P21 Cip1 Protein Research Articles

Role of Krüppel-like Factor 4-p21CIP1 Axis in Breast Cancer Stem-like Cell Inhibition by Benzyl Isothiocyanate.

Cancer chemoprevention by benzyl isothiocyanate (BITC), which is derived from cruciferous vegetables like garden cress, in a transgenic mouse model of breast cancer is associated with inhibition of breast cancer stem-like cells (bCSC), but the molecular regulators of this effect remain elusive. This study demonstrates a protective effect of Krüppel-like factor 4 (KLF4)-p21CIP1 axis in bCSC inhibition by BITC. Exposure of human breast cancer cells (MCF-7, MDA-MB-231, and SUM159) to plasma-achievable concentrations of BITC resulted in a robust induction of KLF4 mRNA and its protein expression as determined by qRT-PCR and Western blotting or confocal microscopy. BITC-mediated suppression of bCSC markers, including aldehyde dehydrogenase 1 activity and mammosphere frequency, was significantly augmented by transient or stable knockdown of KLF4. Western blotting and IHC revealed relatively higher levels of KLF4 protein in mammary tumor sections from BITC-treated mice in comparison with controls, but the difference was insignificant. Analysis of the breast cancer RNA-Seq data from The Cancer Genome Atlas indicated significant positive correlation between expression of KLF4 and that of p21CIP1 (CDKN1A) but not β-Catenin (CTNNB1). Knockdown of p21CIP1 protein also amplified BITC-mediated suppression of bCSC. Finally, KLF4 was recruited to the promoter of p21CIP1 as indicated by chromatin immunoprecipitation assay. These results indicate that induction of KLF4-p21CIP1 axis attenuates inhibitory effect of BITC on bCSC self-renewal. Translational implication of these findings is that breast cancer chemoprevention by BITC may be augmented with a combination regimen involving BITC and an inhibitor of KLF4.

MiR-424 coordinates multilayered regulation of cell cycle progression to promote esophageal squamous cell carcinoma cell proliferation.

BackgroundDysregulation of the cell cycle has been implicated in esophageal squamous cell carcinoma (ESCC) progression. This study aimed to evaluate the role of miR-424 in cell cycle regulation and ESCC proliferation.MethodsThe role of miR-424 in cell proliferation was evaluated in vitro and in vivo. Transcriptional activation of miR-424 was determined using chromatin immunoprecipitation, and binding of miR-424 to targets was verified using miRNA ribonucleoprotein complex immunoprecipitation.FindingsmiR-424 was upregulated and correlated with poor survival in ESCC patients. Repression or overexpression of miR-424 respectively decreased or increased ESCC cell proliferation in vitro and in vivo. miR-424 expression is transcriptionally regulated by E2F1 and increased during G1/S transition. Knockdown or overexpression of miR-424 respectively inhibited or promoted both G1/S and G2/M cell cycle transitions in ESCC cells, and these effects were mediated by two newly identified miR-424 targets, PRKCD and WEE1, respectively. Consequently, elevation of PRKCD by miR-424 knockdown led to enhanced stability of the p21Cip1 protein via increased activation of PRKCD and downstream p38 MAPK and JNK signaling to block CDK2 activation and G1/S transition, while elevated WEE1 maintained CDC2 in an inactive state to block G2/M transition. However, circLARP4 could sponge the binding of miR-424 to PRKCD, thus compromising the regulation of G1/S progression by miR-424.InterpretationmiR-424 coordinates a previously unknown, multilayered regulation of ESCC cell cycle progression to promote ESCC proliferation, and may be used as a novel prognostic marker and an effective therapeutic target for ESCCs.FundNational Natural Science Foundation of China.

Abstract 5523: Allele-specific restoration of biological function of the mutant p53 by Baylis-Hillman adducts

