PI3K Akt Inhibitor Research Articles

Abstract 20: Adiponectin increases VEGF expression and promotes angiogenesis in human chondrosarcoma

Abstract Vascular endothelial growth factor (VEGF) is an angiogenic mediator in tumors and has been implicated in the pathogenesis and progression of cancer. Adiponectin is a protein hormone secreted by adipose tissue. We recently found that adiponectin increased metastasis in human chondrosarcoma. However, the effect of adiponectin on VEGF expression and angiogenesis in human chondrosarcoma cells is mostly unknown. Here we found that adiponectin promoted VEGF expression in human chondrosarcoma and subsequently increased migration and tube formation in endothelial progenitor cells (EPCs). Pretreatment of cells for 30 min with PI3K inhibitors (LY294002 and wortmannine), Akt inhibitor, mTOR inhibitor (Rapamycin) or HIF-1α inhibitor abolished adiponectin-induced VEGF expression, EPC migration and tube formation. In addition, transfection of cells with PI3K, Akt, mTOR, or HIF-1α siRNA also reduced adiponectin-induced VEGF expression, EPC migration and tube formation. Knockdown adiponectin decreased VEGF expression in vitro as well as angiogenesis effects the chick chorioallantoic membrane and matrigel-plug nude mice model in vivo. In addition, using xenograft tumor angiogenesis model, knockdown adiponectin significantly reduced tumor growth and tumor angiogenesis. Finally, we analyzed from chondrosarcoma patients by immunohistochemical staining and found that the expression of adiponectin and VEGF was significantly higher in chondrosarcoma tissue than normal cartilage. Taken together this study showed that adiponectin mediates VEGF expression and angiogenesis of human chondrosarcoma cells. One of the mechanisms underlying adiponectin induced VEGF expression and angiogenesis was activation of PI3K, Akt, mTOR and HIF1-α pathways. Citation Format: Jhao-Sheng Shih, Chih-Hsin Tang. Adiponectin increases VEGF expression and promotes angiogenesis in human chondrosarcoma. [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 20. doi:10.1158/1538-7445.AM2014-20

Abstract 5217: The role of miR-526b in COX-2 mediated breast cancer progression via EP4 signaling

Abstract A minor tumour cell subset, known as stem-like cells (SLCs), appears to defy conventional therapy, causing relapse. Thus, identification of SLC-specific therapeutic targets and markers is urgently needed. Our laboratory has established that aberrant expression of COX-2 promotes breast cancer progression via multiple mechanisms including SLC induction owing to activation of the PGE receptor EP4 which signals via the canonical cAMP/PKA, and non-canonical PI3K-AKT pathways. Differential gene and microRNA (miR) expression micro arrays in human breast cancer cell lines stably transfected to over-express COX-2 revealed up-regulation of miR-526b. We sought to determine if miR-526b is induced by EP4 activation and whether it promotes breast cancer progression, including SLC stimulation. COX-2-ve human breast cancer cell lines MCF-7 (ER+, HER-2-) and SKBR-3 (ER-, HER-2+) were stably transfected to over-express miR-526b (MCF7-526b and SKBR3-526b), and functional assays were performed. The cells exhibited enhanced proliferation, migration and invasiveness, and increased tumoursphere-forming efficiency on ultra-low attachment plates (in vitro surrogate of SLC phenotype), compared to empty vector-transfected and parental controls. These results indicate that miR-526b is a COX-2 induced oncogenic microRNA. In order to assess the roles of miR-526b in vivo, MCF7-526b, SKBR3-526b, or empty vector control cells were injected into the tail vein of NOD/SCID/GUSB-null female mice. Both miR-526b over-expressing cell lines revealed a significantly increased ability to form proliferative lung colonies at 4 weeks identified with HLA staining and EdU proliferation marker. To examine the role of EP4 activation in miR-526b up-regulation, MCF-7 cells cultured as monolayers or tumourspheres were treated with PGE2 or an EP4 agonist, PGE1OH. Both ligands induced a significant over-expression of miR-526b in both culture conditions. Conversely, MCF7-COX-2 cells treated with either an EP4 specific antagonist (ONO-AE3-208) or a COX-2 inhibitor (NS-398) displayed significant reduction in miR-526b expression, as quantified with qPCR. To investigate the role of PI3K-AKT pathway of EP4 signaling in miR-526b expression, PI3K was stimulated with PGE2 or PGE1OH in MCF7 cells, and inhibited with LY294002 or Wortmannin in MCF7-COX-2 cells. MiR-526b expression levels quantified with qPCR revealed significant increases and declines, respectively, with PI3K-AKT pathway stimulators and inhibitors applied to the above cell lines. Together these results suggest an important association of miR-526b with EP4-induced SLC stimulation and breast cancer progression, and the possibility that miR-526b may be used as a biomarker for monitoring patients and personalizing therapy. (Supported by the CBCF, Ontario chapter and the OICR funds to PKL. EL holds a CBCF and CIHR-STP Graduate Fellowship and MM a TBCRU and CIHR-STP Post Doctoral fellowship). Citation Format: Erin O. Landman, Mousumi Majumder, Ling Liu, Peeyush K. Lala. The role of miR-526b in COX-2 mediated breast cancer progression via EP4 signaling. [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 5217. doi:10.1158/1538-7445.AM2014-5217

