Inhibition Of PI3K-AKT Signaling Research Articles

Abietane Diterpenoids Isolated from Torreya nucifera Disrupt Replication of Influenza Virus by Blocking the Phosphatidylinositol-3-Kinase (PI3K)-Akt and ERK Signaling Pathway.

Although vaccines and antiviral drugs are available, influenza viruses continue to pose a significant threat to vulnerable populations globally. With the emergence of drug-resistant strains, there is a growing need for novel antiviral therapeutic approaches. We found that 18-hydroxyferruginol (1) and 18-oxoferruginol (2) isolated from Torreya nucifera exhibited strong anti-influenza activity, with 50% inhibitory concentration values of 13.6 and 18.3 μM against H1N1, 12.8 and 10.8 μM against H9N2, and 29.2 μM (only compound 2) against H3N2 in the post-treatment assay, respectively. During the viral replication stages, the two compounds demonstrated stronger inhibition of viral RNA and protein in the late stages (12-18 h) than in the early stages (3-6 h). Moreover, both compounds inhibited PI3K-Akt signaling, which participates in viral replication during the later stages of infection. The ERK signaling pathway is also related to viral replication and was substantially inhibited by the two compounds. In particular, the inhibition of PI3K-Akt signaling by these compounds inhibited viral replication by sabotaging influenza ribonucleoprotein nucleus-to-cytoplasm export. These data indicate that compounds 1 and 2 could potentially reduce viral RNA and viral protein levels by inhibiting the PI3K-Akt signaling pathway. Our results suggest that abietane diterpenoids isolated from T. nucifera may be potent antiviral candidates for new influenza therapies.

MIR497HG-Derived miR-195 and miR-497 Mediate Tamoxifen Resistance via PI3K/AKT Signaling in Breast Cancer.

Tamoxifen is commonly used for the treatment of patients with estrogen receptor-positive (ER+) breast cancer, but the acquired resistance to tamoxifen presents a critical challenge of breast cancer therapeutics. Recently, long noncoding RNA MIR497HG and its embedded miR-497 and miR-195 are proved to play significant roles in many types of human cancers, but their roles in tamoxifen-resistant breast cancer remain unknown. The results indicate that MIR497HG deficiency induces breast cancer progression and tamoxifen resistance by inducing downregulation of miR-497/195. miR-497/195 coordinately represses five positive PI3K-AKT regulators (MAP2K1, AKT3, BCL2, RAF1, and CCND1), resulting in inhibition of PI3K-AKT signaling, and PI3K-AKT inhibition in tamoxifen-resistant cells restored tamoxifen responsiveness. Furthermore, ER α binds the MIR497HG promoter to activate its transcription in an estrogen-dependent manner. ZEB1 interacts with HDAC1/2 and DNMT3B at the MIR497HG promoter, resulting in promoter hypermethylation and histone deacetylation. The findings reveal that ZEB1-induced MIR497HG depletion contributes to breast cancer progression and tamoxifen resistance through PI3K-AKT signaling. MIR497HG can be used as a biomarker for predicting tamoxifen sensitivity in patients with ER+ breast cancer.

Abstract 5582: Modulation of intracellular kinase signaling to improve TIL manufacturing for adoptive cell therapy

Abstract Tumor-infiltrating lymphocytes (TILs) has demonstrated significant clinical benefits for patients with solid tumor. A robust expansion process is needed in order to produce a large number of cells for clinical usage. We modulated the PI3K-AKT signaling pathway in TILs isolated from cervical and ovarian cancer patients and found the inhibition of either PI3K or AKT can promote TIL proliferation during the REP stage. Inhibiting PI3K-AKT led to an increase of the effector CD8+T cell population with higher CD25+ and CD28+ in the CD8+ population, more TCF-1+ and CD39-CD69- T cells in both CD4+ and CD8+ T cell population, and downregulated exhaustion markers CD39+ and TIM-3+ in CD8+ T cell population. This inhibition significantly increased cytotoxicity when cocultured with tumor cell lines and patient-derived tumor samples. Moreover, dual knockout of ATK1 and ATK2 promoted TIL proliferation, long-term survival and decreased apoptosis of TIL, which were further confirmed by the RNA-seq results. Thus, inhibition of PI3K-AKT signaling represents a promising strategy to generate large numbers of TIL with enhanced stemness, activation and cytotoxicity, and could be used to support adoptive cell therapy to treat solid tumors. Citation Format: Zixiao Shi, Sheng Yao, Peipei Zhao, Fei Li, Di Zhou, Jun Cui, Jingwei Sun, Yarong Liu. Modulation of intracellular kinase signaling to improve TIL manufacturing for adoptive cell therapy [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 5582.