Abstract p53 is the most frequently mutated gene in human cancer, and the p53R175 mutant is the third most frequently found missense mutant that confers resistance to anticancer drugs and apoptosis. Therefore, small-molecule reactivation of mutant p53 function(s) represents an important and novel anticancer strategy. We have previously reported that induction of oxidative protein modifications primarily underlies the mechanistic conversion of mutant to wt or wt-like p53 forms (Eur J Medchem 107:233, 2016; Int J Oncol 48:1426, 2016). Here, we describe the identification of Baylis-Hillman (BH) adducts as potent and novel mutant p53 reactivators through cell-based, high-throughput small-molecule screening procedures. An extensive structure-activity relationship studies indicated that carbon-carbon double bond and the presence of an electron-withdrawing substituent at the aromatic ring were essential for the activity. Cell lines used in this study includes SKBR3, AU565 (p53R175H), MCF7 (p53 wt), MDA-MB-231 (p53del), MDA-MB-468 (p53R273H) and MCF10a normal breast epithelial cells. BH adducts displayed preferential cytotoxicity against cancer cells expressing the allele-specific R175H mutation (SKBR3 and AU565) in comparison with the wt p53 or p53-null cancer cell lines. Additionally, the BH adducts did not show significant cytotoxicity against normal cells (MCF10a). Further, we developed isogenic cell lines expressing the p53R175H in the p53-null H1299 lung cancer cells and evaluated their sensitivities towards BH adducts; these models again verified the greater drug efficacy. To prove the hypothesis the higher efficacy of BH adduct stems from the reactivation of mutant p53, the conformation-specific antibodies (Pab1620 for wt and Pab420 for mutant) were employed. Immunoprecipitation and immunostaining using confocal microscopy showed that BH adduct treatment of p53R175H cells resulted in a marked reciprocal loss of mutant protein and gradual increase of wt-like protein. EMSA revealed a time-dependent restoration of DNA-binding of the mutant p53, which was accompanied by a distinct upregulation of pro-apoptotic signaling pathways mediated by PUMA, Bax, p21cip1 and MDM2 proteins. A strong G2/M arrest and significant apoptosis were observed with BH adduct in a time and concentration-dependent manner. Other data showed that BH adduct restores the wt-structure and function by covalently binding with the mutant p53R175H. In conclusion, BH adduct is an attractive lead compound for further development because of its potent p53 reactivation of nonfunctional mutant protein (supported by CPRIT grants RP130266 and RP170207 to KSS). Citation Format: Surendra R. Punganuru, Viswanath Arutla, Hanumantha Rao Madala, Kalkunte S. Srivenugopal. Allele-specific restoration of biological function of the mutant p53 by Baylis-Hillman adducts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5523.

Abstract 509: Upregulation of miR-424 promotes cell cycle progression and cell proliferation in esophageal squamous cell carcinoma

Abstract Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignancies of the gastrointestinal tract. miR-424 was one of the most significantly upregulated miRNAs in the ESCCs compared with normal esophageal tissues (NEs) identified by our previous miRNA arrays. The effects of miR-424 in tumor development and progression are conflicting according to previous reports till now. Here, we characterized the role of miR-424 in ESCC cell cycle progression and proliferation regulation. Expression of miR-424 was determined using qRT-PCR analysis. Cell proliferation assay by CCK-8 and the xenograft tumor model were used to determine ESCC proliferation in vitro and in vivo. Cell cycle distribution was analyzed by flow cytometry after synchronizing and releasing cells from G0/G1 phase, the onset of S-phase, or mitosis. Targets of miR-424 were validated using luciferase assays. The presence of E2F1-binding sites in the putative promoter of miR-424 gene was investigated using Chromatin immunoprecipitation (ChIP). As a result, miR-424 expression was elevated in ESCC specimens than in their normal counterparts. High miR-424 expression was a predictor of poor survival for Stage II-III ESCC patients undergoing surgery. Consistent with clinical data, knockdown miR-424 function in ESCC cells remarkably suppressed ESCC proliferation in vitro and tumor formation in mice model, while opposite results were observed after overexpressing miR-424. Cell cycle analyses revealed that miR-424 knockdown negatively regulates both G1/S and G2/M transition in ESCC cells, which were mediated by two newly identified miR-424 targets PRKCD and WEE1, respectively. During G1/S transition, increased level of PRKCD protein in KYSE410-miRZip-424 cells resulted in increased activation of PRKCD and downstream p38MAPK and JNK, which enhanced the stability of p21Cip1 protein. p21Cip1 proceeded to block CDK2 activation during G1/S transition. On the other hand, level of CDC2 phosphorylation at Tyr15, the inactive state of CDC2, was increased as a result of increased WEE1 expression by miR-424 knockdown when cells were at the G2/M border. Further evaluation of miR-424 expression in various cell cycle phases showed that miR-424 expression increased along with pri-miR-424 and pre-miR-424 when cell cycle progressed from G0/G1- to S-phase in ESCC cells. ChIP results demonstrated that G1/S transcriptional activating regulator E2F1 was enriched in regions of the putative miR-424 promoter containing the predicted E2F1-binding sites. Here, our results proved that miR-424 functions as an onco-miRNA in ESCCs. Activation of E2F1 when ESCC cells at the G1/S border induced the transcription of miR-424, which in turn targeted the expression of key genes to promote cell cycle progression and cell proliferation of ESCCs. Citation Format: Jing Wen, Xiuying Xie, Jianhua Fu. Upregulation of miR-424 promotes cell cycle progression and cell proliferation in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 509.

S-phase Specific Downregulation of Human O6-Methylguanine DNA Methyltransferase (MGMT) and its Serendipitous Interactions with PCNA and p21cip1 Proteins in Glioma Cells

