Xanthine dehydrogenase (XDH) is the rate-limiting enzyme in purine catabolism by converting hypoxanthine to xanthine and xanthine to uric acid.The altered expression and activity of XDH are associated with the development and prognosis of multiple types of cancer, while its role in lung adenocarcinoma (LUAD) remains unknown.Herein, we demonstrated that XDH was highly expressed in LUAD and was significantly correlated with poor prognosis.Though inhibition of XDH displayed moderate effect on the viability of LUAD cells cultured in the complete medium, it significantly attenuated the survival of starved cells.Similar results were obtained in XDH-knockout cells.Nucleosides supplementation rescued the survival of starved LUAD cells upon XDH inhibition, while inhibition of purine nucleoside phosphorylase abrogated the process, indicating that nucleoside degradation is required for the XDH-mediated survival of LUAD cells.Accordingly, metabolic flux revealed that ribose derived from nucleoside fueled key carbon metabolic pathways to sustain the survival of starved LUAD cells.Mechanistically, down-regulation of XDH suppressed unfolded protein response (UPR) and autophagic flux in starved LUAD cells.Inhibition of XDH decreased the level of amino acids produced by autophagic degradation, which was accompanied with down-regulation of mTORC1 signaling.Supplementation of amino acids including glutamine or glutamate rescued the survival of starved LUAD cells upon knockout or inhibition of XDH.Finally, XDH inhibitors potentiated the anti-cancer activity of 2-deoxy-D-glucose that induced UPR and/or autophagy in vitro and in vivo.In summary, XDH plays a crucial role in the survival of starved LUAD cells and targeting XDH may improve the efficacy of drugs that induce UPR and autophagy in the therapy of LUAD.

Tamoxifen, an important endocrine therapeutic agent, is widely used for the treatment of estrogen receptor positive (ER+) breast cancer. However, de novo or acquired resistance prevents patients from benefitting from endocrine approaches and necessitates alternative treatments. In this study, we report that small heat protein beta-8 (HSPB8) may serve as an important molecule in tamoxifen resistance. HSPB8 expression is enhanced in MCF-7 cells resistant to tamoxifen (MCF-7/R) compared to parent cells. Moreover, high expression of HSPB8 associates with poor prognosis in ER+ breast cancer patients but not in patients without classification. Stimulating ER signaling by heterogeneous expression of ERa or 17β-estradiol promotes HSPB8 expression and reduces the cell population in G1 phase. In contrast, blockage of ER signaling by tamoxifen down-regulates the expression of HSPB8. In addition, knocking down HSPB8 by specific siRNAs induces significant cell cycle arrest at G1 phase. AZD8055 was found to be more potent against the proliferation of MCF-7/R cells than that of parent cells, which was associated with down-regulation of HSPB8. We found that the anti-proliferative activity of AZD8055 was positively correlated with the HSPB8 expression level in ER+ breast cancer cells. Thus, AZD8055 was able to overcome tamoxifen resistance in breast cancer cells, and the expression of HSPB8 may predict the efficacy of AZD8055 in ER+ breast cancer. This hypothesis deserves further investigation.

PI3Kα-selective inhibitor BYL719 is currently in phase II/III clinical trial for the treatment of breast cancer, but highly variable response has been observed among patients. We sought to discover predictive biomarker for the efficacy of BYL719 by dissecting the proliferative signaling pathway mediated by PI3K in breast cancer. BYL719 concurrently inhibited the phosphorylation of AKT and ERK in PIK3CA-mutated human breast cancer cells. PI3K-regulated ERK phosphorylation was independent of canonical PDK1/AKT/mTOR pathway, while it was associated with RAF/MEK. Hyper-activation of EGFR or RAS abrogated inhibition of ERK phosphorylation by BYL719. Furthermore, hyper-activation of receptor tyrosine kinases (RTKs) including EGFR, c-MET, FGFR and HER3 but not IGF-1R restored ERK phosphorylation and cell viability suppressed by BYL719, suggesting the discriminative functions of RTKs in cell signaling and proliferation. By profiling 22 breast cancer cell lines, we found that BYL719 was more potent in cell lines where phosphorylation of both AKT and ERK was attenuated than those where only AKT phosphorylation was inhibited. The potency of BYL719 was further found to be significantly correlated with the expression profile of RTKs in breast cancer cells. Specifically, overexpression of EGFR, c-MET and/or FGFR1 forecasted resistance, while overexpression of IGF-1R and/or HER2 predicted sensitivity to BYL719 in breast cancer cells. Similar correlation between BYL719 efficacy and expression profile of RTKs was found in patient-derived xenograft models of breast cancer. Thus, inhibition of ERK phosphorylation by PI3Kα inhibitor BYL719 contributes to its antitumor efficacy and is determined by the converged signaling from RTKs. The expression profile of RTKs in breast cancer tissue could be potentially developed as a predictive biomarker for the efficacy of PI3Kα inhibitors.