The RNA-binding protein Cpeb4 is a novel positive regulator of osteoclast differentiation

Cytoplasmic polyadenylation element binding (CPEB) proteins are RNA-binding proteins involved in translational regulation of the specific target mRNAs and control function of various organs including brain, liver and hematopoietic system. However, the role of CPEB proteins during osteoclast differentiation remains unclear. Here we show that Cpeb4 is required for RANKL-induced osteoclast differentiation in mouse macrophage-derived RAW264.7 cell line. Cpeb4 mRNA and protein levels are upregulated at the late stage of osteoclast differentiation. Immunofluorescence analysis revealed that Cpeb4 is translocated from cytoplasm to nuclear bodies in response to RANKL stimulation. Inhibition of PI3K-Akt signaling or calcium-NFAT pathways using chemical inhibitors suppressed nuclear localization of Cpeb4. Loss-of-function analysis showed that shRNA-mediated Cpeb4 depletion strongly impaired TRAP-positive osteoclast formation and expression of key differentiation markers including Acp5, Ctsk, Nfatc1 and Dcstamp. These results suggest that Cpeb4 is a positive regulator in osteoclastogenesis downstream of RANKL signaling.

Rituximab-Induced CD20-Mediated Signals and Suppression of PI3K-AKT Pathway Cooperates to Inhibit B-Cell Lymphoma Growth By Down-Regulation of Myc.

Rituximab, anti-CD20 monoclonal antibody, shows anti-lymphoma effects by either activating host immune mechanism against lymphoma cells such as ADCC and CDC or suppressing signal(s) important for growth of B-cell lymphoma. Accumulated evidence shows de-regulated PI3K-AKT pathway is important therapeutic target in B-cell lymphoma. Although studies suggested suppression of PI3K-AKT pathway is possibly critical in rituximab-induced anti-lymphoma signals, details of molecular mechanisms covering CD20-mediated signals and PI3K-AKT pathway are still unknown.We analyzed signals in B-cell lymphoma cell line, SD07, which had bi-allelic deletions of both CD20(MS4A1) and PTEN gene. Briefly, SD07 was established from a patient with diffuse large B-cell lymphoma(DLBCL)who showed refractory CD20-negative relapse(Eur.J. Haematol. 2012).We transiently restored protein expression of either or both of CD20 and PTEN by electroporation of expression plasmid having cDNA of those genes in SD07. In this experiment, expression of both proteins were clearly detected by western blot until 72 hours after transfection. CD20 protein was also detectable on cell surface of SD07 cells by flow cytometric(FCM) analysis at comparable levels of those of Daudi cells after transfection on CD20 cDNA.Our previous study showed expression of both c-myc RNA and protein, as judged by FCM, was induced by transient transfection of CD20 cDNA in SD07 cells. Western blot analysis also showed significant increase of myc protein 24hrs after transfection (vector=13.7±0.2, CD20=14.5±0.4, P<0.05, arbitrary density unit) and the increase was not affected by the addition of rituximab(20µg/ml)in culture. In contrast transfection of PTEN cDNA produced significant decrease of myc protein (PTEN=11.2±0.1, P<0.01). Myc protein levels was comparable between cells transfected with CD20 plus PTEN and PTEN alone. However in the presence of rituximab, myc protein expression was markedly downregulated in SD07 cells at both 24 hours(vector=13.7±0.2,CD20+PTEN=8.3±0.1,P=0.0008)and 48hours(vector=12.4±0.3 vs CD20+PTEN=11.2±0.1,P=0.04).24 hours after transfection, de-phosphorylation and down-regulation of retinoblastoma(RB) protein expression was evident only in SD07 cells transfected with both CD20 and PTEN, but not with either alone.Rituximab also augmented down-regulation of RB protein in SD07 cells transfected with both CD20 and PTEN.After transfection, in vitro cell growth of SD07, as evaluated by MTT, was as follows;vector=4.6±0.3, CD20=4.8±0.3(P=NS), PTEN=4.1±0.2(P<0.05), CD20+PTEN=3.6±0.2(P<0.01), CD20+PTEN+rituximab=2.7±0.2(P<0.01),OD570nm, days3)72 hours after transfection, by FCM, SD07 transfected with both CD20 and PTEN showed significant increase of subG1 DNA content potion compared with cells transfected with either alone(vector=2.5±0.2%,CD20=5.3±0.2%,PTEN=8.8 ±0.5%,CD20+PTEN= 16.1±0.6, P<0.01). Furthermore, addition of rituximabsignificantly increase of cells in subG1 in SD07 with transfected with both CD20 and PTEN(18.6±0.5%,P<0.01), suggesting suppression of PI3K-AKT is important for mediateing rituximab-induced apoptotis.Accordance with proliferative effect of CD20 inSD07, transfection of CD20 alone produced significant increase of phosphorylation of AktSer473(vector=11.2±2.3, CD20=13.3±2.3,pAkt/total Akt,P<0.05). Co-transfection of PTEN with CD20 suppress the CD20-induced phosphorylation of Akt in SD07 cells. (CD20+PTEN=12.8±2.3,P=NS).In summary, although data is limited in a cell line, it clearly shows rituximab-induced CD20-mediated signals and suppression of PI3K-AKT pathway by tumor suppressor PTEN cooperatively inhibits growth and induce apoptosis of DLBCL cell line, SD07. Myc protein is critical molecules in this particular cooperative pathways. The present result partly agree studies which showed that inhibition of PI3K-AKT signaling and down-regulation of myc is important in PTEN-induced cytotoxicity in GCB(germinal center B cell)type DLBCL. In addition, we show here myc is not only for PTEN, but also involved in rituximab-induced anti-lymphoma effect. The present result provide a rationale for combination of rituximab and PI3K inhibitor in the treatment of DLBCL. In this context, myc is a candidate of the therapeutic target in rituximab-based immunochemotherapy for B cell lymphoma. DisclosuresNo relevant conflicts of interest to declare.

