Down-regulation Of phospho-Akt Research Articles

Quatramer™ encapsulation of dual-targeted PI3-Kδ/HDAC6 inhibitor, HSB-510, suppresses growth of breast cancer.

Multiple studies have shown that the progression of breast cancer depends on multiple signaling pathways, suggesting that therapies with multitargeted anticancer agents will offer improved therapeutic benefits through synergistic effects in inhibiting cancer growth. Dual-targeted inhibitors of phosphoinositide 3-kinase (PI3-K) and histone deacetylase (HDAC) have emerged as promising cancer therapy candidates. However, poor aqueous solubility and bioavailability limited their efficacy in cancer. The present study investigates the encapsulation of a PI3-Kδ/HDAC6 dual inhibitor into hybrid block copolymers (polylactic acid-methoxy polyethylene glycol; polylactic acid-polyethylene glycol-polypropylene glycol-polyethylene glycol-polylactic acid) (HSB-510) as a delivery system to target PI3-Kδ and HDAC6 pathways in breast cancer cells. The prepared HSB-510 showed an average diameter of 96 ± 3 nm, a zeta potential of -17 ± 2 mV, and PDI of ˂0.1 with a slow and sustained release profile of PI3-Kδ/HDAC6 inhibitors in a nonphysiological buffer. In vitro studies with HSB-510 have demonstrated substantial growth inhibition of breast cancer cell lines, MDA-MB-468, SUM-149, MCF-7, and Ehrlich ascites carcinoma (EAC)as well as downregulation of phospho-AKT, phospho-ERK, and c-Myc levels. Importantly, bi-weekly treatment of Balb/c wild-type mice harboring EAC cells with HSB-510 at a dose of 25 mg/kg resulted in significant tumor growth inhibition. The treatment with HSB-510 was without any significant effect on the body weights of the mice. These results demonstrate that a novel Quatramer encapsulation of a PI3-Kδ/HDAC6 dual inhibitor (HSB-510) represents an approach for the successful targeting of breast cancer and potentially other cancer types.

Silencing of FAM111B inhibits tumor growth and promotes apoptosis by decreasing AKT activity in ovarian cancer.

Ovarian cancer is the most lethal gynecological tumor in women worldwide. FAM111B (family with sequence similarity 111 member B) is an oncoprotein associated with multiple cancers, but its biological functions in ovarian cancer remain elusive. In this study, FAM111B was overexpressed in ovarian cancer tissues and cell lines. Functional studies in vitro revealed that silencing of FAM111B inhibited ovarian cancer cell proliferation, invasion, and migration, as well as increased cell apoptosis. Furthermore, FAM111B silencing arrested the ovarian cancer cell cycle at the G1/S phase. Furthermore, western blot assays demonstrated that silencing of FAM111B resulted in downregulation of phospho-AKT (p-AKT) protein expression, as well as upregulation of p53 and caspase-1 protein expression. The xenograft animal model of ovarian cancer demonstrated that FAM111B silencing inhibited tumor growth, enhanced cell apoptosis, and inhibited Ki-67 and proliferating cell nuclear antigen (PCNA) protein expression in vivo. Conversely, the overexpression of FAM111B exhibited opposite effects on the ovarian cancer xenograft. It was previously established that inactivating AKT inhibited ovarian cancer progression. This study found that silencing of FAM111B inhibits tumor growth and promotes apoptosis by decreasing AKT activity in ovarian cancer. Caspase-1 and p53 signaling also influenced the function of FAM111B in SKOV3 cells. Collectively, our results demonstrate that silencing of FAM111B is a potential therapeutic strategy against ovarian cancer.

Abstract 5406: Novel quatramer nano-formulation of dual-targeted PI3-Kδ/HDAC6 inhibitor, HSB-510, suppresses growth of triple negative breast cancer

Abstract Triple negative breast cancer (TNBC) is an aggressive subtype with limited treatment options and a worse clinical outcome as compared with other breast cancer subtypes. Multiple studies have shown that progression of TNBC depends on more than one signalling pathways, suggesting that the therapies with multi-targeted anti-cancer agents will offer improved therapeutic benefit through synergistic effects in inhibiting cancer growth. Dual-targeted inhibitors of phosphoinositide 3-kinase (PI3-K) and histone deacetylase (HDAC) have emerged as promising cancer therapy candidates. However, poor aqueous solubility and bioavailability limited their efficacy in cancer and accordingly, improved delivery systems are needed. The present studies have investigated the encapsulation of PI3-Kδ/HDAC6 dual inhibitor into hybrid block co-polymers (polylactic acid-methoxy polyethylene glycol; polylactic acid-polyethylene glycol-polypropylene glycol-polyethylene glycol-polylactic acid) (HSB-510) as a delivery system to target PI3-Kδ and HDAC6 pathways in TNBC cells. The prepared HSB-510 Quatramer nano-formulation showed an average diameter of 96 ±3 nm, the zeta potential of -17±2 mV, and PDI of ˂0.1 with a slow and sustained release profile of PI3-Kδ/HDAC6 inhibitors in a non-physiological buffer. In vitro studies with HSB-510 have demonstrated substantial growth inhibition of TNBC cell lines, MDA-MB-468, SUM-149 and Ehrlich ascites carcinoma (EAC) as well as downregulation of phospho-AKT, phospho-ERK and c-MYC levels. Importantly, bi-weekly treatment of BALB/c wild-type mice harbouring EAC cells (syngeneic mouse model) with HSB-510 at a dose of 25 mg/kg resulted in significant (76%) tumor growth inhibition (TGI). The treatment with HSB-510 was without any significant effect on the body weights of the mice. These results demonstrate that a novel Quatramer nano-formulation of a novel PI3-Kδ/HDAC6 dual inhibitor (HSB-510) represents an approach for successful targeting of TNBC and potentially other cancer types. Citation Format: Sachchidanand Tiwari, Suiyang Liu, Mohammad Anees, Neha Mehrotra, Ashish Thakur, Gregory Tawa, Gurmit Grewal, Richard M. Stone, Surender M. Kharbanda, Harpal Singh. Novel quatramer nano-formulation of dual-targeted PI3-Kδ/HDAC6 inhibitor, HSB-510, suppresses growth of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5406.