mTOR inhibition led to activation of upstream receptor tyrosine kinases (RTKs) and AKT, which may attenuate the efficacy of mTOR kinase inhibitors. We sought to discover efficient drug combination with mTOR inhibitors by elucidating the survival feedback loops induced by mTOR inhibition in breast cancer. The feedback signaling upon treatment of mTOR inhibitor AZD8055 was determined and the combinatorial activity of AZD8055 and HSP90 inhibitor AUY922 in cell signaling and proliferation were detected. Treatment of breast cancer T47D cells with AZD8055 induced activation of AKT and phosphatidylinositol 3‐kinase (PI3K), which was accompanied with increase in expression of multiple upstream proteins including EGFR, HER2, HER3 and IRS‐1. Different RTKs were revealed to be responsible for the reactivation of AKT by AZD8055 in different breast cancer cell lines. Down‐regulation of these proteins differentially enhanced the antiproliferative activity of AZD8055. AZD8055 and AUY922 displayed synergistic effect against a panel of human breast cancer cells irrespective their genotype, which was associated with enhanced cell cycle arrest and inhibition of DNA synthesis. AUY922 destabilized multiple tested tyrosine kinases and abrogated activation of AKT induced by AZD8055. AZD8055 also inhibited up‐regulation of HSP70 and HSP27 upon AUY922 treatment. Cotreatment of these two drugs demonstrated synergistic activity against triple negative MDA‐MB‐468 xenograft without enhanced toxicity. The combination of AZD8055 and AUY922 demonstrated synergistic activity against various types of breast cancer and established a mechanistic rationale for a combination approach using catalytic mTOR kinase inhibitor and HSP90 inhibitor in the treatment of breast cancer.

Abstract Mammalian target of rapamycin (mTOR) has been validated as a promising target for cancer therapy. Several mTOR kinase inhibitors displaying potent antitumor activity due to their capability to inhibit both mTORC1 and mTORC2 have entered clinical trials. It has been recently reported that long term inhibition of mTOR led to activation of upstream receptor tyrosine kinases and Akt, which may attenuate the efficacy of mTOR kinase inhibitors. As a number of receptor tyrosine kinases and AKT are client proteins of Hsp90, we hypothesized that combination of inhibitors targeting mTOR and Hsp90 respectively would potentiate the anticancer activity. By calculating the Combination Index, mTOR kinase inhibitor AZD8055 and Hsp90 inhibitor AUY922 displayed synergistic effect against a panel of human breast cancer cells irrespective their ER, HER2 or PI3K status. The combination of sub-optimal concentration of these two drugs exerted significant antiproliferative activity, which is associated with enhanced cell cycle arrest but not induction of apoptosis. Down-regulation of either raptor or rictor by specific shRNA reinforced the antitumor activity of AUY922, indicating restraint of both mTORC1 and mTORC2 contribute to the synergistic activity of AZD8055 with AUY922. In T47D cells,long time exposure to AZD8055 led to restoration of phosphorylated AKT at T308 and GSK3β at S9, indicating reactivation of AKT, which was abrogated by AUY922 co-treatment. Moreover, phosphorylation of AKT at S473 was more significantly inhibited when cells were co-treated with AZD8055 and AUY922 than single agent treatment. We further revealed that AUY922 prevented feedback activation of PI3K upon AZD8055 treatment. As PI3K is not a client protein of Hsp90, block of PI3K activation by AUY922 might be due to down-regulation of upstream tyrosine kinase receptors. Accordingly, though the mRNA levels of EGFR, HER2, HER3 and HER4 increased after either single agent treatment or co-treatment, AUY922 not only inhibited the expression of tested tyrosine kinases, but more importantly, abrogated activation of HER2/HER3 induced by AZD8055 . In summary, the combination of catalytic mTOR inhibitor and Hsp90 inhibitor demonstrated synergistic activity against breast cancer cells, which is due to inhibition of tyrosine kinases expression and abrogation of feedback activation of HER2/HER3. As both AZD8055 and AUY922 are in clinical trials for breast cancer therapy, this study established a mechanistic rationale for a combination approach using AZD8255 and AUY922 in the treatment of breast cancer. Citation Format: Simeng Chen, Chenliang Guo, Jian Ding, Linghua Meng. Hsp90 inhibitor AUY922 abrogates mTOR inhibitor AZD8055-induced up-regulation of HER2/HER3 and potentiates antiproliferative activity in human breast cancer cells. [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 909. doi:10.1158/1538-7445.AM2013-909