Genetic depletion of glutathione peroxidase-1 potentiates nephrotoxicity induced by multiple doses of cocaine via activation of angiotensin II AT1 receptor

ABSTRACTWe investigated the possible roles of angiotensin II type 1 receptor (AT1R) and oxidative stress responsive nuclear factor κB (NFκB) in renal damage caused by multiple doses of cocaine in glutathione peroxidase (GPx)-1 gene-depleted mice. Treatment with cocaine resulted in significant increases in malondialdehyde, protein carbonyl, and pro-apoptotic Bax expression and decreases in the ratio of glutathione (GSH) and its oxidized form (GSSG), GSH-dependent enzymes, and anti-apoptotic factors in the kidney. These alterations were more pronounced in GPx-1 knockout (−/−) mice than in wild type (WT) mice. Notably, the AT1R antagonist losartan protected against the renal toxicity induced by cocaine, whereas the NFκB inhibitor pyrrolidine dithiocarbamate was not protective. The toxicity was more pronounced in GPx-1 (−/−) mice than in WT mice. The protective effect afforded by losartan against cocaine toxicity appeared to be more sensitive in GPx-1 (−/−) mice than that in WT mice. These losartan-mediated protective effects were inhibited by the phosphatidyl-inositol-3-kinase (PI3K) inhibitor LY294002, indicating that losartan provides significant protection from cocaine-induced renal toxicity through PI3K/Akt signaling. Our results suggest that genetic inhibition of GPx-1 potentiates cocaine-induced renal damage via activation of AT1R by inhibition of PI3K-Akt signaling, and that AT1R can be a therapeutic target against renal toxicity induced by cocaine.

Relationship of EGFR-independent activation of PI3K in KRAS wild-type colorectal cancers and cetuximab resistance.