Whether the antimutagenic DNA repair protein MGMT works solo in human cells and if it has other cellular functions is not known. Here, we show that human MGMT associates with PCNA and in turn, with the cell cycle inhibitor, p21cip1 in glioblastoma and other cancer cell lines. MGMT protein was shown to harbor a nearly perfect PCNA-Interacting Protein (PIP box) motif. Isogenic p53-null H1299 cells were engineered to express the p21 protein by two different procedures. Reciprocal immunoprecipitation/western blotting, Far-western blotting, and confocal microscopy confirmed the specific association of MGMT with PCNA and the ability of p21 to strongly disrupt the MGMT-PCNA complexes in tumor cells. Alkylation DNA damage resulted in a greater colocalization of MGMT and PCNA proteins, particularly in HCT116 cells deficient in p21 expression. p21 expression in isogenic cell lines directly correlated with markedly higher levels of MGMT mRNA, protein, activity and greater resistance to alkylating agents. In other experiments, four glioblastoma cell lines synchronized at the G1/S phase using either double thymidine or thymidine-mimosine blocks and subsequent cycling consistently showed a loss of MGMT protein at mid- to late S-phase, irrespective of the cell line, suggesting such a downregulation is fundamental to cell cycle control. MGMT protein was also specifically degraded in extracts from S-phase cells and evidence strongly suggested the involvement of PCNA-dependent CRL4Cdt2 ubiquitin-ligase in the reaction. Overall, these data provide the first evidence for non-repair functions of MGMT in cell cycle and highlight the involvement of PCNA in MGMT downregulation, with p21 attenuating the process.

Activation of TNF-α/NF-κB axis enhances CRL4BDCAF11 E3 ligase activity and regulates cell cycle progression in human osteosarcoma cells.

Cullin 4B, a member of the Cullins, which serve as scaffolds to facilitate the assembly of E3 ligase complexes, is aberrantly expressed in many cancers, including osteosarcoma. Recently, we observed that CUL4B forms the CRL4BDCAF 11 E3 ligase, which specifically ubiquitinates and degrades the cyclin‐dependent kinase (CDK) inhibitor p21Cip1 in human osteosarcoma cells. However, the underlying mechanisms regarding the aberrant expression of CUL4B and the upstream members of this signaling pathway are mostly unknown. In this study, we demonstrate that nuclear factor kappaB (NF‐κB) is a direct modulator of CUL4B expression. The CUL4B promoter is responsive to several NF‐κB subunits, including RelA, RelB, and c‐Rel, but not to p50 or p52. Additional studies reveal that the tumor necrosis factor alpha (TNF‐α)/NF‐κB axis pathway is activated in human osteosarcoma cells. This activation causes both CUL4B and NF‐κB subunits to become abundant in the nucleus of human osteosarcoma cells. The down‐regulation of individual genes, including TNFR1, RelA, RelB, c‐Rel, and CUL4B, or pairs of them, including TNFR1 + RelA,TNFR1 + RelB,TNFR1 + c‐Rel, and RelA+CUL4B, has similar effects on cell growth inhibition, colony formation, cell invasion, and in vivo tumor formation, whereas the overexpression of CUL4B in these knockdown cells significantly reverses their phenotypes. The inhibition of the TNF‐α/NF‐κB pathway greatly attenuates CRL4BDCAF 11 E3 ligase activity and causes the accumulation of p21Cip1, thereby leading to cell cycle arrest at the S phase. Taken together, our results support a model in which the activation of the TNF‐α/NF‐κB axis contributes to an increase in CRL4BDCAF 11 activity and a decrease in p21Cip1 protein levels, thereby controlling cell cycle progression in human osteosarcoma cells.

A diphenyldiselenide derivative induces autophagy via JNK in HTB-54 lung cancer cells.

Symmetric aromatic diselenides are potential anticancer agents with strong cytotoxic activity. In this study, the in vitro anticancer activities of a novel series of diarylseleno derivatives from the diphenyldiselenide (DPDS) scaffold were evaluated. Most of the compounds exhibited high efficacy for inducing cytotoxicity against different human cancer cell lines. DPDS 2, the compound with the lowest mean GI50 value, induced both caspase‐dependent apoptosis and arrest at the G0/G1 phase in acute lymphoblastic leucemia CCRF‐CEM cells. Consistent with this, PARP cleavage; enhanced caspase‐2, ‐3, ‐8 and ‐9 activity; reduced CDK4 expression and increased levels of p53 were detected in these cells upon DPDS 2 treatment. Mutated p53 expressed in CCRF‐CEM cells retains its transactivating activity. Therefore, increased levels of p21CIP1 and BAX proteins were also detected. On the other hand, DPDS 6, the compound with the highest selectivity index for cancer cells, resulted in G2/M cell cycle arrest and caspase‐independent cell death in p53 deficient HTB‐54 lung cancer cells. Autophagy inhibitors 3‐methyladenine, wortmannin and chloroquine inhibited DPDS 6‐induced cell death. Consistent with autophagy, increased LC3‐II and decreased SQSTM1/p62 levels were detected in HTB‐54 cells in response to DPDS 6. Induction of JNK phosphorylation and a reduction in phospho‐p38 MAPK were also detected. Moreover, the JNK inhibitor SP600125‐protected HTB‐54 cells from DPDS 6‐induced cell death indicating that JNK activation is involved in DPDS 6‐induced autophagy. These results highlight the anticancer effects of these derivatives and warrant future studies examining their clinical potential.