Background: The cross-talk between CXCL12/CXCR4 axis and PI3K/mTOR pathway in migration of gastric carcinoma cells is unknown. Results: p110β provided a conduit for CXCL12-stimulated signaling and targeting PI3K/mTOR blocked CXCL12-activated cell motility. Conclusion: Targeting PI3K/mTOR pathway inhibited CXCL12-mediated cell migration. Significance: Drugs targeting mTOR pathway may be used for the therapy of metastatic gastric cancer expressing high levels of CXCL12. CXCL12/CXCR4 plays an important role in metastasis of gastric carcinoma. Rapamycin has been reported to inhibit migration of gastric cancer cells. However, the role of mTOR pathway in CXCL12/CXCR4-mediated cell migration and the potential of drugs targeting PI3K/mTOR pathway remains unelucidated. We found that CXCL12 activated PI3K/Akt/mTOR pathway in MKN-45 cells. Stimulating CHO-K1 cells expressing pEGFP-C1-Grp1-PH fusion protein with CXCL12 resulted in generation of phosphatidylinositol (3,4,5)-triphosphate, which provided direct evidence of activating PI3K by CXCL12. Down-regulation of p110β by siRNA but not p110α blocked phosphorylation of Akt and S6K1 induced by CXCL12. Consistently, p110β-specific inhibitor blocked the CXCL12-activated PI3K/Akt/mTOR pathway. Moreover, CXCR4 immunoprecipitated by anti-p110β antibody increased after CXCL12 stimulation and Gi protein inhibitor pertussis toxin abrogated CXCL12-induced activation of PI3K. Further studies demonstrated that inhibitors targeting the PI3K/mTOR pathway significantly blocked the chemotactic responses of MKN-45 cells triggered by CXCL12, which might be attributed primarily to inhibition of mTORC1 and related to prevention of F-actin reorganization as well as down-regulation of active RhoA, Rac1, and Cdc42. Furthermore, rapamycin inhibited the secretion of CXCL12 and the expression of CXCR4, which might form a positive feedback loop to further abolish upstream signaling leading to cell migration. Finally, we found cells expressing high levels of cxcl12 were sensitive to rapamycin in its activity inhibiting migration as well as proliferation. In summary, we found that the mTOR pathway played an important role in CXCL12/CXCR4-mediated cell migration and proposed that drugs targeting the mTOR pathway may be used for the therapy of metastatic gastric cancer expressing high levels of cxcl12.