385 Background: Therapies targeting receptor tyrosine kinases (RTKs) are typically effective only when they lead to simultaneous inhibition of PI3K-AKT and MEK-ERK signaling. Cetuximab is a monoclonal antibody against EGFR that has been approved for treatment of metastatic colorectal cancer (CRC). Studies have suggested that its benefit is restricted to patients with KRAS wildtype (WT) CRC. However, even in KRAS WT CRC, the response rate to single-agent cetuximab is low, and the mechanistic basis for this is not well-understood. Methods: The ability of cetuximab to inhibit PI3K-AKT and MEK-ERK signaling in a panel of KRAS WT CRC cell lines was assessed by immunoblotting. Paired biopsies from CRC patients obtained pre-treatment and after 3 weeks of cetuximab were evaluated by immunohistochemistry and proteomic techniques to assess inhibition of PI3K activity. We preformed immunoprecipitations (IPs) of the regulatory subunit of PI3K from cell lines and primary CRCs to identify RTKs involved in PI3K activation. Cetuximab-based therapeutic combinations were evaluated in KRAS WT CRC cell lines and xenograft models. Results: Cetuximab led to effective inhibition of MEK-ERK signaling (&gt;75% reduction in phospho-ERK), but led to incomplete inhibition of PI3K-AKT signaling (0-50% reduction in phospho-AKT) in all KRAS WT CRC cell lines tested. Analysis of paired biopsies from patients with KRAS WT CRC obtained pre-treatment and after 3 weeks of cetuximab demonstrated that the PI3K pathway remained active in many patients, despite cetuximab therapy. PI3K regulatory subunit IPs from cell lines and primary CRCs revealed multiple RTK inputs to PI3K, most notably from IGFIR. Combined treatment with cetuximab and an IGFIR inhibitor or a PI3K inhibitor led to improved efficacy in vitro and to tumor regressions in KRAS WT CRC xenograft models. Conclusions: Cetuximab fails to inhibit PI3K signaling in some KRAS WT CRCs. Additional RTKs other than EGFR play a role in PI3K activation in these cancers, which may contribute to cetuximab resistance. Combining cetuximab with targeted therapies directed against the PI3K pathway may lead to improved efficacy in KRAS WT CRC, and should be evaluated in future clinical trials.

Menin and GIP are inversely regulated by food intake and diet via PI3/AKT signaling in the proximal duodenum

Background and Aims:Ingestion of food stimulates the secretion of incretin peptides glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 to ensure the proper absorption and storage of nutrients. Menin is the 67 kDa protein product of the MEN1 gene recently reported to have a role in metabolism. In this study, we will determine the regulation of menin in the proximal duodenum by food intake and diet in correlation with GIP levels in the proximal duodenum of mice after an 18 h fast followed by 4 and 7 h refeeding and 3 months of high-fat diet.Methods:A dual luciferase assay was used to determine GIP promoter activity and ELISA was used to measure the levels of GIP after inhibition of menin through small interfering RNA (siRNA) and exposure to MAPK and AKT inhibitors. Colocalization of menin and GIP were determined by immunofluorescence.Results:Menin and GIP expression are regulated by fasting, refeeding and diet in the proximal duodenum. Overexpression of menin in STC-1 cells significantly inhibited GIP mRNA and promoter activity, whereas menin siRNA upregulated GIP levels. Inhibition of GIP expression by the PI3/AKT inhibitor, LY294002, was abrogated in STC-1 cells with reduced menin levels, whereas the MAPK inhibitor, UO126, inhibited the expression of GIP independent of menin. Exposure of STC-1 cells to GIP reduced menin expression in a dose-dependent manner via PI3K-AKT signaling.Conclusion:Feeding and diet regulates the expression of menin, which inversely correlates with GIP levels in the proximal duodenum. In vitro assays indicate that menin is a negative regulator of GIP via inhibition of PI3K-AKT signaling. We show menin colocalizing with GIP in K cells of the proximal gut and hypothesize that downregulation of menin may serve as a mechanism by which GIP is regulated in response to food intake and diet.