47IN - The Impact of Targeted Therapy in Cervical and Endometrial Cancer

ABSTRACT Endometrial (EC) and Cervical Cancer (CC) are the most common gynecological tumors. Although the majority of EC are diagnosed when localized to the uterus, up to 25% of cases recur. In CC the risk of recurrence is 10–20% in early stages and up 70% in locally advanced cases. Unfortunately, in these gynecologic tumors, the systemic therapies are limited in efficacy for recurrent and metastatic disease and new therapies are in need. Increasing understanding of molecular biology is enabling the development of new therapies, which target pathways crucial to tumor proliferation. The most promising targeted therapies including the PI3K/AKT/mTOR pathway and angiogenesis inhibition. The PI3K pathway is often deregulated in gynecologic cancer (PIK3CA mutations up to 33% of CC; 39% of EC) and is highly druggable leading the development of large number of agents. The rapamycin analogs (rapalogs) directly bind and inhibit mTORC1. From more than a half dozen clinical trials of the rapalogs as single agents, it can be concluded that rapalogs have a modest antitumor activity in EC. Objective response rates range from 0%–30% depending upon the clinical population. A molecular biomarker that predicts the clinical benefit of rapalogs has yet to be identified. In CC results of a phase II study of single-agent temsirolimus have recently been reported showing disappointing results. Newer PI3K pathway agents, including pan-PI3K, dual PI3K/mTOR, AKT, and catalytic mTOR inhibitors are being tested in EC. Angiogenesis plays a significant role in tumor progression and an association between VEGF over-expression and poorer prognosis has been established in these both gynecologic tumors. Bevacizumab, a humanized monoclonal antibody against VEGF has clinical activity as single agent in recurrent disease in both tumors. Apart, incorporating Bevacizumab to first line chemotherapy for metastatic or recurrent CC improves overall survival compared to chemotherapy alone in this poor prognosis patients, leading a new standard of care. Phase II studies based on other antiangionenic therapies as multitargeted tyrosine-kinase inhibitors (Sorafenib, Sunitinib, Pazopanib) and VEGF-Trap have shown a modest activity . We should try to identify and develop new target therapies to improve the outcomes in these malignancies. Disclosure: The author has declared no conflicts of interest.

AKT regulates NPM dependent ARF localization and p53mut stability in tumors.

Nucleophosmin (NPM) is known to regulate ARF subcellular localization and MDM2 activity in response to oncogenic stress, though the precise mechanism has remained elusive. Here we describe how NPM and ARF associate in the nucleoplasm to form a MDM2 inhibitory complex. We find that oligomerization of NPM drives nucleolar accumulation of ARF. Moreover, the formation of NPM and ARF oligomers antagonizes MDM2 association with the inhibitory complex, leading to activation of MDM2 E3-ligase activity and targeting of p53. We find that AKT phosphorylation of NPM-Ser48 prevents oligomerization that results in nucleoplasmic localization of ARF, constitutive MDM2 inhibition and stabilization of p53. We also show that ARF promotes p53 mutant stability in tumors and suppresses p73 mediated p21 expression and senescence. We demonstrate that AKT and PI3K inhibitors may be effective in treatment of therapeutically resistant tumors with elevated AKT and carrying gain of function mutations in p53. Our results show that the clinical candidate AKT inhibitor MK-2206 promotes ARF nucleolar localization, reduced p53(mut) stability and increased sensitivity to ionizing radiation in a xenograft model of pancreatic cancer. Analysis of human tumors indicates that phospho-S48-NPM may be a useful biomarker for monitoring AKT activity and in vivo efficacy of AKT inhibitor treatment. Critically, we propose that combination therapy involving PI3K-AKT inhibitors would benefit from a patient stratification rationale based on ARF and p53(mut) status.