Altersolanol B, a fungal tetrahydroanthraquinone, inhibits the proliferation of estrogen receptor-expressing (ER+) human breast adenocarcinoma by modulating PI3K/AKT, p38/ERK MAPK and associated signaling pathways

The present study focused on the apoptosis-inducing effects and cellular signal-modulating properties of altersolanol B (AB), a minor fungal tetrahydroanthraquinone (THAQ) metabolite, in the estrogen receptor positive (ER+) human breast adenocarcinoma cell line, MCF-7. AB demonstrated approximately 4-fold greater antiproliferative activity in ER+ MCF-7 cells (IC50 5.5 μM) compared to the ER-negative (triple-negative) MDA-MB-231 (IC50 21.3 μM). The viability of normal breast fibrocystic epithelial cells, MCF-10A, was unaffected. AB induced intrinsic apoptosis in MCF-7 cells; it triggered the activation of caspase 9 and poly (ADP-ribose) polymerase (PARP), upregulated the expression of pro-apoptotic Bax, and downregulated the expression of anti-apoptotic Bcl-2. AB induced cell cycle arrest at G0/G1, as indicated by the downregulation of key checkpoint proteins operating at the G0/G1 phase of the cell cycle (cyclin D1, CDK4 and CDK2). The observed increase in p21Waf1/Cip1 and p53 expression may facilitate cell cycle arrest, and the subsequent induction of apoptosis. AB lacked significant effects on intracellular ROS levels, while it down-regulated nuclear factor erythroid 2-related factor 2 (Nrf2), and the Nrf2-dependent antioxidant enzyme, heme oxygenase-1. The compound disrupted AKT signaling through the downregulation of phospho-AKT and phospho-FOXO1, and the upregulation of PTEN, a phosphatase and tumor suppressor that negatively regulates the PI3K/AKT pathway. AB also disrupted the phosphorylation of AKT-controlled eukaryotic translation initiation factor, 4E-BP1, and GSK-3β, both of which are aberrantly regulated in human cancer. The AB-dependent downregulation of NF-κB was corroborated by the inhibition of TNFα-induced NF-κB activity as monitored in a luciferase reporter. The NF-κB inhibitory activity of AB was 3-fold more potent than that of the standard inhibitor, N-p-Tosyl-l-phenylalanine chloromethyl ketone. In addition to reducing the pro-survival effects of NF-кB, the inhibition of AKT phosphorylation by AB may also lead to FOXO1-mediated growth arrest and apoptosis. AB upregulated the expression of phospho-MKK4 and phospho-p38, and downregulated the expression of phospho-MEK1/2 and phospho-ERK1/2 indicating opposing effects on the two important oncogenic signaling cascades that are aberrantly activated in many cancers. AB disrupted both the AKT and ERK1/2 signaling pathways leading to apoptosis in ER+ MCF-7 cells through mitochondria-associated mechanisms coupled with the potent inhibition of NF-кB activation. The clinical limitations of multi-agent combination therapy that targets multiple pathways in cancer may potentially be circumvented by using a single molecule, such as AB, that inhibits both AKT and ERK1/2 signaling. Our preliminary study suggested that the THAQ pharmacophore, with its disrupted conjugated ring system and relative redox inactivity, may possess greater mechanistic advantage against ER+ breast cancer when compared to the fully conjugated ring systems of the anthraquinone that possess intrinsic redox activity and DNA interacting ability. This study supports the continued investigation of THAQs as lead molecules in anticancer drug discovery and development.

Alkaloids from Piper nigrum Synergistically Enhanced the Effect of Paclitaxel against Paclitaxel-Resistant Cervical Cancer Cells through the Downregulation of Mcl-1.

In the current study, nine amide alkaloids, including two new dimeric amides and a new natural product, were identified from Piper nigrum. Among them, seven compounds sensitized paclitaxel-resistant cervical cancer cells HeLa/PTX to paclitaxel. Piperine was a major component obtained from Piper nigrum, and its sensitization mechanism was investigated. Combination treatment enhanced cell apoptosis, which was mediated by downregulation of phospho-Akt and Mcl-1. Piperine (50 μM) combined with paclitaxel (200 nM) downregulated Mcl-1 protein expression with a decrease of 35.9 ± 9.5% ( P < 0.05). Moreover, overexpression of Mcl-1 attenuated the inhibitory effect of this combination. Furthermore, combination treatments of six dimeric amide alkaloids and paclitaxel all downregulated Mcl-1 protein expression with a decrease ranging from 23.5 ± 9.7% to 41.7 ± 7.2% ( P < 0.05). We reveal, for the first time, that dimeric amide alkaloids from plants possess a remarkable sensitization effect on cancer cells to paclitaxel.