Abstract 1247: ARHI downregulates PI3K and Ras-MAP signaling and induces autophagy by promoting integrin and EGFR membrane protein degradation

Abstract Autophagy selectively degrades cellular proteins, protein aggregates and organelles, providing energy in a nutrient-deprived environment. We have reported that autophagy can be induced by re-expression of ARHI (DIRAS3), a maternally imprinted Ras-related tumor suppressor gene that is down regulated in more than 60% of ovarian cancers. Whereas prolonged expression of ARHI in cultured cells induces autophagic cell death, ARHI expression in xenografts sustains dormant cancer cell survival for weeks. When ARHI levels were reduced in dormant cells, xenografts grew rapidly. Inhibition of ARHI-induced autophagy with chloroquine dramatically reduces regrowth of xenografts, suggesting that autophagy contributes to the survival of dormant cells. Re-expression of ARHI inhibits both PI3K and Ras-MAPK signaling. Growth factors (VEGF, IL-8, IGF) and ECM components (fibronectin) found in the cancer microenvironment can rescue ovarian cancer cells from ARHI-induced autophagic cell death in culture, associated with partial reversal of ARHI-mediated inhibition of PI3K signaling. Consequently, we have hypothesized that autophagy together with growth factors and ECM in the microenvironment are all required to maintain dormancy, where autophagy provides energy for xenografts and survival factors prevent autophagic death by partially reversing inhibition of PI3K-AKT signaling. The mechanism by which ARHI regulates PI3K and Ras-MAPK signaling has not been fully elucidated. Here, we show that re-expression of ARHI in SKOv3 ovarian cancer cells inhibits expression of integrin β1 and EGFR. ARHI expression enhanced EGF-mediated receptor endocytosis and colocalization of EGFR with Rab11 and Rab7, markers for multivesicular endosomes and lysosomes, respectively. Our results suggest that ARHI enhances membrane receptor turnover, in part, by facilitating the internalization and association of membrane receptors with vesicular trafficking proteins targeted for degradation in the lysosome. ARHI-mediated downregulation of membrane receptors reduces both PI3K-AKT and Ras-MAPK signaling, ultimately contributing to the inhibition of tumor growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1247. doi:1538-7445.AM2012-1247

Relationship of incomplete inhibition of PI3K pathway signaling and efficacy of cetuximab in KRAS wild-type colorectal cancers.

462 Background: Therapies targeting receptor tyrosine kinases (RTKs) are typically effective only when they lead to simultaneous downregulation of PI3K-AKT and MEK-ERK signaling. Cetuximab is a monoclonal antibody directed against EGFR that has been approved for the treatment of metastatic colorectal cancer, and several studies have suggested that its benefit is restricted to patients with KRAS wildtype cancers. However, even in KRAS wildtype colorectal cancers, the response rate to single-agent cetuximab is low, and the mechanistic basis for this is not well-understood. Methods: The ability of cetuximab to downregulate PI3K-AKT and MEK-ERK signaling in a panel of KRAS wildtype colorectal cancer cell lines was assessed by immunoblotting. We preformed immunoprecipitations of the regulatory subunit of PI3K on lysates derived from cell lines and primary colorectal tumor specimens to identify the RTKs involved in PI3K activation. Cetuximab-based therapeutic combinations were evaluated in KRAS wildtype cell lines and tumor xenograft models. Results: Cetuximab led to effective inhibition of MEK-ERK signaling (&gt;75% reduction in phospho-ERK), but led to incomplete inhibition of PI3K-AKT signaling (0-50% reduction in phospho-AKT) in all KRAS wildtype colorectal cell lines tested. PI3K regulatory subunit immunoprecipitations revealed multiple RTK inputs to PI3K in cell lines and primary tumors, most notably from IGFIR. Combined treatment with cetuximab and an IGFIR inhibitor or a PI3K inhibitor led to improved efficacy in vitro and in tumor xenograft models. Conclusions: Cetuximab fails to completely downregulate PI3K-AKT signaling in KRAS wildtype colorectal cancer cell lines. Biochemical analysis of cell lines and primary colorectal tumors reveals that additional RTKs other than EGFR play a role in PI3K activation in these cancers. Therefore, combining cetuximab with targeted therapies directed against the PI3K pathway may lead to improved efficacy in KRAS wildtype colorectal cancers.