Statins upregulate cystathionine γ-lyase transcription and H2S generation via activating Akt signaling in macrophage

Hydrogen sulfide (H2S), the third gaseous transmitter, is implicated in various pathophysiologic processes. In the cardiovascular system, H2S exerts effects of cardioprotection, vascular tone regulation, and atherogenesis inhibition. Recent studies demonstrated that atorvastatin, the inhibitor of 3-hydroxyl-3-methyl coenzyme A reductase, affected H2S formation in kidney and other organs. However, the underlying mechanisms are not fully understood. In this study, we examined the effects of three different statins (fluvastatin, atorvastatin and pravastatin) on H2S formation in raw264.7 macrophages. There was a remarkable rise in H2S level in fluvastatin- and atorvastatin-stimulated macrophages, while pravastatin failed to show any significant effect on it. Moreover, fluvastatin and atorvastatin enhanced the mRNA and protein expression of cystathionine γ-lyase (CSE) in dose- and time-dependent manners. Fluvastatin also markedly enhanced the CSE activity. However, fluvastatin did not alter the mRNA or protein expression of another H2S-producing enzyme 3-mercaptopyruvate sulfurtransferase. Blockade of CSE with its inhibitor dl-propargylglycine (PAG) or siRNA markedly reduced the H2S level in fluvastatin-stimulated macrophages. In addition, fluvastatin elevated Akt phosphorylation, which occurred as early as 15min after treatment, peaked at 1h, and lasted at least 3h. Both PI3K inhibitor LY294002 (10μM) and Akt inhibitor perifosine (10μM) were able to reverse the increases of CSE mRNA and H2S production in fluvastatin-stimulated macrophages. Last, we showed that fluvastatin reduced the mRNA levels of pro-inflammatory molecules such as IL-1β and MCP-1 in LPS-treated macrophages, which were completely reversed by CSE inhibitor PAG. Taken together, the findings demonstrate that statins may up-regulate CSE expression/activity and subsequently elevate H2S generation by activating Akt signaling pathway and also imply that CSE–H2S pathway plays a critical role in the anti-inflammation elicited by statins.

HER3 Limits the Antitumor Activity of PI3K–AKT Inhibitors in TNBC

Abstract Combined inhibition of EGFR and HER3 sensitizes TNBC to PI3K–AKT pathway inhibitors.

MicroRNA-7 inhibits tumor metastasis and reverses epithelial-mesenchymal transition through AKT/ERK1/2 inactivation by targeting EGFR in epithelial ovarian cancer.

Epidermal growth factor receptor (EGFR) overexpression and activation result in increased proliferation and migration of solid tumors including ovarian cancer. In recent years, mounting evidence indicates that EGFR is a direct and functional target of miR-7. In this study, we found that miR-7 expression was significantly downregulated in highly metastatic epithelial ovarian cancer (EOC) cell lines and metastatic tissues, whereas the expression of, EGFR correlated positively with metastasis in both EOC patients and cell lines. Overexpression of miR-7 markedly suppressed the capacities of cell invasion and migration and resulted in morphological changes from a mesenchymal phenotype to an epithelial-like phenotype in EOC. In addition, overexpression of miR-7 upregulated CK-18 and β-catenin expression and downregulated Vimentin expression, accompanied with EGFR inhibition and AKT/ERK1/2 inactivation. Similar to miR-7 transfection, silencing of EGFR with this siRNA in EOC cells also upregulated CK-18 and β-catenin expression and downregulated Vimentin expression, and decreased phosphorylation of both Akt and ERK1/2, confirming that EGFR is a target of miR-7 in reversing EMT. The pharmacological inhibition of PI3K-AKT and ERK1/2 both significantly enhanced CK-18 and β-catenin expression and suppressed vimentin expression, indicating that AKT and ERK1/2 pathways are required for miR-7 mediating EMT. Finally, the expression of miR-7 and EGFR in primary EOC with matched metastasis tissues was explored. It was showed that miR-7 is inversely correlated with EGFR. Taken together, our results suggested that miR-7 inhibited tumor metastasis and reversed EMT through AKT and ERK1/2 pathway inactivation by reducing EGFR expression in EOC cell lines. Thus, miR-7 might be a potential prognostic marker and therapeutic target for ovarian cancer metastasis intervention.

