bound the actin-capping protein CapZA2. TNKS1BP1 depletion dissociated CapZA2 from the cytoskeleton, leading to cofilin phosphorylation and enhanced cell invasion. Tankyrase overexpression increased cofilin phosphorylation, dissociated CapZA2 from cytoskeleton, and enhanced cell invasion in a PARP activity-dependent manner. In clinical samples of pancreatic cancer, TNKS1BP1 expression was reduced in invasive regions. We propose that the tankyrase-TNKS1BP1 axis constitutes a posttranslational modulator of cell invasion whose aberration promotes cancer malignancy. Cancer Res; 77(9); 2328-38. ©2017 AACR.

Epithelial–mesenchymal transition promotes SOX2 and NANOG expression in bladder cancer

Bladder cancer is the most common malignant tumor of the urothelium and is classified into non-muscle-invasive bladder cancer (NMIBC) and muscle-invasive bladder cancer (MIBC). Stemness markers such as SOX2 and NANOG are frequently overexpressed in various aggressive cancers, including MIBC; epithelial–mesenchymal transition (EMT) has been proposed as a potential trigger of stemness in cancers. To determine whether cancer stemness is acquired via EMT in bladder cancer, we studied the effect of EMT on the expression of SOX2 and NANOG in bladder cancer cell lines. We also analyzed their expression in clinical tissue samples. Our results revealed that a potent EMT inducer (transforming growth factor β1) reduced the expression of the epithelial marker E-cadherin and increased expression of both SOX2 and NANOG in epithelial-type bladder cancer cells. As for clinical bladder cancer samples, in NMIBC, E-cadherin expression was slightly diminished, and the expression of both SOX2 and NANOG was negligible. In contrast, in MIBC, E-cadherin expression was highly and heterogeneously diminished, while the expression of both SOX2 and NANOG was increased. We also noticed that either E-cadherin or SOX2 (or NANOG) was expressed (ie, in a manner exclusive of each other). In addition, the concentration of E-cadherin showed a significant negative correlation with tumor grade and stage, while expression of SOX2 and NANOG positively correlated with those clinicopathological parameters. These findings suggest that EMT promotes stemness of bladder cancer cells, contributing to tumor aggressiveness. This EMT–cancer stemness axis may also play an important role in the pathogenesis of NMIBC and MIBC.