Abstract 4690: Inhibition of PI3K-AKT signaling causes a FOXO-dependent induction of receptor tyrosine kinase expression and phosphorylation in cancer

Abstract Hyperactivation of the PI3K-AKT-mTOR pathway is a common mechanism for oncogenic signaling in tumors and multiple inhibitors of this pathway are now in clinical development. As the PI3K-AKT-mTOR pathway is known to be tightly regulated by negative feedback, we hypothesized that these drugs might relieve feedback inhibition, reactivate upstream signaling and limit their antitumor effects. We tested the effects of inhibitors of PI3K and AKT kinase on receptor tyrosine kinase signaling in a panel of cell lines derived from multiple tumor types. PI3K-AKT pathway inhibition resulted in increased expression and phosphorylation of a common set of receptor tyrosine kinases, including IGF-1R and insulin receptor and HER3. Induction of RTK expression was dependent on activation of members of the Forkhead transcription factor family in response to AKT inhibition. We found that depletion of FOXO proteins blocked the rise in HER3, IGF-1R, and insulin receptor protein and phosphorylation caused by AKT inhibition. Expression of a constitutively activated form of one of the FOXO proteins resulted in increased expression of these receptors. Moreover, immunoprecipitation of FOXO proteins revealed that they are preferentially associated with the promoters of these receptors upon AKT inhibition. To determine the consequences of induction of these RTKs, we utilized HER kinase inhibitors to block activation of the receptors. In multiple xenograft tumor models, PI3K-AKT inhibition resulted in potent RTK induction in vivo and combined inhibition of the HER kinases and AKT resulted in improved antitumor effects with tumor regressions observed in several cases. We conclude that the PI3K-AKT pathway feedback regulates RTK expression through a FOXO-dependent induction of RTK transcripts. This reactivation of upstream RTK signaling is likely to attenuate the effects of AKT inhibition. Blockade of feedback-upregulated RTKs may significantly improve the antitumor effects of PI3K-AKT inhibitors and is a potential combination strategy for PI3K-AKT driven cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4690. doi:10.1158/1538-7445.AM2011-4690

AKT Inhibition Relieves Feedback Suppression of Receptor Tyrosine Kinase Expression and Activity

Activation of the PI3K-AKT pathway in tumors is modulated by negative feedback, including mTORC1-mediated inhibition of upstream signaling. We now show that AKT inhibition induces the expression and phosphorylation of multiple receptor tyrosine kinases (RTKs). In a wide spectrum of tumor types, inhibition of AKT induces a conserved set of RTKs, including HER3, IGF-1R, and insulin receptor. This is in part due to mTORC1 inhibition and in part secondary to a FOXO-dependent activation of receptor expression. PI3K-AKT inhibitors relieve this feedback and activate RTK signaling; this may attenuate their antitumor activity. Consistent with this model, we find that, in tumors in which AKT suppresses HER3 expression, combined inhibition of AKT and HER kinase activity is more effective than either alone.

Targeting Epidermal Growth Factor Receptor–Associated Signaling Pathways in Non–Small Cell Lung Cancer Cells: Implication in Radiation Response

Several studies have shown solid evidence for the potential value of targeting epidermal growth factor receptor (EGFR) signaling to enhance the antitumor activity of radiation. However, therapeutic resistance has emerged as an important clinical issue. Here, we investigated whether strategies for targeting EGFR-associated downstream signaling would radiosensitize a panel of non-small cell lung cancer cell lines. Inhibition of K-RAS using RNA interference attenuated downstream signaling and increased radiosensitivity of A549 and H460 cells, whereas inhibition of EGFR did not. A549 cells harboring a K-RAS mutation at codon V12 were radiosensitized by small interfering RNA (siRNA) targeting this codon. H460 cells having mutation at codon V61 was radiosensitized by siRNA targeting of this mutation. K-RAS siRNA did not radiosensitize H1299 cells possessing wild-type K-RAS. Inhibition of the phosphoinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin pathway led to significant radiosensitization of the two cell lines, whereas selective inhibition of extracellular signal-regulated kinase signaling did not. Inhibitors targeting the PI3K-AKT-mTOR pathway also abrogated G(2) arrest following irradiation and induced gammaH2AX foci formation. A dual inhibitor of class I PI3K and mammalian target of rapamycin effectively increased the radiosensitivity of A549 and H460 cells. Inhibition of PI3K-AKT signaling was associated with the downregulation of DNA-PKs. Although apoptosis was the primary mode of cell death when cells were pretreated with LY294002 or AKT inhibitor VIII, cells pretreated with rapamycin or PI-103 showed mixed modes of cell death, including apoptosis and autophagy. Our results suggest possible mechanisms for counteracting EGFR prosurvival signaling implicated in radioresistance and offer an alternative strategy for overcoming resistance to EGFR inhibitors used in combination with irradiation.