Abstract 3069: The S-phase checkpoint function of MGMT provides an unexpected rational route to profoundly increase the efficacy of anticancer antimetabolites in cell culture and xenograft models

Abstract MGMT is an anti-mutagenic DNA repair protein highly expressed not only in brain tumors, but also breast cancers, melanoma and colon cancers, and confers drug resistance. Recently, we showed that human MGMT has non-repair functions with vital roles in cell cycle; MGMT underwent PCNA-dependent degradation in the S-phase along with p21cip1 and CDT1 proteins. MGMT inactivation by O6-benzylguanine (BG) or a pool of shRNAs in asynchronous or G1/S-phase synchronized cells led to the activation of a checkpoint resulting in a significant inhibition of DNA synthesis and greatly diminished progression of cells into S-phase. We hypothesized that the replication stress caused by MGMT inactivation will provide a means to combine the S-phase specific drugs for synergistic cytotoxicity. We screened the sensitivity of a panel of colon cancer and glioblastoma cell lines with this combination using MTT and clonogenic growth assays. Annexin V/PI and acridine orange/ EtBr cell stainings were used to quantify apoptosis. MGMT inhibition/depletion significantly reduced the IC50 (5 to 10-fold) for different antimetabolites (5-FU, gemcitabine, Ara-C) and enhanced the overall apoptotic cell death (>5-fold). Nevertheless, the synergistic effects strongly depended on time and administration schedule of these two agents. Thus, a pretreatment of BG or MGMT shRNA followed by 5-FU for or a simultaneous treatment of the two were far less cytotoxic than the antimetabolite first followed by BG or shRNA as in the post-treatment mode. While the pre or simultaneous MGMT inhibition significantly prolonged the antimetabolite-induced S-phase arrest, the BG-post-treatments resulted in the abrogation of antimetabolite-induced cell cycle block and rapid progression into premature mitosis and apoptosis. We observed an increased activation of checkpoint kinases (pChk1 or pChk2) during pre or simultaneous treatments. In contrast, the post-MGMT inhibition interfered with S-phase checkpoint activity and had an abnormal CDK2 activation. Finally, we verified the therapeutic potentiation of antimetabolites by BG using the HT29-luc2 subcutaneous xenografts developed in nude mice. BG (60mg/Kg), 5-FU (30 mg/Kg), gemcitabine (100 mg/Kg) and Ara-C (40 mg/Kg/dose) were administered through i.p. route once a week for 7 weeks. Tumor growth revealed a 50 to 70% more reduction in combination groups (antimetabolites+BG) compared with drugs alone. No significant changes in mouse body weight, liver functions, and whole blood counts were noted. A marked downregulation of ki-67 and increased levels of apoptotic markers (cleaved caspase-3, cleaved PARP) in was evident in tumor tissues. Collectively, our data suggest that MGMT inhibition is a rational strategy not only for alkylating agents but also for antimetabolites as well (supported by CPRIT RP130266 & RP170207 grants to KSS). Citation Format: AGM Mostofa, Debasish Basak, Hanumantha Madala, Surendra R. Punganuru, Kalkunte S. Srivenugopal. The S-phase checkpoint function of MGMT provides an unexpected rational route to profoundly increase the efficacy of anticancer antimetabolites in cell culture and xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3069. doi:10.1158/1538-7445.AM2017-3069

Abstract 2570: Induction of mild oxidative stress as a strategy for reactivation of mutant p53 proteins: KSS72, a small molecule derived from ethacrynic acid restores the biological functions of R248W/Q mutant

Abstract p53 gene mutations in 50% of human cancers drive the emergence of oncogenic genomes and confer resistance to anticancer drugs & apoptosis. Therefore, a pharmacological reactivation of p53 mutant proteins to their wild-type forms has emerged as an intense research area. We have shown that induction of oxidative protein modifications primarily underlie the mechanistic conversion of mutant to wt or wt-like p53 forms (Eur J Medchem 107:233, 2016; Int J Oncol 48:1426, 2016). R248W/Q DNA contact mutations in the p53 core are highly frequent and cause functional inactivation. No small molecules reactivating this p53 mutation are available. Here, we describe the design and synthesis of a small molecule KSS72 using a cell-based screening and its potency in reactivating the R248 p53 mutants. KSS72 is a non-diuretic hydrophobic analog [1-(2,3-dichloro-4-methoxyphenyl)-2-methylenebutan-1-one] of ethacrynic acid (Edecrin) obtained by removing the carboxylate side chain. KSS-72 retained the ability to bind with GSH, inhibit GSTs and induce marked redox-imbalance in tumor cells. KSS72 displayed preferential cytotoxicity (3-4 fold more cell killing) against cancer cells expressing the allele specific R248W mutation (MiaPaca-2) in comparison with the wt p53 or p53-null cancer cell lines. Further, we developed isogenic cell lines expressing the R248W and R248Q mutants in the p53-null H1299 lung cancer cells and evaluated their sensitivity towards KSS72; these models again verified the greater drug efficacy. To prove the hypothesis the higher efficacy of KSS72 stems from the reactivation of mutant p53, the conformation-specific antibodies (Pab1620 for wt and Pab420 for mutant) were employed. Immunoprecipitation and immunostaining using confocal microscopy showed that KSS72 treatment of R248W or R248Q p53-cells results in a marked reciprocal loss of mutant protein and gradual increase of wt-like protein. EMSA revealed a time-dependent restoration of DNA-binding of the mutant p53, which was accompanied by a distinct upregulation of pro-apoptotic signaling pathways mediated by PUMA, Bax, p21cip1 and MDM2 proteins. A strong G2/M arrest and significant apoptosis were observed. Other data showed that KSS72 restores the wt structure and function by covalently binding the p53R248 protein. Pharmacokinetics showed that KSS72 has excellent oral bioavailability with a half-life of 3.5 h. In MiaPaca-2 xenografts developed in nude mice, there was a marked tumor growth suppression with KSS72 as a single drug. In conclusion, KSS72 is an attractive lead compound for further development because p53 reactivation occurred without discernible organ toxicities after oral doses and a wide therapeutic window. The redox-sensitive cysteines involved in the p53 reactivation are being identified by mass spectrometry (supported by CPRIT grants RP130266 & RP170207 to KSS). Citation Format: Surendra R. Punganuru, Hanumantha Rao Madala, Kalkunte S. Srivenugopal. Induction of mild oxidative stress as a strategy for reactivation of mutant p53 proteins: KSS72, a small molecule derived from ethacrynic acid restores the biological functions of R248W/Q mutant [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2570. doi:10.1158/1538-7445.AM2017-2570

