Youichirou Matsuzaki

Histone deacetylase inhibitors activate INK4d gene through Sp1 site in its promoter.

Histone deacetylase (HDAC) inhibitors are known to arrest human tumor cells at the G1 phase of the cell cycle and activate the cyclin-dependent kinase inhibitor, p21WAF1/Cip1. However, several studies have suggested the existence of a p21WAF1/Cip1-independent molecular pathway. We report here that HDAC inhibitors activate a member of the INK4 family, the INK4d gene, causing G1 phase arrest, in the human T cell leukemia cell line, Jurkat. One of the major Trichostatin A (TSA)-responsive elements is a specific Sp1 binding site in the INK4d promoter. Electrophoretic mobility-shift assay revealed that Sp1 and Sp3 can specifically interact with this Sp1 binding site. Furthermore, using chromatin immunoprecipitation assay, we demonstrated that HDAC2 was present in the INK4d proximal promoter region in the absence, but not the presence, of TSA. Taken together, these results suggest that treatment with TSA transcriptionally activates INK4d by releasing HDAC2 from the histone–DNA complex at the INK4d promoter. Using a p21WAF1/Cip1-deleted human colorectal carcinoma cell line, HCT116 p21 (−/−), we show that upregulation of p19INK4d by TSA is associated with inhibition of cell proliferation. Moreover, mouse embryo fibroblasts lacking Ink4d were resistant to the growth inhibitory effects of TSA as compared to their wild-type counterpart. Our findings suggest that p19INK4d in addition to p21WAF1/Cip1 is an important molecular target of HDAC inhibitors inducing growth arrest.

Sesamin, a lignan of sesame, down‐regulates cyclin D1 protein expression in human tumor cells

Sesamin is a major lignan constituent of sesame and possesses multiple functions such as antihypertensive, cholesterol‐lowering, lipid‐lowering and anticancer activities. Several groups have previously reported that sesamin induces growth inhibition in human cancer cells. However, the nature of this growth inhibitory mechanism remains unknown. The authors here report that sesamin induces growth arrest at the G1 phase in cell cycle progression in the human breast cancer cell line MCF‐7. Furthermore, sesamin dephosphorylates tumor‐suppressor retinoblastoma protein (RB). It is also shown that inhibition of MCF‐7 cell proliferation by sesamin is correlated with down‐regulated cyclin D1 protein expression, a proto‐oncogene that is overexpressed in many human cancer cells. It was found that sesamin‐induced down‐regulation of cyclin D1 was inhibited by proteasome inhibitors, suggesting that sesamin suppresses cyclin D1 protein expression by promoting proteasome degradation of cyclin D1 protein. Sesamin down‐regulates cyclin D1 protein expression in various kinds of human tumor cells, including lung cancer, transformed renal cells, immortalized keratinocyte, melanoma and osteosarcoma. Furthermore, depletion of cyclin D1 protein using small interfering RNA rendered MCF‐7 cells insensitive to the growth inhibitory effects of sesamin, implicating that cyclin D1 is at least partially related to the antiproliferative effects of sesamin. Taken together, these results suggest that the ability of sesamin to down‐regulate cyclin D1 protein expression through the activation of proteasome degradation could be one of the mechanisms of the antiproliferative activity of this agent. (Cancer Sci 2007; 98: 1447–1453)

p15INK4b in HDAC inhibitor‐induced growth arrest

Histone deacetylase (HDAC) inhibitors arrest human tumor cells at the G1 phase of the cell cycle and activate the cyclin‐dependent kinase inhibitor, p21WAF1/Cip1. However, several studies have suggested the existence of a p21WAF1/Cip1‐independent molecular pathway. We report here that HDAC inhibitors, trichostatin A (TSA) and sodium butyrate, activate the p15INK4b gene, a member of the INK4 gene family, through its promoter in HaCaT cells. Furthermore, we show that up‐regulation of p15INK4b by TSA is associated with cell growth inhibition of HCT116 p21 (−/−) cells. Our findings suggest that p15INK4b is one of the important molecular targets of HDAC inhibitors.