Targeting HER2 signaling pathway for radiosensitization: Alternative strategy for therapeutic resistance

Several studies have indicated the potential value of targeting HER-2 signaling to enhance the anti-tumor activity of ionizing radiation. However, therapeutic resistance resulting from several factors, including activation of the downstream pathway, represents a major obstacle to treatment. Here, we investigated whether inhibitors targeting downstream of HER-2 signaling would radiosensitize SKBR3 breast cancer cells that exhibit overamplification of HER2. Selective inhibition of MEK-ERK signaling using pharmacologic inhibitors (PD98059, UO126) did not increase the radiosensitivity of SKBR3 cells. Selective inhibition of the PI3K-AKT-mTOR pathway using pharmacologic inhibitors (LY294002, AKT inhibitor VIII, Rapamycin) significantly attenuated expression of p-AKT and p-70S6K, respectively and radiosensitized SKBR3 cells. MCF-7 cells those did not over-express HER-2, showed less radiosensitization compared to SKBR3 cells by inhibition of this pathway. Pre-treatment with these inhibitors also caused significant abrogation of typical G2 arrest following ionizing radiation and induced marked prolongation of γH2AX foci indicating impairment of DNA damage repair. A dual inhibitor of Class I PI3K and mTOR, PI103 effectively radiosensitized SKBR3 cells and showed significant prolongation of γH2AX foci. Inhibition of PI3K-AKT signaling was associated with down-regulation of DNA-PKs, respectively. While apoptosis was the major mode of cell death when the cells were pretreated with LY294002 or AKT inhibitor VIII, the cells were pretreated by rapamycin or PI103 showed mixed mode of cell death including autophagy. Our results suggest possible mechanisms to counteract the HER-2 prosurvival signaling implicated in radioresistance, and offer an alternative strategy to overcome resistance to HER-2 inhibitors combined with radiation.

Gab2-Mediated Signaling Promotes Melanoma Metastasis

Metastatic melanoma is a disease with a poor prognosis that currently lacks effective treatments. Critical biological features of metastasis include acquisition of migratory competence, growth factor independence, and invasive potential. In an attempt to identify genes that contribute to melanoma pathogenesis, a genome-wide search using bacterial artificial chromosome array comparative genomic hybridization and single nucleotide polymorphism arrays in a series of 64 metastatic melanoma samples and 20 melanoma cell lines identified increased copy numbers of Gab2 located on 11q14.1. Gab2 is an adaptor protein that potentiates the activation of the Ras-Erk and PI3K-Akt pathways and has recently been implicated in human cancer; however, its role in melanoma has not been explored. In this study, we found that Gab2 was either amplified (approximately 11%) and/or overexpressed (approximately 50%) in melanoma. Gab2 protein expression correlated with clinical melanoma progression, and higher levels of expression were seen in metastatic melanomas compared with primary melanoma and melanocytic nevi. We found that overexpression of Gab2 potentiates, whereas silencing of Gab2 reduces, migration and invasion of melanoma cells. Gab2 mediated the hyperactivation of Akt signaling in the absence of growth factors, whereas inhibition of the PI3K-Akt pathway decreased Gab2-mediated tumor cell migration and invasive potential. Gab2 overexpression resulted in enhanced tumor growth and metastatic potential in vivo. These studies demonstrate a previously undefined role for Gab2 in melanoma tumor progression and metastasis.