Abstract 2178: Growth inhibition of the crude extracts of Musa basjoo in human colon carcinoma cells

Abstract Musa basjoo (MB) is a tropical evergreen tree growing mainly in subtropical or tropical countries. MB has been used globally as a folk medicine such as antipyretic, diuretic, and hemostatic drugs for centuries but evidence-based biological activities and molecular mechanism of action of MB are unknown. Therefore, in the current study we examined whether the crude extracts of MB exert anticancer activity in HT29 and HCT116 human colon carcinoma cell lines. Dried leaves of MB samples were extracted with acetone or methanol. Crude extracts of MB were then dissolved in dimethylsulfoxide (DMSO) and used for the following experiments. Growth inhibition was determined by colony or MTT assays in these cell lines. Cells were treated with increasing concentrations (12.5 to 200 μg/mL in colony assays, 25 to 400 μg/mL in MTT assays) of acetone/methanol extracts of MB in DMEM/5%FBS. Untreated control cells were treated with DMSO alone. Crude extracts of MB inhibited the growth of cells with IC50 values of 118 µg/mL (acetone extract, HT29), 75 µg/mL (acetone extract, HCT116), >200 µg/mL (methanol extract, HT29), 141 µg/mL (methanol extract, HCT116) in colony assays, and with IC50 values of 137 µg/mL (acetone extract, HT29), 73 µg/mL (acetone extract, HCT116), 240 µg/mL (methanol extract, HT29), 248 µg/mL (methanol extract, HCT116) in MTT assays. Acetone extract was used in the flowcytometry and western blot analyses because it showed stronger growth inhibition than methanol extract in both cell lines. Flowcytometry analysis indicated that when HT29 and HCT116 cells were treated with 100 µg/mL acetone extract of MB for 96h, the percentage of cells in G1 increased by 5.4% and this was associated with a concomitant decrease of cells in the S and G2-M phases of the cell cycle. Acetone extract of MB did not cause subG1 fraction in either HT29 or HCT116 cell lines. The results indicate that acetone extract of MB causes carcinoma cells to arrest in the G1 phase. We then performed western blot analysis to determine whether treatment of carcinoma cells with acetone extract of MB alters cellular levels of the G1 cell cycle control proteins cyclin D1, cdk4 and the cell cycle inhibitor protein p21CIP1. When HT29 and HCT116 cells were treated with 50 and 100 µg/mL acetone extract of MB for 96h, there was a marked decrease in the levels of expression of the cyclin D1 and cdk4 proteins and a marked increase in the levels of expression of the p21CIP1 protein. Thus, a decrease in cyclin D1 may cooperate with the induction of p21CIP1 to arrest cells in G1 and thereby further contribute to MB-induced growth inhibition. Taken together, the crude extracts of MB contain active component(s) that exert growth inhibition of human colon carcinoma cells. The current study is the first systematic examination of the anticancer activity of MB and may provide a novel approach to the chemoprevention and/or chemotherapy of human colon carcinoma. Citation Format: Harutoshi Matsumoto, Saeko Ando, Katsumi Fukamachi, Mitsuru Futakuchi, Kazunori Kimura, Naoki Yoshimi, Masumi Suzui. Growth inhibition of the crude extracts of Musa basjoo in human colon carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2178. doi:10.1158/1538-7445.AM2017-2178

Inactivation of EGFR/AKT signaling enhances TSA-induced ovarian cancer cell differentiation.