The mechanism of Demethy1zeylasteral (ZST93) on autophagy in human pancreatic cancer cells

Objective To explore the mechanism of the novel tripterygium wilfordii hook F(TWHF) extract Demethylzeylasteral (ZST93) on autophagy of human pancreatic cancer (PC) cells. Methods Autophagy levels induced by ZST93 were evaluated by immunofluorescence and transmission electron microscopy in human pancreatic cancer cell lines. The activity levels of extracellular signal-regulated kinase1/2 (ERK1/2) and protein kinase B/mammalian target of rapamycin (Akt/mTOR) pathways were determined by Western blot in PC cells treated with different concentrations of ZST93. Immunofluorescence and transmission electron microscopy were used to evaluate the autophagy levels induced by ZST93 after inhibition of ERK1/2 pathway with PD 98059. Results Low concentrations of ZST93 could induce autophagy in PC cells. The LC3 expression were elevated to 3.4 and 19 times to control groups in MIA PaCa-2 and PANC-1 cells, respectively. And the number of autophagic vacuole were increased to 13 and 7 times to control groups, respectively. The knock-down of Beclin1 effectively inhibited the autophagy process induced by ZST93, with that LC3 expression reduced to 1.9 (P<0.05) and 2 (P<0.01) times to control groups in MIA PaCa-2 and PANC-1 cells, respectively, and the number of autophagic vacuole reduced to 2 (P<0.01) and 1 time (P<0.01) to control groups, respectively. The autophagy induced by low concentrations of ZST93 on PC cells was associated with the significant up-regulation of phospho-ERK1/2 (p-ERK1/2) and down-regulation of phospho-Akt (p-Akt) and phospho-mTOR (p-mTOR) expression. After introduction of low concentrations of ZST93, p-ERK/ERK was increased from 1.03±0.11 to 1.84±0.13 (P<0.01), while p-Akt/Akt and p-mTOR/mTOR were reduced from 0.98±0.07 to 0.51±0.05 (P<0.01) and from 1.00±0.03 to 0.63±0.02 (P<0.01), respectively, in MIA PaCa-2 cells. In PANC-1 cells, p-ERK/ERK was increased from 1.15±0.13 to 1.86±0.09 (P<0.01), while p-Akt/Akt and p-mTOR/mTOR were reduced from 1.02±0.06 to 0.43±0.05 (P<0.01) and from 0.99±0.02 to 0.61±0.01 (P<0.01), respectively. Treatment with high concentrations of ZST93 did not show such effects. Moreover, ERK1/2 pathway inhibitor PD 98059 could effectively inhibit the elevated level of autophagy induced by low-dose ZST93. Conclusion Low concentrations of ZST93 could induce Beclin1-dependent autophagy by activating ERK1/2 pathway and inhibiting Akt/mTOR pathway. Key words: Demethylzeylasteral; Autophagy; Pancreatic cancer; Extracellular signal-regulated kinase1/2 pathway; Protein kinase B/mammalian target of rapamycin pathway

Inhibitory effect of Au@Pt-NSs on proliferation, migration, and invasion of EJ bladder carcinoma cells: involvement of cell cycle regulators, signaling pathways, and transcription factor-mediated MMP-9 expression.

BackgroundAlthough the diverse biological properties of nanoparticles have been studied intensively, research into their mechanism of action is relatively rare. In this study, we investigated the molecular mechanisms of the anticancer activity of heterometallic Au@Pt-nanoseeds (NSs) against bladder cancers.Materials and methodsMode of action of Au@Pt-NSs was investigated through MTT assay, flow cytometry analysis, Western immunoblots, real-time qPCR, wound-healing migration and invasion assays, zymography, and electrophoretic mobility shift assay (EMSA).ResultsTreatment with Au@Pt-NSs significantly inhibited the proliferation of EJ cells in a dose-dependent manner by inducing G1 phase cell cycle arrest. Among the regulators associated with the G1 cell cycle phase, CDK2, CDK4, cyclin D1, cyclin E, and p21WAF1 were shown to participate in the inhibitory pathways of Au@Pt-NSs. In addition, treatment with Au@Pt-NSs led to upregulation of phospho-p38 MAPK and downregulation of phospho-AKT in EJ cells. Interestingly, Au@Pt-NSs inhibited the migratory and invasive potential of the cells, which was attributed to the suppression of the enzymatic activity of matrix metalloproteinase-9 (MMP-9). Using MMP-9-specific oligonucleotides, we showed that transcription factors such as NF-κB and Sp-1 were responsible for the MMP-9-mediated metastatic potential of EJ cells.ConclusionAu@Pt-NSs significantly limited the progression, migration, and invasion of bladder cancer EJ cells. Our data represent a novel insight into developing cisplatin-like chemotherapeutic reagents with fewer side effects and provide useful information on molecular markers to monitor patients under Au@Pt-NSs-based chemotherapy.

Nrf2 protects against acute lung injury and inflammation by modulating TLR4 and Akt signaling.

Lung injury, which is associated with systemic inflammatory responses, is a common problem with significant morbidity and mortality. Here, we examined the involvement of toll-like receptor 4 (TLR4) and nuclear factor erythroid 2-related factor 2 (Nrf2) on intestinal ischemia-reperfusion (I/R)-induced lung injury in vivo and in vitro. Analysis of lung tissues in Nrf2-knockout mice by western blotting, immunohistochemistry, and TUNEL assay, and analysis of bronchoalveolar lavage fluid showed that Nrf2 deficiency upregulated TLR4, enhanced I/R-induced lung injury, apoptosis, inflammation, and autophagy, and increased the I/R-induced inactivation of Akt. In mouse lung epithelial cells subjected to oxygen and glucose deprivation/reperfusion (OGD/Rep), Nrf2 silencing increased the OGD/Rep-induced upregulation of TLR4 and MyD88 and downregulation of HO-1, and exacerbated OGD/Rep-induced apoptosis, autophagy, and the downregulation of phospho-Akt. TLR4 silencing and Akt inhibition experiments indicated that OGD/Rep-induced cell death by suppressing Akt signaling, and Nrf2 protected lung cells by modulating TLR4 and Akt signaling. These results indicated that the Nrf2/TLR4/Akt axis plays a role in inflammation-associated lung damage, suggesting potential therapeutic targets for the treatment of lung injury.