Osteocalcin attenuates high fat diet-induced impairment of endothelium-dependent relaxation through Akt/eNOS-dependent pathway

BackgroundRecent studies have demonstrated a protective effect of osteocalcin (OCN) on glucose homeostasis and metabolic syndrome. However, its role in vascular function remains unknown. This study investigated the contribution of OCN to the pathogenesis of endothelial dysfunction in the thoracic aorta of apolipoprotein E-deficient (ApoE-KO) mice.MethodsEight-week-old ApoE–KO mice were given chow or high fat diet (HFD) for 12 weeks with or without daily intraperitoneal injection of OCN. Intraperitoneal glucose tolerance test (IPGTT), insulin tolerance test (ITT),measurement of serum lipid profiles and blood pressure were carried out. Endothelium-dependent relaxation (EDR) was measured by wire myography. Human umbilical vein endothelial cells (HUVECs) were used to study the role of OCN on eNOS levels in vitro. PI3K inhibitor (LY294002) and Akt inhibitor V were used ex-vivo to determine whether PI3K/Akt/eNOS contributes to the beneficial effect of OCN for the vascular or not.ResultsDaily injections of OCN can significantly improve lipid metabolism, glucose tolerance and insulin sensitivity in ApoE-KO mice. In ApoE-KO mice fed with HFD, the OCN-treated mice displayed an improved acetylcholine-stimulated EDR compared to the vehicle-treated group. In addition, compared to vehicle-treated HUVECs, OCN-treated HUVECs displayed increased activation of the Akt-eNOS signaling pathway, as evidenced by significantly higher levels of phosphorylated Akt and eNOS. Furthermore, a similar beneficial effect of OCN on thoracic aorta was observed using ex vivo organ culture of isolated mouse aortic segment. However, this effect was attenuated upon co-incubation with PI3K inhibitor or Akt inhibitor V.ConclusionsOur study demonstrates that OCN has an endothelial-protective effect in atherosclerosis through mediating the PI3K/Akt/eNOS signaling pathway.

VEGF Increases Paracellular Permeability in Brain Endothelial Cells via Upregulation of EphA2

Neurological disorders are associated with an increase in the permeability of human brain microvascular endothelial cells (HBMEC). Our previous findings have indicated that EphA2 could increase the permeability of HBMEC. Recent evidence has linked EphA2 and vascular endothelial growth factor (VEGF) to abnormalities in the vascular response. However, it is unclear whether EphA2 is involved in the VEGF-induced changes in the permeability of HBMEC. Here, changes in permeability were determined by measuring transendothelial electrical resistance (TEER) and the flux of FITC-dextran. We found that knockdown of EphA2 in HBMEC abolished the VEGF-induced reduction in TEER and increase in flux of fluorescent dextran. Moreover, VEGF-induced redistribution of ZO-1 and the recruitment of detergent-soluble occludin and claudin-5 were also prevented. Further results showed that VEGF increased EphA2 expression in a time- and dose-dependent manner, which was inhibited by a neutralizing antibody against VEGFR2 or SU1498. VEGF-induced EphA2 expression was suppressed in the brain endothelium following treatments with the PI3K inhibitor LY294002, Akt inhibitor or transfection with the dominant-negative PI3K mutants (Δp110). Similar results were obtained when ERK1/2 activation was inhibited by PD98059 or ERK1/2 siRNA transfection. Our data suggest that VEGF upregulates the expression of EphA2 in HBMEC through binding to VEGFR2 and subsequently activating the intracellular PI3K/Akt and ERK1/2 signaling pathways, which contribute to an increase in paracellular permeability. These data reveal a novel role for VEGF as a regulator of EphA2 expression in the brain endothelial cells and provide insights into the molecular mechanisms of VEGF-mediated changes in paracellular permeability.

Src mediates ERK reactivation in gefitinib resistance in non-small cell lung cancer

To study epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) resistance mechanisms, we established a novel gefitinib-resistant lung cancer cell line derived from an EGFR-mutant non-small cell lung cancer cell line (PC-9) pretreated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (designated PC9-GR). We found that gefitinib substantially suppressed the EGFR signaling pathway, whereas ERK was reactivated after several hours in PC9-GR but not in PC-9. The combination of gefitinib with ERK inhibition (by U0126) restored gefitinib susceptibility in PC9-GR, but PI3K-Akt inhibition with LY294002 did not. Although the levels of phosphorylated Src were up-regulated simultaneously with ERK reactivation, neither ERK suppression using U0126 nor an ERK-specific siRNA induced Src phosphorylation. Furthermore, dual inhibition of EGFR and Src restored gefitinib sensitivity in PC9-GR in vitro and in vivo. In conclusion, our results indicate that Src-mediated ERK reactivation may play a role in a novel gefitinib resistance mechanism, and that the combined use of gefitinib with a Src inhibitor may be a potent strategy to overcome this resistance.