Ovarian tumor is one of the most lethal gynecologic cancers, but differentiation therapy for this cancer is poorly characterized. Here, we show that thrichostatin A (TSA), the well known inhibitor of histone deacetylases (HDACs), can induce cell differentiation in HO8910 ovarian cancer cells. TSA-induced cell differentiation is characterized by typical morphological change, increased expression of the differentiation marker FOXA2, decreased expression of the pluripotency markers SOX2 and OCT4, suppressing cell proliferation, and cell cycle arrest in the G1 phase. TSA also induces an elevated expression of cell cycle inhibitory protein p21Cip1 along with a decrease in cell cycle regulatory protein cyclinD1. Significantly, blockage of epidermal growth factor receptor(EGFR) signaling pathway with specific inhibitors of this signaling cascade promotes the TSA-induced differentiation of HO8910 cells. These results imply that the EGFR cascade inhibitors in combination with TSA may represent a promising differentiation therapy strategy for ovarian cancer.

Cdk5 Directly Targets Nuclear p21CIP1 and Promotes Cancer Cell Growth.

The significance of Cdk5 in cell-cycle control and cancer biology has gained increased attention. Here we report the inverse correlation between the protein levels of Cdk5 and p21CIP1 from cell-based and clinical analysis. Mechanistically, we identify that Cdk5 overexpression triggers the proteasome-dependent degradation of p21CIP1 through a S130 phosphorylation in a Cdk2-independent manner. Besides, the evidence from cell-based and clinical analysis shows that Cdk5 primarily regulates nuclear p21CIP1 protein degradation. S130A-p21CIP1 mutant enables to block either its protein degradation or the increase of cancer cell growth caused by Cdk5. Notably, Cdk5-triggered p21CIP1 targeting primarily appears in S-phase, while Cdk5 overexpression increases the activation of Cdk2 and its interaction with DNA polymerase δ. The in vivo results show that Cdk2 might play an important role in the downstream signaling to Cdk5. In summary, these findings suggest that Cdk5 in a high expression status promotes cancer growth by directly and rapidly releasing p21CIP1-dependent cell-cycle inhibition and subsequent Cdk2 activation, which illustrates an oncogenic role of Cdk5 potentially applied for future diagnosis and therapy. Cancer Res; 76(23); 6888-900. ©2016 AACR.

11a-N-Tosyl-5-deoxi-pterocarpan, LQB-223, a novel compound with potent antineoplastic activity toward breast cancer cells with different phenotypes.

Multidrug resistance is the major obstacle for successful treatment of breast cancer, prompting the investigation of novel anticancer compounds. In this study, we tested whether LQB-223, an 11a-N-Tosyl-5-deoxi-pterocarpan newly synthesized compound, could be effective toward breast cancer cells. Human breast cell lines MCF-7, MDA-MB-231, HB4a and MCF-7 Dox(R) were used as models for this study. Cell culture, MTT and clonogenic assay, flow cytometry and Western blotting were performed. The LQB-223 decreased cell viability, inhibited colony formation and induced an expressive G2/M arrest in breast cancer cells. There was an induction in p53 and p21(Cip1) protein levels following treatment of wild-type p53 MCF-7 cells, which was not observed in the mutant p53 MDA-MB-231 cell line, providing evidence that the compound might act to modulate the cell cycle regardless of p53 status. In addition, LQB-223 resulted in decreased procaspase levels and increased annexin V staining, suggesting that the apoptotic cascade is also triggered. Importantly, LQB-223 treatment was shown to be less cytotoxic to non-neoplastic breast cells than docetaxel and doxorubicin. Strikingly, exposure of doxorubicin-resistant MCF-7-Dox(R) cells to LQB-223 resulted in suppression of cell viability and proliferation in levels comparable to MCF-7. Of note, MCF-7-Dox(R) cells have an elevated expression of the P-glycoprotein efflux pump when compared to MCF-7. Together, these results show that LQB-223 mediates cytotoxic effects in sensitive and resistant breast cancer cells, while presenting low toxicity to non-neoplastic cells. The new compound might represent a potential strategy to induce toxicity in breast cancer cells, especially chemoresistant cells.

Effects of a novel carbocyclic analog of pyrrolo[2,3-d]pyrimidine nucleoside on pleiotropic induction of cell death in prostate cancer cells with different androgen responsiveness.

Prostate cancer is the most frequently diagnosed cancer and is one of the leading causes of male cancer death in the world. Recently, in the course of our screening for a novel anticancer compound, we synthesized carbocyclic analogs of pyrrolo[2,3-d]pyrimidine nucleoside; compounds 5, and 6. In the current study, we report the effects of compound 5 on pleiotropic induction of cell death via up-regulation of AR-associated p21(Cip1) protein in prostate cancer cells with different androgen responsiveness, such as LNCaP (androgen-dependent and -sensitive), LNCaP(C4-2) (androgen-independent and -sensitive; androgen-refractory), and DU145 (androgen-independent and -insensitive) cells. The treatment of LNCaP cells with 6 μM compound 5 for 24 h stimulated the androgen receptor (AR) activity and dramatically up-regulated transcription (56-fold) of p21(Cip1), which, in turn, induces typical apoptosis in the cells. However, induction of apoptosis through up-regulation (23-fold) of AR-associated p21(Cip1) achieved in LNCaP(C4-2) cells was possible by intensive cell treatment with compound 5 (9 μM, 48 h), because the cells are less sensitive and independent to androgen than LNCaP cells. Furthermore, 6 μM compound 5-treated DU145 cells, which exhibit extremely low AR activation due to no androgen responsiveness and dependency, showed neither up-regulation of p21(Cip1) nor apoptotic induction. Instead, a different type of cell death, autophagy-like death through the LC3B-associated autophagosome formation, was obviously induced in DU145 cells. Taken together, our results suggest that pleiotropic induction of prostate cancer cell death by compound 5 is determined by how efficiently and how abundantly androgen-dependent activation of the AR occurs, whereas compound 6 shows no induction of apoptosis in LNCaP cells.