Abstract 2287: Final results of the pharmacodynamic (PD) data of PQR309-001, a first-in-human trial of a combined PI3K/mTOR inhibitor in advanced solid tumors

Abstract Background: PQR309 is a novel, oral, balanced pan-class 1 PI3K, mTORC1 and mTORC2 inhibitor. PQR309-001 was a first-in-man dose escalation study evaluating PQR309 in patients with advanced solid tumours. The primary objective of the study was to establish the maximum tolerated dose (MTD) of PQR309, its safety and tolerability. Secondary objectives included characterization of PQR309 pharmacokinetics (PK) and pharmacodynamics (PD). Methods: We investigated PQR309 treatment-induced PD, including changes of 16 PI3K-mTOR and MAPK associated phospho-proteins, and 84 mRNAs in serial tumor biopsies obtained from patients enrolled in study PQR309-001. 13 patients were eligible for PD analysis after histologic evaluation of fresh-frozen tissue. A solid-phase assay with phospho-specific antibodies was used for the investigation of PI3K/mTOR and MAPK signalling. RNA analysis was performed with an RTK/PI3K-specific RNA array. Results: Under treatment with PQR309, phospho-Akt (p_T308 and p_S473), phospho-mTOR and phospho-S6 were significantly downregulated compared to baseline (p&amp;lt;0.05, Wilcoxon signed-rank test). Moreover, patients with radiographic tumor shrinkage (n = 7) had significantly stronger phospho-Akt (p_T308), phospho-mTOR and phospho-S6 reduction than patients whose tumor was growing (n = 6) (p&amp;lt;0.05, Mann-Whitney test). Importantly, ERK1/2 phosphorylation, as the readout of MAPK activity, was decreased overall with no statistically significant difference between shrinking and growing tumors. The analysis of mRNAs showed a trend towards upregulation of PDGFRA (3.2 times, not statistically significant) after 21 days of therapy with PQR309. Intriguingly, no consistent changes of other PI3K-related RNAs was observed. Conclusion: PD assessments in tumour tissue samples from patients treated with PQR309 demonstrated a reduction of phosphorylation of direct targets for PQR309 (PI3K and mTOR) as well as downstream effectors of mTOR. Furthermore, the data indicate that downregulation of phospho-Akt (p_T308) and mTORC1 activity may be predictive of tumor shrinkage while upregulation of PDGFRA may represent a mechanism of resistance against PI3K/mTOR inhibition. Citation Format: Andreas Wicki, Vincent Prêtre, Reto Ritschard, Nicholas Brown, Vincent Bize, Thomas Fabbro, Natasa Cmiljanovic, Sasa Dimitrijevic, Deborah Schmitz, Michael Stumm, Rebecca Kristeleit. Final results of the pharmacodynamic (PD) data of PQR309-001, a first-in-human trial of a combined PI3K/mTOR inhibitor in advanced solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2287.

Effects of RAF inhibitors on PI3K/AKT signalling depend on mutational status of the RAS/RAF signalling axis

Targeted therapies within the RAS/RAF/MEK/ERK signalling axis become increasingly popular, yet cross-talk and feedbacks in the signalling network lead to unexpected effects. Here we look systematically into how inhibiting RAF and MEK with clinically relevant inhibitors result in changes in PI3K/AKT activation. We measure the signalling response using a bead-based ELISA, and use a panel of three cell lines, and isogenic cell lines that express mutant forms of the oncogenes KRAS and BRAF to interrogate the effects of the MEK and RAF inhibitors on signalling. We find that treatment with the RAF inhibitors have opposing effects on AKT phosphorylation depending on the mutational status of two important oncogenes, KRAS and BRAF. If these two genes are in wildtype configuration, RAF inhibitors reduce AKT phosphorylation. In contrast, if BRAF or KRAS are mutant, RAF inhibitors will leave AKT phosphorylation unaffected or lead to an increase of AKT phosphorylation. Down-regulation of phospho-AKT by RAF inhibitors also extends to downstream transcription factors, and correlates with apoptosis induction. Our results show that oncogenes rewire signalling such that targeted therapies can have opposing effects on parallel pathways, which depend on the mutational status of the cell.

Abstract 1490: MiR-21 has strong prognostic implications and functions as an oncogenic miR by modulating PI3K/Akt pathway at multiple levels in diffuse large B cell lymphoma