Ursolic acid induces U937 cells differentiation by PI3K/Akt pathway activation

AimUrsolic acid (UA), a pentacyclic triterpenoid, is used as an anti-inflammatory and anti-cancer agent. There were few studies on the effects of UA on differentiation, and this is the first time to elucidate the potential effect and molecular mechanism of UA on inducing differentiation in the human leukemia cell line U937. MethodsWright-Giemsa staining, nitroblue tetrazolium reduction assay and flow cytometric analysis were utilized to demonstrate the differentiation of U937 cells induced by UA. Western blotting and immunofluorescence assay were used to investigate the possible mechanism. ResultsIt was found that UA could induce the differentiation of U937cells and Akt-activity was significantly increased during differentiation. Additionally, LY294002, a PI3K-Akt inhibitor, could block the differentiation of U937 cells induced by UA. ConclusionUA could induce the differentiation of U937 cells by activating the PI3K/Akt pathway, and it could be a potential candidate as a differentiation-inducing agent for the therapy of leukemia.

Resveratrol down-regulates a glutamate-induced tissue plasminogen activator via Erk and AMPK/mTOR pathways in rat primary cortical neurons

Resveratrol (3,5,4'-trihydroxy-trans-stilbene, RSV) is a polyphenolic compound present in a variety of plant species (including grapes) that produces a myriad of biological activities including anti-inflammatory, antioxidant and neuroprotective effects. In this study, we investigate the effects of resveratrol on the basal and glutamate-stimulated expression and activity of a tissue plasminogen activator (tPA) that plays neuromodulatory or neurotoxic roles in many different neurological situations. Under basal conditions, resveratrol decreased the tPA expression and activity without affecting the tPA mRNA level in rat primary cortical neurons. RSV induced AMPK phosphorylation and inhibited mTOR phosphorylation. Inhibition of AMPK phosphorylation using compound C prevented resveratrol-induced down-regulation of tPA activity. This suggested that AMPK/mTOR-dependent translational inhibition contributes to the down-regulation of the tPA. Under glutamate-stimulated conditions of rat primary cortical neurons, tPA activity and expression were increased along with increased tPA mRNA expression but afterward treatment of RSV inhibited the glutamate-induced increase in tPA activity and expression and tPA mRNA expression. Glutamate stimulation induced activation of Akt and MAPK pathways as well as mTOR which were inhibited by RSV. Interestingly, the Erk pathway inhibitor U0126, but neither PI3K-Akt inhibitor LY294002 nor p38 inhibitor SB203580, mimicked the inhibitory action of RSV on glutamate-induced tPA up-regulation. This suggested the essential role of Erk in the transcriptional up-regulation of tPA expression, which is targeted by RSV. Glutamate stimulation induced neuronal cell death as determined by PI staining and MTT assay. However, RSV protected the cultured rat primary cortical neurons from glutamate-induced cell death as paralleled with the changes in tPA expression. These results suggested that RSV can modulate tPA activity under basal and stimulated conditions by both translational and transcriptional mechanisms. The regulation of the tPA by RSV provides additional therapeutic targets on top of the growing number of molecular substrates of RSV's action in the brain.

Inactivating CUX1 mutations promote tumorigenesis.

A major challenge for cancer genetics is to determine which low frequency somatic mutations are drivers of tumorigenesis. Here we interrogate the genomes of 7,651 diverse human cancers to identify novel drivers and find inactivating mutations in the homeodomain transcription factor CUX1 (cut-like homeobox 1) in ~1-5% of tumors. Meta-analysis of CUX1 mutational status in 2,519 cases of myeloid malignancies reveals disruptive mutations associated with poor survival, highlighting the clinical significance of CUX1 loss. In parallel, we validate CUX1 as a bona fide tumor suppressor using mouse transposon-mediated insertional mutagenesis and Drosophila cancer models. We demonstrate that CUX1 deficiency activates phosphoinositide 3-kinase (PI3K) signaling through direct transcriptional downregulation of the PI3K inhibitor PIK3IP1 (phosphoinositide-3-kinase interacting protein 1), leading to increased tumor growth, while exposing susceptibility to PI3K-AKT inhibition. Thus, our complementary approaches identify CUX1 as a new pan-driver of tumorigenesis and uncover a potential strategy for treating CUX1-mutant tumors.