Indole-3-carbinol activates the cyclin-dependent kinase inhibitor p15INK4b gene

Indole‐3‐carbinol (I3C) is a naturally occurring compound found in vegetables such as broccoli and cauliflower, and has been shown to arrest human tumor cells in the G1 phase of the cell cycle. However, the molecular mechanism responsible for this effect has not been sufficiently elucidated. We report here that I3C activates the cyclin‐dependent kinase (CDK) inhibitor p15INK4b gene through its promoter, accompanied by cell growth inhibition in HaCaT cells. Treatment with I3C almost did not affect the expressions of the other CDK inhibitors such as p19INK4d, p21WAF1 and p27Kip1. These results suggest that p15INK4b is an important molecular target of I3C among CDK inhibitors.

Identification of JTP‐70902, a p15INK4b‐inductive compound, as a novel MEK1/2 inhibitor

The INK4 family members p16INK4a and p15INK4b negatively regulate cell cycle progression by inhibition of cyclin‐dependent kinase (CDK) 4/6. Loss of p16INK4a functional activity is frequently observed in tumor cells, and is thought to be one of the primary causes of carcinogenesis. In contrast, despite the biochemical similarity to p16INK4a, the frequency of defects in p15INK4b was found to be lower than in p16INK4a, suggesting that p15INK4b‐inductive agents may be useful for tumor suppression. Here we report the discovery of a novel pyrido‐pyrimidine derivative, JTP‐70902, which exhibits p15INK4b‐inducing activity in p16INK4a‐inactivated human colon cancer HT‐29 cells. JTP‐70902 also induced another CDK‐inhibitor, p27KIP1, and downregulated the expression of c‐Myc and cyclin D1, resulting in G1 cell cycle arrest. MEK1/2 was identified by compound‐immobilized affinity chromatography as the molecular target of JTP‐70902, and this was further confirmed by the inhibitory activity of JTP‐70902 against MEK1/2 in kinase assays. JTP‐70902 suppressed the growth of most colorectal and some other cancer cell lines in vitro, and showed antitumor activity in an HT‐29 xenograft model. However, JTP‐70902 did not inhibit the growth of COLO320 DM cells; in these, constitutive extracellular signal‐regulated kinase phosphorylation was not detected, and neither p15INK4b nor p27KIP1 induction was observed. Moreover, p15INK4b‐deficient mouse embryonic fibroblasts were found to be more resistant to the growth‐inhibitory effect of JTP‐70902 than wild‐type mouse embryonic fibroblasts. These findings suggest that JTP‐70902 restores CDK inhibitor‐mediated cell cycle control by inhibiting MEK1/2 and exerts a potent antitumor effect. (Cancer Sci 2007; 98: 1809–1916)

ZD1839 induces p15INK4b and causes G1 arrest by inhibiting the mitogen-activated protein kinase/extracellular signal–regulated kinase pathway

Inactivation of the retinoblastoma protein pathway is the most common abnormality in malignant tumors. We therefore tried to detect agents that induce the cyclin-dependent kinase inhibitor p15INK4b and found that ZD1839 (gefitinib, Iressa) could up-regulate p15INK4b expression. ZD1839 has been shown to inhibit cell cycle progression through inhibition of signaling pathways such as phosphatidylinositol 3′-kinase-Akt and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascades. However, the mechanism responsible for the differential sensitivity of the signaling pathways to ZD1839 remains unclear. We here showed that ZD1839 up-regulated p15INK4b, resulting in retinoblastoma hypophosphorylation and G1 arrest in human immortalized keratinocyte HaCaT cells. p15INK4b induction was caused by MAPK/ERK kinase inhibitor (PD98059), but not by Akt inhibitor (SH-6, Akt-III). Moreover, mouse embryo fibroblasts lacking p15INK4b were resistant to the growth inhibitory effects of ZD1839 compared with wild-type mouse embryo fibroblasts. Additionally, the status of ERK phosphorylation was related to the antiproliferative activity of ZD1839 in human colon cancer HT-29 and Colo320DM cell lines. Our results suggest that induction of p15INK4b by inhibition of the MAPK/ERK pathway is associated with the antiproliferative effects of ZD1839. [Mol Cancer Ther 2007;6(5):1579–1587]