Abstract MicroRNA (miR)-21, -17-92 cluster and -155 have been known to play important roles in the development and progression of various cancer types. However, effects of their expression level on the patient's prognosis in diffuse large B cell lymphoma (DLBCL) have not been fully elucidated. Thus, we investigated the association of miR-21, -17-92 cluster and -155 expressions with clinicopathologic features and prognosis in patients with DLBCL. Quantitative real-time reverse transcriptase-polymerase chain reaction analysis was performed to evaluate the expression level of miR-21, -17-92 cluster and -155 in 200 cases of DLBCL, and 11 tonsils as control, using formalin-fixed paraffin-embedded tissues. Role of miR-21 in DLBCL was also analyzed using several DLBCL cell lines. MiR-21, -17-92 cluster and -155 expressions were significantly increased in DLBCL compared with control (p=0.012, p=0.001 and p&amp;lt;0.0001, respectively). MiR-21 up-regulation was more frequently observed with higher stage (p=0.004) and higher International Prognostic Index (IPI) (p=0.043) and miR-17-92 cluster up-regulation was more common with older age (p=0.019) and higher performance status (PS) (p=0.012). Moreover, miR-21 and -17-92 cluster up-regulations were significantly associated with decreased progression free survival (PFS) (p=0.001 and p=0.014, respectively) and overall survival (OS) (p=0.002 and p=0.012, respectively). In the Cox proportional hazard model, overexpression of miR-21 was found to be an independently poor predictor for OS (HR=1.72; p=0.043). Importantly, high miR-21 expression showed stronger prediction for OS in rituximab treated (HR=2.62; p=0.017) and germinal center B-like (HR=11.91; p&amp;lt;0.001) subgroups. MiR-21 expression was inversely correlated with PTEN and FOXO1 expression in DLBCL cell lines. PTEN and FOXO1 expressions were increased by inhibition of miR-21 in SU-DHL4 and SU-DHL5 cell lines. Inhibition of miR-21 suppressed cell proliferation and sensitized these cells to doxorubicin cytotoxicity, which was accompanied by down-regulation of phospho-Akt. In addition, Bim, a transcriptional target of FOXO1, was increased by miR-21 inhibitor at the transcriptional level. Expression and functional activity of MDR1, which is known to be up-regulated by mTOR, were suppressed by miR-21 inhibitor. Together these data suggest that miR-21 might contribute to activation of PI3K/Akt pathway by down-regulating PTEN at an upstream level, and moreover suppress the expression of FOXO1, a tumor suppressive transcription factor at a downstream level, putatively in a direct and indirect manner. In conclusion, up-regulation of miR-21 is considered to play an important oncogenic role in DLBCL and have a strong prognostic implication. Citation Format: Heounjeong Go, Ji-Young Jang, Soo Jeong Nam, Young-Goo Kim, Jin Ho Paik, Tae Min Kim, Dae Seog Heo, Chul-Woo Kim, Yoon Kyung Jeon. MiR-21 has strong prognostic implications and functions as an oncogenic miR by modulating PI3K/Akt pathway at multiple levels in diffuse large B cell lymphoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1490. doi:10.1158/1538-7445.AM2014-1490

CacyBP/SIP protein is important for the proliferation of human glioma cells.

Recently, calcyclin-binding protein or Siah-1-interacting protein (CacyBP/SIP), a component of a novel ubiquitinylation pathway, could regulate the β-catenin degradation (Fukushima et al., Immunity 2006, 24, 29-39). However, the potential role of CacyBP/SIP itself in human glioma cells has not been clarified. Here, we found that CacyBP/SIP was expressed highly in human glioma tissues. Silencing of CacyBP/SIP by short-hairpin RNA severely suppressed the proliferation of human glioma cell U251, which was at least partly mediated by downregulation of phospho-Akt (p-Akt) and phospho-β-catenin (p-β-catenin) as well as upregulation of p53 and p21. Furthermore, overexpression of CacyBP/SIP obviously promoted the proliferation of human glioma U251, which exhibited the exactly contrary trend in the expression of p-Akt, p-β-catenin, p53, and p21. Taken together, these findings suggest that CacyBP/SIP plays important roles in the proliferation of human glioma cell which might be involved in the development of human glioma.

Antitumor Activities Of An Oral Selective HSP90α/β Inhibitor, TAS-116, In Combination With Bortezomib In Multiple Myeloma

Heat-shock protein 90 (HSP90) is a molecular chaperone considered to be attractive therapeutic target in many cancers, including multiple myeloma (MM), since HSP90 inhibition triggers downregulation /degradation of numerous client proteins to trigger apoptosis. In this study, we examined the anti-MM activities of a novel and orally bioavailable HSP90α/β inhibitor TAS-116 (TAIHO PHARMACEUTICAL CO., LTD., Japan) in MM. TAS-116 triggered downregulation of known client proteins associated with significant growth inhibition in MM cell lines and patient MM, without cytotoxicity in normal peripheral blood mononuclear cells. Importantly, TAS-116 showed cytotoxicity even in 17-allylamino-17-demethoxygeldanamycin (17-AAG)-resistant acute B cell leukemia cell line NALM6. TAS-116 induced cell cycle arrest, associated with downregulation of cell cycle regulatory proteins; followed by apoptosis, evidenced by increased annexin V-positive staining, as well as cleavage of caspases and PARP. Since the bone marrow (BM) microenvironment plays a crucial role in MM pathogenesis including drug resistance, we further examined the effect of TAS-116 in the presence of BM stromal cells (BMSCs). TAS-116 induced significant cytotoxicity in MM cells even in the presence of BMSCs, associated with downregulation of phospho-Akt and phospho-ERK. In addition, neither exogenous IL-6 nor IGF-1 was able to protect against TAS-116. Importantly, TAS-116 did not induce cytotoxicity in BMSCs. Our previous studies have shown that combined HSP90 inhibitor and proteasome inhibitor treatment induces synergistic MM cell death; therefore we similarly examined whether TAS-116 enhances bortezomib-induced cytotoxicity. TAS-116 enhanced bortezomib-induced cytotoxicity in MM.1S and RPMI-8226 cells, associated with decreased phospho-Akt, phospho-RelA (p65), phospho-IkBa, and phospho-IKKα/β; as well as increased CHOP and PARP cleavage. These results suggest that TAS-116 blocks bortezomib-induced canonical NF-κB pathway and augments bortezomib-induced endoplasmic reticulum (ER) stress. Finally, we examined the in vivo efficacy of TAS-116 in combination with bortezomib using a xenograft murine model of human MM. Although both monotherapy of TAS-116 and bortezomib were effective, TAS-116 in combination with bortezomib significantly enhanced growth inhibition (80%, p&lt;0.05) and prolonged overall survival (29 vs 57 days, p&lt;0.001). Immunohistochemical analysis of harvested human MM confirmed a significant increase in cleaved caspase-3- and TUNEL-positive cells in TAS-116 animals. These treatments were well tolerated, and no significant body weight loss of the animals was observed. Taken together, our studies show that the novel and selective HSP90α/β inhibitor TAS-116 blocks MM cell growth both in vitro and in vivo, providing the preclinical framework for clinical evaluation of this agent to improve patient outcome in MM. Disclosures: Hideshima: Acetylon Pharmaceuticals: Consultancy. Ohkubo:TAIHO PHARMACEUTICAL Co.,LTD: Employment. Richardson:Millenium: Consultancy; Celgene: Consultancy; Johnson &amp; Johnson: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Munshi:Celgene: Consultancy; Novartis: Consultancy; Millennium: Consultancy. Utsugi:TAIHO PHARMACEUTICAL CO.,LTD: Employment. Anderson:celgene: Consultancy; onyx: Consultancy; gilead: Consultancy; sanofi aventis: Consultancy; oncopep: Equity Ownership; acetylon: Equity Ownership.