Pan-Mammalian Target of Rapamycin (mTOR) Inhibitor AZD8055 Primes Rhabdomyosarcoma Cells for ABT-737-induced Apoptosis by Down-regulating Mcl-1 Protein

The PI3K/mammalian Target of Rapamycin (mTOR) pathway is often aberrantly activated in rhabdomyosarcoma (RMS) and represents a promising therapeutic target. Recent evaluation of AZD8055, an ATP-competitive mTOR inhibitor, by the Preclinical Pediatric Testing Program showed in vivo antitumor activity against childhood solid tumors, including RMS. Therefore, in the present study, we searched for AZD8055-based combination therapies. Here, we identify a new synergistic lethality of AZD8055 together with ABT-737, a BH3 mimetic that antagonizes Bcl-2, Bcl-xL, and Bcl-w but not Mcl-1. AZD8055 and ABT-737 cooperate to induce apoptosis in alveolar and embryonal RMS cells in a highly synergistic fashion (combination index < 0.2). Synergistic induction of apoptosis by AZD8055 and ABT-737 is confirmed on the molecular level, as AZD8055 and ABT-737 cooperate to trigger loss of mitochondrial membrane potential, activation of caspases, and caspase-dependent apoptosis that is blocked by the pan-caspase inhibitor Z-VAD-fmk. Similar to AZD8055, the PI3K/mTOR inhibitor NVP-BEZ235, the PI3K inhibitor NVP-BKM120 and Akt inhibitor synergize with ABT-737 to trigger apoptosis, whereas no cooperativity is found for the mTOR complex 1 inhibitor RAD001. Interestingly, molecular studies reveal a correlation between the ability of different PI3K/mTOR inhibitors to potentiate ABT-737-induced apoptosis and to suppress Mcl-1 protein levels. Importantly, knockdown of Mcl-1 increases ABT-737-induced apoptosis similar to AZD8055/ABT-737 cotreatment. This indicates that AZD8055-mediated suppression of Mcl-1 protein plays an important role in the synergistic drug interaction. By identifying a novel synergistic interaction of AZD8055 and ABT-737, our findings have important implications for the development of molecular targeted therapies for RMS.

IGF-1-Induced Enhancement of PRNP Expression Depends on the Negative Regulation of Transcription Factor FOXO3a

The conformational conversion of the cellular prion protein (PrPC) into its β-sheet-rich scrapie isoform (PrPSc) causes fatal prion diseases, which are also called transmissible spongiform encephalopathies (TSEs). Recent studies suggest that the expression of PrPC by the PRNP gene is crucial for the development of TSEs. Therefore, the identification of the exogenous and endogenous stimulating factors that regulate PRNP expression would help to understand the pathogenesis of TSEs. Here, we demonstrate that forkhead box O3a (FOXO3a) negatively regulates PRNP expression by binding to the PRNP promoter, which is negatively regulated by insulin-like growth factor 1 (IGF-1). Our results show that the IGF-1-induced enhancement of PRNP mRNA and protein levels is due to the activation of the PI3K-Akt signaling pathway. The activation of Akt then induces the phosphorylation of FOXO3a, leading to its translocation from the nucleus to the cytoplasm and preventing its binding to the PRNP promoter. Treatment with the PI3K-Akt inhibitor LY294002 induces the nuclear retention of FOXO3a, which leads to a decrease in PRNP expression. We present a new IGF-1-PI3K-Akt-FOXO3a pathway, which influences PRNP expression. The results of this work are vital for understanding the function of PrPC and for future therapeutic approaches to human TSEs.