Activation of protein kinase C promotes human cancer cell growth through downregulation of p18 INK4c

p18INK4c, a member of INK4 family of cyclin-dependent kinase inhibitors, negatively regulates the cyclin D-cyclin-dependent kinase 4/6 complexes which promote G1/S transition by phosphorylating the retinoblastoma tumor-suppressor gene product. Several recent studies using p18INK4c-null mice revealed that the p18INK4c plays an important role in cell proliferation and tumor development. We report here that 12-O-tetradecanoylphorbol-13-acetate (TPA), widely used as a protein kinase C (PKC) activator, suppresses the expression of p18INK4c through its promoter, accompanied by the induction of human cancer cell growth. Reduction of p18INK4c using small interfering RNA (siRNA) also enhanced cell growth, suggesting that p18INK4c is a critical target of TPA. Ro 31-8425, a potent and highly specific PKC inhibitor abrogated the suppressive effect of TPA on p18INK4c gene expression. However, the expression of dominant-negative c-Jun (TAM-67) did not inhibit the action of TPA on p18INK4c. These findings suggest that activation of PKC promotes human cancer cell growth through downregulation of p18INK4c in an AP-1 activation-independent manner. These results suggest that the accelerated cellular proliferation of some human tumors caused by enhanced PKC activity at least partially involves the suppression of p18INK4c, which is a ubiquitously expressed cyclin-dependent kinase inhibitor.

Oct‐1 is involved in the transcriptional repression of the p15INK4b gene

p15INK4b functions as a tumor suppressor and implicated in cellular senescence. Here, we show that the Oct‐1 binding site in the human p15INK4b gene promoter functions as a silencer. Oct‐1 specifically interacts with this binding site in vitro and in vivo and SMRT and HDAC1 are present in the p15INK4b proximal promoter region. Moreover, mouse embryo fibroblasts (MEFs) lacking Oct‐1 have shown significantly increased levels of p15INK4b protein compared to their normal counterparts. Treatment with a histone deacetylase (HDAC) inhibitor has activated the expression of p15INK4b in wild‐type MEFs but has no effect in MEFs lacking Oct‐1, suggesting that Oct‐1 represses p15INK4b gene expression in an HDAC‐dependent manner. Finally, we show that the expression of Oct‐1 protein significantly decreases, whereas p15INK4b protein significantly increases with the cellular aging process. Taken together, these results suggest that Oct‐1 is an important transcriptional repressor for p15INK4b gene and the transcriptional repression of the p15INK4b gene by Oct‐1 may be one of the regulatory mechanisms of cellular senescence.

Trichostatin A activates p18INK4c gene: differential activation and cooperation with p19INK4d gene

We have reported that histone deacetylase (HDAC) inhibitors activate a member of the INK4 family, the p19INK4d gene, causing G1 phase arrest. We report here that HDAC inhibitor, Trichostatin A, activates another member of the INK4 family, the p18INK4c gene, through its promoter in Jurkat cells. Interestingly, the activation patterns of the p18INK4c gene were different from those of p19INK4d. Furthermore, mouse embryo fibroblasts lacking p18Ink4c or p18Ink4c/p19Ink4d were resistant to the growth inhibitory effects of TSA as compared to their wild‐type counterpart. Our findings suggest that p18INK4c is involved in TSA‐mediated cell growth inhibition and cooperates with p19INK4d.

INK4 Family —A promising target for ‘gene-regulating chemoprevention’ and ‘molecular-targeting prevention’ of cancer

Inactivation of the p16INK4a gene is one of the most frequent defects that contribute to oncogenesis in human cancer, since it is a tumor-suppressor gene. Therefore, functional restoration of p16INK4a is one of the most effective methods for cancer prevention. We proposed the concept of ‘gene-regulating chemoprevention’ and ‘molecular-targeting prevention’ of cancer, which assumes that transcriptional regulation by drugs on tumor-suppressor genes or functionally similar genes to the tumor-suppressor genes contributes to the prevention of human malignancies. The p16INK4a homologs p15INK4b, p18INK4c and p19INK4d have been recently identified, and these four members constitute the INK4 family of proteins. All directly bind to cyclin D-cyclin dependent kinase (CDK) 4/6 and are therefore specific inhibitors of these complexes. We recently showed that histone deacetylase (HDAC) inhibitors, promising chemopreventive and chemotherapeutical agents, induce p15INK4b and p19INK4d gene expression and cause growth arrest, suggesting that both genes are important molecular targets for HDAC inhibitors. Furthermore, we found that 12-O-tetradecanoylphorbol-13-acetate (TPA), which is widely used as a tumor promoter and protein kinase C activator, promotes human cancer cell growth through the down-regulation of p18INK4c gene expression. This suggests that a mouse two-stage carcinogenesis model using TPA might partially represent the most common human carcinogenesis pathway related to RB. Our results suggest that the INK4 family consists of attractive and promising molecular targets for the ‘gene-regulating chemoprevention’ and ‘molecular-targeting prevention’ of cancer.