TAS-117, a Novel Selective Akt Inhibitor Demonstrates Significant Growth Inhibition in Multiple Myeloma Cells in Vitro and in Vivo

AbstractAbstract 942The PI3K/Akt pathway mediates multiple myeloma (MM) cell growth and drug resistance, and targeting this molecule is a promising therapeutic option. In this study, we examined anti-MM activities of TAS-117 (TAIHO PHARMACEUTICAL CO., LTD., JAPAN), a selective potent Akt inhibitor in MM cell lines including MM.1S, MM.1R, OPM1 and H929 cells with high level of baseline Akt phosphorylation. TAS-117 induced significant growth inhibition in these cell lines, associated with downregulation of phosphorylation (Ser473 and Thr308) of Akt and downstream molecule FKHR/FKHRL1, without cytotoxicity in normal peripheral blood mononuclear cells. TAS-117 triggered G0/G1 arrest followed by apoptosis, evidenced by increased annexin V-positive cells, in both MM.1S and H929 cell lines. Apoptosis was further confirmed by cleavage of caspase-8, -3 and PARP. Interestingly, TAS-117 also induced: autophagy, evidenced by increased LC3-II; as well as endoplasmic reticulum (ER) stress, confirmed by induction of phospho-eIF2α, phospho-IRE1α and a molecular chaperone BiP/GRP78. Since the bone marrow (BM) microenvironment plays a crucial role in MM cell pathogenesis including drug resistance, we further examined the effect of TAS-117 in the presence of BM stromal cells (BMSCs). TAS-117 induced significant cytotoxicity in MM cells even in the presence of BMSCs, associated with downregulation of phospho-Akt. Importantly, TAS-117 inhibited secretion of IL-6 from BMSCs, and exogenous IL-6 and IGF-1 did not block cytotoxicity induced by this agent. We have previously shown the bortezomib activates Akt, and that Akt inhibition with bortezomib triggers synergistic MM cell cytotoxicity. TAS-117 enhanced bortezomib-induced cytotoxicity in MM.1S cells, associated with increased CHOP followed by PARP cleavage, suggesting that TAS-117 augments bortezomib-induced ER stress and apoptotic signaling. TAS-117 also enhanced cytotoxicity induced by other therapeutic agents (ie, rapamycin, dexamethasone, 17-AAG) in MM.1S cells. Finally, we examined anti-MM activities of TAS-117 in a xenograft murine model. Oral administration of TAS-117 for 14 days significantly inhibited growth of H929 plasmacytoma and was well tolerated. Taken together, the novel and selective Akt inhibitor TAS-117 blocks MM cell growth in vitro and in vivo, providing the preclinical framework for clinical evaluation of this agent to improve patient outcome in MM. Disclosures:Shimomura:TAIHO PHARMACEUTICAL CO., LTD.: Employment. Utsugi:TAIHO PHARMACEUTICAL CO., LTD.: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene, Millennium, BMS, Onyx: Membership on an entity's Board of Directors or advisory committees; Acetylon, Oncopep: Scientific Founder, Scientific Founder Other.

Targeting notch pathway enhances rapamycin antitumor activity in pancreas cancers through PTEN phosphorylation

BackgroundPancreas cancer is one of most aggressive human cancers with the survival rate for patients with metastatic pancreas cancer at 5-6 months. The poor survival demonstrates a clear need for better target identification, drug development and new therapeutic strategies. Recent discoveries have shown that the role for Notch pathway is important in both development and cancer. Its contribution to oncogenesis also involves crosstalks with other growth factor pathways, such as Akt and its modulator, PTEN. The mounting evidence supporting a role for Notch in cancer promotion and survival suggests that targeting this pathway alone or in combination with other therapeutics represents a promising therapeutic strategy.ResultsUsing a pancreas cancer tissue microarray, we noted that Jagged1, Notch3 and Notch4 are overexpressed in pancreas tumors (26%, 84% and 31% respectively), whereas Notch1 is expressed in blood vessels. While there was no correlation between Notch receptor expression and survival, stage or tumor grade, Notch3 was associated with Jagged1 and EGFR expression, suggesting a unique relationship between Notch3 and Jagged1. Inhibition of the Notch pathway genetically and with gamma-secretase inhibitor (GSI) resulted in tumor suppression and enhanced cell death. The observed anti-tumor activity appeared to be through Akt and modulation of PTEN phosphorylation. We discovered that transcriptional regulation of RhoA by Notch is important for PTEN phosphorylation. Finally, the mTOR inhibitor Rapamycin enhanced the effect of GSI on RhoA expression, resulting in down regulation of phospho-Akt and increased in vitro tumor cytotoxity.ConclusionsNotch pathway plays an important role in maintaining pancreas tumor phenotype. Targeting this pathway represents a reasonable strategy for the treatment of pancreas cancers. Notch modulates the Akt pathway through regulation of PTEN phosphorylation, an observation that has not been made previously. Furthermore, we discovered that this regulation is dependent on RhoA/Rock1 activation. Enhanced phospho-Akt suppression when GSI is combined with rapamycin suggests that targeting both pathways will lead to a greater efficacy in the treatment of patients with pancreas cancer.