Dermatologic toxicities to targeted cancer therapy: shared clinical and histologic adverse skin reactions

Dermatologic toxicities (DT) to targeted cancer therapy may present as inflammatory dermatoses, keratoses, and as benign and malignant squamous proliferations. Published reports of DT with cancer therapy with epidermal growth factor receptor (EGFR), tyrosine kinase (TK), MEK, PI3K, AKT, and BRAF inhibitors were reviewed. DT associated with targeted cancer therapy demonstrated similar reactions and may be grouped as (i) DT as cutaneous inflammation, and (ii) DT as cutaneous epithelial proliferation. EGFR inhibitor, cetuximab, and MEK inhibitors, selumetinib and trametinib, demonstrated papulopustular rash with a suppurative folliculitis in 83%, 93%, and 80% of the patients on therapy, respectively. Common DT with EGFR inhibitors erlotinib and tyrosine kinase inhibitor sorafenib were hand-foot skin reactions in 30-60% of patients on therapy. PI3K inhibitor BKM-120 and AKT inhibitor MK2206 produced maculopapular eruptions seen as dermal hypersensitivity reaction on the skin biopsy. RAF inhibitors vemurafenib and sorafenib were associated with a variety of cutaneous epithelial proliferations (keratosis pilaris, seborrheic keratosis, verruca vulgaris, actinic keratosis, keratoacanthoma, and squamous cell carcinoma. Various anticancer agents may target similar cellular compounds and/or cell signaling pathways thus share similar clinical and histologic features of DT. The knowledge of the overlap of DT with different types of targeted cancer therapy will assist in evaluation of cutaneous reactions.

Thioredoxin-1 mediates hypoxia-induced pulmonary artery smooth muscle cell proliferation

Pathological pulmonary artery smooth muscle cell (PASMC) proliferation contributes to pulmonary vascular remodeling in pulmonary hypertensive diseases associated with hypoxia. Both the hypoxia-inducible factor (HIF) and phosphatidylinositol 3-kinase (PI3K)/serine/threonine kinase (Akt) pathways have been implicated in hypoxia-induced PASMC proliferation. Thioredoxin-1 (Trx1) is a ubiquitously expressed protein that is involved in redox-dependent signaling via HIF and PI3K-Akt in cancer. The role of Trx1 in PASMC proliferation has not been elucidated. The present studies tested the hypothesis that Trx1 regulates hypoxia-induced PASMC proliferation via HIF and/or PI3K- and Akt-dependent mechanisms. Following exposure to chronic hypoxia, our data indicate that Trx1 activity is increased in adult murine lungs. Furthermore, hypoxia-induced increases in cellular proliferation are correlated with increased Trx1 expression, HIF activation, and Akt activation in cultured human PASMC. Both small-interfering RNA-mediated knockdown and pharmacological Trx1 inhibition attenuated hypoxia-induced PASMC proliferation, HIF activation, and Akt activation. While Trx1 knockdown suppressed hypoxia-induced PI3K-Akt activation in PASMC, PI3K-Akt inhibition prevented hypoxia-induced proliferation but had no effect on hypoxia-induced increases in Trx1 or HIF activation. Thus, our findings indicate that Trx1 contributes to hypoxia-induced PASMC proliferation by modulating HIF activation and subsequent PI3K-Akt activation. These novel data suggest that Trx1 might represent a novel therapeutic target to prevent hypoxic PASMC proliferation.

Abstract 3684: Differential mechanisms of green tea polyphenols-induced apoptosis in human prostate cancer cells: Role of p53.

Abstract Tumor suppressor gene p53 is frequently inactivated in a variety of human cancers including prostate cancer and is associated with therapeutic resistance. We have previously demonstrated that green tea polyphenols (GTP) induces apoptosis in prostate cancer cells irrespective of p53 status (Carcinogenesis 33:377-84, 2012). However, the molecular mechanisms underlying these observations are not properly understood. In the present study we investigated the mechanisms of GTP-induced apoptosis in human prostate cancer cells with and without functional p53. Human prostate cancer LNCaP cells were stably transfected with short hairpin-53 (LNCaPshp53) and control vector (LNCaPshV), exposed to GTP in dose and time dependent manner. GTP exposure induced p53 stabilization and activation of downstream targets p21/waf1 and Bax in dose-dependent manner specifically in LNCaPshV cells. However, GTP-induced FAS upregulation through the activation of c-jun N-terminal kinase resulting in FADD phosphorylation, caspase-8 activation and truncation of BID leading to apoptosis was common in both LNCaPshV and LNCaPshp53 cells. In parallel, treatment of cells with GTP resulted in inhibition of survival pathway, mediated by Akt deactivation and loss of BAD phosphorylation more prominently in LNCaPshp53 cells. These distinct routes of cell death converged to a common pathway leading to loss of mitochondrial transmembrane potential, cytochrome c release and activation of terminal caspases resulting in PARP cleavage. GTP-induced apoptosis was attenuated with JNK inhibitor, SP600125 in both cell lines; whereas PI3K-Akt inhibitor, LY294002 resulted in increased cell death in LNCaPshp53 cells, establishing the role of two distinct pathways of GTP-mediated apoptosis. Furthermore, GTP exposure resulted in the inhibition of class I HDAC protein, accumulation of acetylated histone H3 in total cellular chromatin resulting in increased accessibility to bind with the promoter sequences of p21/waf1 and Bax irrespective of p53 status of cells, consistent with the effects elicited by HDAC inhibitor, trichostatin A. These results demonstrate that GTP induces prostate cancer cell death by two distinct mechanisms irrespective of p53 status, thus identifying for the first time through defined molecular targets for their chemopreventive and/or therapeutic actions. Citation Format: Karishma Gupta, Vijay S. Thakur, Natarajan Bhaskaran, Akbar Nawab, Melissa A. Babcook, Mark W. Jackson, Sanjay Gupta. Differential mechanisms of green tea polyphenols-induced apoptosis in human prostate cancer cells: Role of p53. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3684. doi:10.1158/1538-7445.AM2013-3684