Blockade of the phosphatidylinositol-3-kinase–Akt signaling pathway enhances the induction of apoptosis by microtubule-destabilizing agents in tumor cells in which the pathway is constitutively activated

Constitutive activation of the phosphatidylinositol-3-kinase (PI3K)-Akt signaling pathway is associated with the neoplastic phenotype in many human tumor cell types. Given the antiapoptotic role of this pathway, we examined whether its specific blockade might sensitize human tumor cells to the induction of apoptosis by various anticancer drugs. Although specific blockade of the PI3K-Akt pathway alone with inhibitors such as LY294002 did not induce cell death, it resulted in marked and selective enhancement of the induction of apoptosis by microtubule-destabilizing agents such as vincristine. This effect was apparent only in tumor cells in which the PI3K-Akt pathway is constitutively activated. Blockade of the PI3K-Akt pathway induced the activation of glycogen synthase kinase-3beta, which phosphorylates microtubule-associated proteins such as tau and thereby reduces their ability to bind and stabilize microtubules. The consequent destabilization of microtubules induced by the inhibition of PI3K-Akt signaling appeared to increase their sensitivity to low concentrations of microtubule-destabilizing agents that alone do not lead to the disruption of cytoplasmic microtubules in tumor cells. Such a synergistic effect on microtubule integrity was not apparent for stable microtubules in the neurites of neuronal cells. These results suggest that the administration of a combination of a PI3K-Akt pathway inhibitor and a microtubule-destabilizing agent is a potential chemotherapeutic strategy for the treatment of tumor cells in which this signaling pathway is constitutively activated.

A role for PI3K-Akt signaling in pulmonary metastatic nodule formation of the osteosarcoma cell line, LM8

To search for the intracellular signaling pathway critical for the pulmonary metastasis of osteosarcoma, we examined two osteosarcoma cell lines with different metastatic capability, Dunn and LM8. While parental Dunn is poorly metastatic to lung, LM8, derived from Dunn by in vivo selection through pulmonary metastasis, displays clear capability of pulmonary metastasis. We found that LM8 had higher levels of Akt phosphorylation and Ezrin expression than Dunn. In contrast, no clear difference was observed between Dunn and LM8 in other signaling mediators, including MAPK, SAPK and Stat3. In contrast to Dunn, LM8 secreted higher levels of matrix metalloproteinase-2 and -9, showed higher levels of invasiveness and cell motility, and displayed strong pulmonary metastasis. Inhibition of PI3K-Akt signaling in LM8 by a PI3K inhibitor, LY294002, or by a dominant negative form of Akt, resulted in suppression of MMP secretion, in vitro invasiveness, cell locomotion and in vivo pulmonary metastasis. In contrast, expression of an active form of Akt in Dunn substantially activated its MMP secretion, in vitro invasiveness, cell locomotion and in vivo pulmonary metastasis. Taken together, our results demonstrate, for the first time, an important role of Akt signaling in pulmonary metastasis of osteosarcoma.

Regulation of Fas Expression by STAT3 and c-Jun Is Mediated by Phosphatidylinositol 3-Kinase-AKT Signaling

Cooperation between STAT3 and c-Jun results in suppression of Fas Receptor (FasR) transcription, which is often seen in advanced human tumors. To identify requirements for STAT3-Jun cooperation, we elucidated the role of protein kinases that affect both transcription factors. The phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway was found capable of down-regulating both STAT3- and c-Jun-dependent transcription, resulting in derepression of FasR transcription. Conversely, inhibition of PI3K-AKT signaling via the specific pharmacological inhibitor LY294002 up-regulated AP1/Jun- and STAT-dependent transcriptional activities, resulting in suppression of the FasR promoter activities and decreased FasR surface expression. PI3K-AKT's ability to affect FasR transcription was not observed in c-jun null fibroblasts, suggesting that c-Jun is required for PI3K/AKT-mediated regulation of FasR transcription. Interestingly, the dominant negative form of Rac1 (RacN17) was also efficient in relieving FasR expression, suggesting that the increase in FasR expression following AKT stimuli could be mediated via AKT ability to elicit suppression of Rac1, which in turn decreases JNK activities and c-Jun phosphorylation. Overall, our findings demonstrate that through its negative effects on both c-Jun and STAT3, the PI3K-AKT pathway disrupts cooperation between c-Jun and STAT3, which is required for silencing the FasR promoter, resulting in increased expression of surface FasR and concomitant sensitization to FasL-mediated programmed cell death.