Methylseleninic Acid Superactivates p53-Senescence Cancer Progression Barrier in Prostate Lesions of Pten-Knockout Mouse.

Monomethylated selenium (MM-Se) forms that are precursors of methylselenol, such as methylseleninic acid (MSeA), differ in metabolism and anticancer activities in preclinical cell and animal models from seleno-methionine that had failed to exert preventive efficacy against prostate cancer in North American men. Given that human prostate cancer arises from precancerous lesions such as high-grade prostatic intraepithelial neoplasia (HG-PIN), which frequently have lost phosphatase and tensin homolog (PTEN) tumor suppressor permitting phosphatidylinositol-3-OH kinase (PI3K)-protein kinase B (AKT) oncogenic signaling, we tested the efficacy of MSeA to inhibit HG-PIN progression in Pten prostate-specific knockout (KO) mice and assessed the mechanistic involvement of p53-mediated cellular senescence and of the androgen receptor (AR). We observed that short-term (4 weeks) oral MSeA treatment significantly increased expression of P53 and P21Cip1 proteins and senescence-associated-β-galactosidase staining, and reduced Ki67 cell proliferation index in Pten KO prostate epithelium. Long-term (25 weeks) MSeA administration significantly suppressed HG-PIN phenotype, tumor weight, and prevented emergence of invasive carcinoma in Pten KO mice. Mechanistically, the long-term MSeA treatment not only sustained P53-mediated senescence, but also markedly reduced AKT phosphorylation and AR abundance in the Pten KO prostate. Importantly, these cellular and molecular changes were not observed in the prostate of wild-type littermates which were similarly treated with MSeA. Because p53 signaling is likely to be intact in HG-PIN compared with advanced prostate cancer, the selective superactivation of p53-mediated senescence by MSeA suggests a new paradigm of cancer chemoprevention by strengthening a cancer progression barrier through induction of irreversible senescence with additional suppression of AR and AKT oncogenic signaling.

TrkA is amplified in malignant melanoma patients and induces an anti-proliferative response in cell lines.

BackgroundThe nerve growth factor (NGF) receptor tyrosine-kinase TrkA is a well-known determinant of the melanocytic lineage, through modulation of the MAPK and AKT cascades. While TrkA gene is frequently rearranged in cancers, its involvement in malignant melanoma (MM) development is still unclear.MethodsWe analyzed a dataset of primary cutaneous MM (n = 31) by array comparative genomic hybridization (aCGH), to identify genomic amplifications associated with tumor progression. The analysis was validated by genomic quantitative PCR (qPCR) on an extended set of cases (n = 64) and the results were correlated with the clinical outcome. To investigate TrkA molecular pathways and cellular function, we generated inducible activation of the NGF-TrkA signaling in human MM cell lines.ResultsWe identified amplification of 1q23.1, where the TrkA locus resides, as a candidate hotspot implicated in the progression of MM. Across 40 amplicons detected, segmental amplification of 1q23.1 showed the strongest association with tumor thickness. By validation of the analysis, TrkA gene amplification emerged as a frequent event in primary melanomas (50 % of patients), and correlated with worse clinical outcome. However, experiments in cell lines revealed that induction of the NGF-TrkA signaling produced a phenotype of dramatic suppression of cell proliferation through inhibition of cell division and pronounced intracellular vacuolization, in a way straightly dependent on NGF activation of TrkA. These events were triggered via MAPK activity but not via AKT, and involved p21cip1 protein increase, compatibly with a mechanism of oncogene-induced growth arrest.ConclusionsTaken together, our findings point to TrkA as a candidate oncogene in MM and support a model in which the NGF-TrkA-MAPK pathway may mediate a trade-off between neoplastic transformation and adaptive anti-proliferative response.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1791-y) contains supplementary material, which is available to authorized users.

Role of RUNX2 in Breast Carcinogenesis.