Regulation of the Na+,HCO3‐ cotransporter NBCn1 (SLC4A7) by a constitutively active ErbB2 receptor in MCF‐7 breast cancer cells

Net acid production is greatly increased in tumors due to a shift towards glycolytic metabolism. We previously showed that the Na+,HCO3− cotransporter NBCn1 (SLC4A7) is strongly upregulated in MCF‐7 breast cancer cells upon expression of a truncated, constitutively active ErbB2 receptor, ΔNErbB2. Here, we explore the mechanisms involved in this upregulation. NBCn1 mRNA and protein levels were reduced by inhibition of ERK or Src in ΔNErbB2‐, but not vector‐expressing MCF‐7 cells. PI3K‐Akt inhibition reduced NBCn1 expression in both cell types. NBCn1 expression was increased by stimulation of full‐length ErbB receptors. In luciferase promoter assays, the 1.2 kb genomic region upstream of SLC4A7 increased luciferase activity after ΔNErbB2 expression ~2.5 fold. Promoter deletions identified the ΔNErbB2‐ responsive region and putative transcription factor binding sites. NBCn1 expression was increased by expression of dominant‐negative (DN)‐Sp1 or by Sp1 knockdown, and decreased by knockdown of Krüppel‐like factor 4 (KLF4), while DN‐Sp3 or Sp3 knockdown had no effect. Chromatin immunoprecipitation assay (ChIP) demonstrated Sp1, Sp3 and KLF4 binding to the NBCn1 promoter. In conclusion, we identified an NBCn1 promoter and demonstrated that NBCn1 transcriptional regulation involves Sp1 and KLF4, and that ErbB2‐dependent upregulation of NBCn1 is controlled at least in part by ERK‐ and Src signaling.Funding: Danish Cancer Society, Danish Research Council

NDRG2: a newly identified mediator of insulin cardioprotection against myocardial ischemia–reperfusion injury

The N-myc downstream-regulated gene 2 (NDRG2) is involved in cell apoptosis and survival. Although reported to be highly expressed in the cardiac tissue, the biological function of NDRG2 in the heart remains to be established. Insulin exerts protective effects against myocardial ischemia/reperfusion (I/R) injury through the PI3K/Akt pathway. Here, we examined the changes in phosphorylation of NDRG2, a novel substrate and phosphoprotein of Akt, in insulin-induced protection against myocardial I/R. Rat hearts were subjected to 30min regional ischemia followed by reperfusion with or without insulin at the onset of reperfusion. Reperfusion with insulin inhibited myocardial apoptosis and reduced infarct size, as well as significantly up-regulated myocardial Akt and NDRG2 phosphorylation levels compared with the I/R group. These effects of insulin were blocked by pretreatment with the PI3K inhibitor wortmannin or Akt inhibitor. To further ascertain the role of NDRG2 in insulin-induced cardioprotection, cardiomyocytes were transduced with a lentivirus encoding shRNA targeting NDRG2 (loss-of-function), which rendered the cells more susceptible to I/R injury and significantly blunted the anti-apoptotic effect of insulin. Moreover, the NDRG2 shRNA lentivirus was tested in vivo, and NDRG2 knockdown aggravated myocardial I/R injury and attenuated the insulin-mediated cardioprotection against I/R injury. Taken together, these results suggest a novel role of PI3K/Akt/NDRG2 signaling in the cardioprotective effect of insulin.