Inhibition of activated receptor tyrosine kinases by Sunitinib induces growth arrest and sensitizes melanoma cells to Bortezomib by blocking Akt pathway

Despite the use of multiple therapeutic strategies, metastatic melanoma remains a challenge for oncologists. Thus, new approaches using combinational treatment may be used to try to improve the prognosis of this disease. In this report, we have analyzed the expression of receptor tyrosine kinases (RTKs) in melanoma specimens and in four metastatic melanoma cell lines. Both melanoma specimens and cell lines expressed RTKs, suggesting that they may represent eventual targets for multitargeted tyrosine kinase inhibitor, Suntinib. Sunitinib reduced the proliferation of two melanoma cell lines (M16 and M17) and increased apoptosis in one of them (M16). Moreover, the two metastatic melanoma cell lines harbored an activated receptor (PDGFRα and VEGFR, respectively), and Sunitinib suppressed the phosphorylation of the RTKs and their downstream targets Akt and ribosomal protein S6, in these two cell lines. Similar results were obtained when either PDGFRα or VEGFR2 expression was silenced by lentiviral-mediated short-hairpin RNA delivery in M16 and M17, respectively. To evaluate the interaction between Sunitinib and Bortezomib, median dose effect analysis using MTT assay was performed, and combination index was calculated. Bortezomib synergistically enhanced the Sunitinib-induced growth arrest in Sunitinib-sensitive cells (combination index < 1). Moreover, LY294002, a PI3K inhibitor, sensitized melanoma cells to Bortezomib treatment, suggesting that downregulation of phospho-Akt by Sunitinib mediates the synergy obtained by Bortezomib + Sunitinib cotreatment. Altogether, our results suggest that melanoma cells harboring an activated RTK may be clinically responsive to pharmacologic RTK inhibition by Sunitinib, and a strategy combining Sunitinib and Bortezomib, may provide therapeutic benefit.

The Role of Pea3 Group Transcription Factors in Esophageal Squamous Cell Carcinoma

The transcription factors Pea3, Erm, and Er81 can promote cancer initiation and progression in various types of solid tumors. However, their role in esophageal squamous cell carcinoma (ESCC) has not been elucidated. In this study, we found that the expression levels of Pea3 and Erm, but not that of Er81, were significantly higher in ESCC compared with nontumor esophageal epithelium. A high level of Pea3 expression was significantly correlated with a shorter overall survival in a cohort of 81 patients with ESCC and the subgroup with N1 stage tumor (Wilcoxon-Gehan test, P = 0.016 and P = 0.001, respectively). Pea3 was overexpressed in seven ESCC cell lines compared with two immortalized esophageal cell lines. Pea3 knockdown reduced cell proliferation and suppressed nonadherent growth, migration, and invasion in ESCC cells in vitro. In addition, Pea3 knockdown in ESCC cells resulted in a down-regulation of phospho-Akt and matrix metalloproteinase 13, whereas a significant positive correlation in the expression levels was observed between Pea3 and phospho-Akt (r = 0.281, P < 0.013) and between Pea3 and matrix metalloproteinase 13 in the human specimens (r = 0.462, P < 0.001). Moreover, Pea3 modulated the sensitivity of EC109 cells to doxorubicin, probably via reduced activity of the phosphatidylinositol 3-kinase-Akt-mammalian target of Rapamycin complex 1 pathway on Pea3 knockdown. In conclusion, our results suggest that Pea3 plays an important role in the progression of ESCC.

IGFBP-3 Inhibits the Proliferation of Neural Progenitor Cells

Insulin like growth factor-1 (IGF-1) plays an important role in the proliferation and differentiation of neural progenitor cells. The effects of IGF-1 can be regulated by insulin like growth factor binding protein-3 (IGFBP-3) which can either inhibit or stimulate the proliferation of cells depending on the expression of proteases that can release IGF-1 from IGF1-IGFBP3 complex. Although IGF-1 is essential for the development of brain, both IGFBP-3 and IGF-1 are elevated in the brains of children younger than 6 months of age. Likewise, IGFBP-3 is also upregulated following cerebral ischemia and hypoxia. However, the role of IGFBP-3 in neurogenesis is not clear. Using an in vitro culture system of rat neural progenitor cells, we demonstrate that IGFBP-3 specifically regulates the IGF-1 mediated neural progenitor cell proliferation via down regulation of phospho-Akt, and cyclin D1. In addition, IGFBP-3 also decreased the content of nestin in the neural progenitor cells indicating its potential role in neurogenesis.