RUNX2 is a transcription factor playing the major role in osteogenesis, but it can be involved in DNA damage response, which is crucial for cancer transformation. RUNX2 can interact with cell cycle regulators: cyclin-dependent kinases, pRB and p21Cip1 proteins, as well as the master regulator of the cell cycle, the p53 tumor suppressor. RUNX2 is involved in many signaling pathways, including those important for estrogen signaling, which, in turn, are significant for breast carcinogenesis. RUNX2 can promote breast cancer development through Wnt and Tgfβ signaling pathways, especially in estrogen receptor (ER)-negative cases. ERα interacts directly with RUNX2 and regulates its activity. Moreover, the ERα gene has a RUNX2 binding site within its promoter. RUNX2 stimulates the expression of aromatase, an estrogen producing enzyme, increasing the level of estrogens, which in turn stimulate cell proliferation and replication errors, which can be turned into carcinogenic mutations. Exploring the role of RUNX2 in the pathogenesis of breast cancer can lead to revealing new therapeutic targets.

Extra-Nuclear Signaling Pathway Involved in Progesterone-Induced Up-Regulations of p21cip1 and p27kip1 in Male Rat Aortic Smooth Muscle Cells

Previously, we demonstrated that progesterone (P4) at physiologic levels (5-500 nM) inhibited proliferation in cultured rat aortic smooth muscle cells (RASMCs) through a P4 receptor (PR)-dependent pathway. We also showed that P4-induced cell cycle arrest in RASMCs occurs when the cyclin-CDK2 system is inhibited just as p21cip1 and p27kip1 protein levels are augmented. In the present study, we further investigated the molecular mechanism underlying P4-induced up-regulations of p21cip1 and p27kip1 in RASMCs. We used pharmacological inhibitors as well as dominant negative constructs and conducted Western blot analyses to delineate the signaling pathway involved. Our data suggest that P4 up-regulated the expression of p21cip1 and p27kip1 in RASMCs through increasing the level of p53 protein mediated by activating the cSrc/Kras/Raf-1/AKT/ERK/p38/IκBα/NFκB pathway. The findings of the present study highlight the molecular mechanism underlying P4-induced up-regulations in p21cip1 and p27kip1 in RASMCs.

The effects of two polymorphisms on p21cip1 function and their association with Alzheimer's disease in a population of European descent.

With the exception of ApoE4, genome-wide association studies have failed to identify strong genetic risk factors for late-onset Alzheimer’s disease, despite strong evidence of heritability, suggesting that many low penetrance genes may be involved. Additionally, the nature of the identified genetic risk factors and their relation to disease pathology is also largely obscure. Previous studies have found that a cancer-associated variant of the cell cycle inhibitor gene p21cip1 is associated with increased risk of Alzheimer’s disease. The aim of this study was to confirm this association and to elucidate the effects of the variant on protein function and Alzheimer-type pathology. We examined the association of the p21cip1 variant with Alzheimer’s disease and Parkinson’s disease with dementia. The genotyping studies were performed on 719 participants of the Oxford Project to Investigate Memory and Ageing, 225 participants of a Parkinson’s disease DNA bank, and 477 participants of the Human Random Control collection available from the European Collection of Cell Cultures. The post mortem studies were carried out on 190 participants. In the in-vitro study, human embryonic kidney cells were transfected with either the common or rare p21cip1 variant; and cytometry was used to assess cell cycle kinetics, p21cip1 protein expression and sub-cellular localisation. The variant was associated with an increased risk of Alzheimer’s disease, and Parkinson’s disease with dementia, relative to age matched controls. Furthermore, the variant was associated with an earlier age of onset of Alzheimer’s disease, and a more severe phenotype, with a primary influence on the accumulation of tangle pathology. In the in-vitro study, we found that the SNPs reduced the cell cycle inhibitory and anti-apoptotic activity of p21cip1. The results suggest that the cancer-associated variant of p21cip1 may contribute to the loss of cell cycle control in neurons that may lead to Alzheimer-type neurodegeneration.

Tocotrienol-rich fraction prevents cellular aging by modulating cell proliferation signaling pathways.

Vitamin E has been suggested as nutritional intervention for the prevention of degenerative and age-related diseases. In this study, we aimed to elucidate the underlying mechanism of tocotrienol-rich fraction (TRF) in delaying cellular aging by targeting the proliferation signaling pathways in human diploid fibroblasts (HDFs). Tocotrienol-rich fraction was used to treat different stages of cellular aging of primary human diploid fibroblasts viz. young (passage 6), pre-senescent (passage 15) and senescent (passage 30). Several selected targets involved in the downstream of PI3K/AKT and RAF/MEK/ERK pathways were compared in total RNA and protein. Different transcriptional profiles were observed in young, pre-senescent and senescent HDFs, in which cellular aging increased AKT, FOXO3, CDKN1A and RSK1 mRNA expression level, but decreased ELK1, FOS and SIRT1 mRNA expression level. With tocotrienol-rich fraction treatment, gene expression of AKT, FOXO3, ERK and RSK1 mRNA was decreased in senescent cells, but not in young cells. The three down-regulated mRNA in cellular aging, ELK1, FOS and SIRT1, were increased with tocotrienol-rich fraction treatment. Expression of FOXO3 and P21Cip1 proteins showed up-regulation in senescent cells but tocotrienol-rich fraction only decreased P21Cip1 protein expression in senescent cells. Tocotrienol-rich fraction exerts gene modulating properties that might be responsible in promoting cell cycle progression during cellular aging.