Histone Deacetylase Inhibitors Potentiate the Lethality of the Irreversible Proteasome Inhibitor Carfilzomib In Mantle Cell Lymphoma Cells In Vitro and In Vivo

AbstractAbstract 3938The proteasome inhibitor (PI) bortezomib (BTZ) is approved for the treatment of refractory mantle cell lymphoma (MCL). However, BTZ achieves clinical responses in only 40% of refractory MCL patients, justifying the search for newer proteasome inhibitors and/or the development of rational combination regimens. Recently we reported that the irreversible PI carfilzomib (CFZ) interacted synergistically with the histone deacetylase inhibitor (HDACI) vorinostat (Vor) in diverse DLBCL cells including GC (germinal center) and ABC (activated B-cell) sub-types both in vitro and in vivo. (Dasmahapatra et al., Blood 2010; 115:4478-87, 2010). Moreover, regimens combining PIs with histone deacetylase inhibitors (HDACIs) have shown promising activity in multiple myeloma. Collectively, these preclinical and clinical findings raise the possibility combining CFZ with HDACIs might be an effective strategy in MCL. To test this possibility, CFZ/HDACI interactions were investigated in diverse MCL cell types, including those resistant to BTZ. Simultaneous treatment (24-48 hr) with sub-lethal CFZ concentrations (2-5 nM) and minimally toxic concentrations of vorinostat (Vor; 1–2 μM), SNDX-275 (1-2 μM) or SBHA (30-50 μM) strikingly increased apoptosis (manifested by annexin V, 7-AAD or TUNEL positivity) in various MCL lines, including Granta, HF-4B, Rec-1, JVM-2, MINO and JVM-12. Interactions were highly synergistic, as determined by Median Dose Effect analysis, with Combination Index values significantly less than 1.0. HDACIs also interacted synergistically with ONX 0912 (formerly PR-047), an orally bioavailable analog of CFZ. The activity of these regimens was associated with a sharp increase in caspase-3 activation, PARP cleavage, mitochondrial damage (loss of ΔΨm, cytoplasmic cytochrome c release), and induction of p21CIP1. Combined CFZ/Vor exposure also markedly induced phosphorylation of JNK and c-Jun, down-regulation of phospho-AKT and phospho-ERK1/2, and induction of γH2A.X, a marker of double-stranded DNA breaks. In contrast to single agent administration (48 hr), where the percentage of apoptotic cells gradually declined following drug washout over the ensuing 4–5 days, extensive apoptosis (e.g. > 80%) persisted in cells co-treated with CFZ/Vor. Combined treatment of MCL cells with CFZ/Vor induced more sustained inhibition of chymotrypsin-like (CT-L) proteasome activity than that observed following single-agent treatment. CFZ alone exhibited partial cross resistance to BTZ- resistant Granta cells (GR-25BR). However, co-administration of CFZ/Vor resulted in the highly synergistic induction of apoptosis in BTZ-resistant cells. Genetic interruption of JNK signaling (e.g., via shRNA knockdown) significantly attenuated CFZ/Vor-mediated apoptosis, indicating that JNK activation plays a functional role in the lethality of this regimen in MCL cells. Combined treatment with CFZ and HDACIs induced G2M arrest in both parental and BTZ-resistant GR-25BR cells, an effect that was not modified by genetic interruption of JNK signaling. The CFZ/Vor regimen also strikingly induced apoptosis in 3 primary human MCL specimens, in contrast to the minimal lethality exhibited toward normal CD34+ cells as previously described (Blood; 115:4478-87, 2010). Finally, in vivo administration of CFZ (IV BIW) and Vor (IP TIW) to Beige-nude-XID mice (NIH-III) inoculated in the flank with Granta cells substantially suppressed tumor growth compared to single agent treatment. Collectively, these findings indicate that combining HDACIs with CFZ synergistically induces apoptosis in MCL cells through a JNK-dependent mechanism in association with G2M arrest and the induction of DNA damage. They also suggest that this strategy, which is active against diverse MCL cell types, including BTZ-resistant and primary MCL cells, and which displays in vivo activity, warrants further examination in MCL. Accordingly, plans for a Phase I CFZ/HDACI trial in NHL, including MCL patients, are currently underway. Supported by Lymphoma SPORE 1P50 CA130805. Disclosures:Friedberg:Genentech: Honoraria.

Abstract 1575: Bortezomib selectively induces apoptosis in hematological malignant cells through down-regulation of phospho-Akt independent of its proteasome inhibitory activity

Abstract Background: Bortezomib, a proteasome inhibitor (PI), has excellent clinical activity in patients with multiple myeloma and mantle cell lymphoma. Our previous study has shown that down-regulation of phospho-Akt (P-Akt) plays a key role in determining the sensitivity of hepatocellular carcinoma cells to bortezomib-induced apoptosis. (Cancer Res, 2008). Here, we further examined whether P-Akt is associated with the drug sensitivity of hematological malignant cells toward bortezomib. Methods: Several hematological malignant cell lines were used for in vitro studies. Apoptosis was examined by both flow cytometry and Western blot. Signal transduction pathways in cells were assessed by Western Blot. Gene silencing was done by small interference RNA (siRNA). Results: Bortezomib induced apoptosis in association with down-regulation of P-Akt in a dose- and time-dependent manner in sensitive cells (HL-60) but not in resistant K562 cells. Down-regulation of Akt1 by small interference RNA overcame the apoptotic resistance to bortezomib in K562 cells whereas a constitutively activated Akt1 protected HL-60 cells from bortezomib-induced apoptosis. Importantly, bortezomib showed the similar inhibition of the proteasome activity in both sensitive and resistant cells, suggesting that bortezomib-induced apoptotic effect may not depend on its proteasome inhibitory effect. Conclusions: Down-regulation of P-Akt plays an important role in bortezomib-induced apoptosis in hematological malignancies. Targeting on Akt signaling, either directly or through upstream regulation, provides an attractive approach to overcome bortezomib resistance in certain hematological malignancies. Supported by VGHTPE 99DHA0100332 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1575.