The mammalian target of rapamycin (mTOR), is deregulated in about 50% of human malignancies and exists in two complexes: mTORC1 and mTORC2. Rapalogs partially inhibit mTORC1 through allosteric binding to mTORC1 and their efficacy is modest as a cancer therapy. A few mTOR kinase inhibitors that inhibit both mTORC1 and mTORC2 have been reported to possess potent anticancer activities. Herein, we designed and synthesized a series of pyrazolopyrimidine derivatives targeting mTOR kinase domain and X-387 was identified as a promising lead. X-387 selectively inhibited mTOR in an ATP-competitive manner while sparing a panel of kinases from the PIKK family. X-387 blocked mTORC1 and mTORC2-mediacted signaling pathway in cell lines with activated mTOR signaling and in rapamycin-resistant cells. Specifically, X-387 inhibited phosphorylation of AKT at T308, which is thought to be a target of PDK1 but not mTOR. Such activity was not due to inhibition of PI3K since X-387 did not inhibit translocation of AKT to the cell membrane. X-387 induced autophagy as observed for other mTOR inhibitors, while induced autophagy is pro-survival since concurrent inhibition of autophagy by 3-MA reinforced the antiproliferative activity of mTOR inhibitors. X-387 also inhibited cell motility, which is associated with decrease in activity of small GTPases such as RhoA, Rac1 and Cdc42. Taken together, X-387 is a promising compound lead targeting mTOR and with a wide spectrum anticancer activity among tumor cell lines. The data also underscores the complexity of the mTOR signaling pathways which are far from being understood.

The central role of Src in tumor progression and metastasis has validated it as an attractive therapeutic target for the treatment of human breast cancer. The aim of this study was to identify potential Src kinase inhibitor, explore its activity, and mechanism of action in human breast cancer. A strategy integrating focused combinatorial library design, virtual screening, chemical synthesis, and high-throughput screening was adopted and a novel 6-hydrazinopurine-based inhibitor of c-Src kinase PH006 was obtained. The kinase enzymatic activities were measured by enzyme-linked immunosorbent assay. The binding mode between PH006 and Src was profiled by surface plasmon resonance approach and molecular simulation. The anti-proliferative activity was evaluated by Sulforhodamin B (SRB) and Colony formation. The anti-invasion and anti-migration activities were assessed by trans-well and wound healing assay. Results indicated that PH006 was an ATP-competitive Src inhibitor, which selectively inhibited c-Src with an IC50 of 0.38 μM among a panel of 14 diverse tyrosine kinases. PH006 potently inhibited c-Src phosphorylation and c-Src-dependent signal transduction, resulting in inhibition of cell proliferation, migration, and invasion in human breast cancer MDA-MB-231 cells. Further study demonstrated that the anti-proliferative activity of PH006 was ascribed to its capability to arrest cells in G1 phase, while its anti-motility activity was related to suppression of MMP2/9 and HGF secretion. Moreover, PH006 exhibited potent activity against tumor growth as well as metastasis of human breast cancer MDA-MB-435 xenograft beard in nude mice, which was accompanied with reduced Src/FAK signaling in tumor tissue. Taken together, PH006 is a novel selective inhibitor of c-Src and possesses potent activity against breast cancer growth and metastasis, which could be potentially developed as a lead candidate against breast cancers with elevated Src tyrosine kinase activity.

Dihydroartemisinin (DHA), the main active metabolite of artemisinin derivatives, is among the artemisinin derivatives possessing potent anti-malarial and anti-cancer activities. In the present study, we found that DHA displayed significant anti-proliferative activity in human colorectal carcinoma HCT116 cells, which may be attributed to its induction of G1 phase arrest and apoptosis. To further elucidate the mechanism of action of DHA, a proteomic study employed two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed. Glucose-regulated protein 78 (GRP78), which is related with endoplasmic reticulum stress (ER stress), was identified to be significantly up-regulated after DHA treatment. Further study demonstrated that DHA enhanced expression of GRP78 as well as growth arrest and DNA-damage-inducible gene 153 (GADD153, another ER stress-associated molecule) at both mRNA and protein levels. DHA treatment also led to accumulation of GADD153 in cell nucleus. Moreover, pretreatment of HCT116 cells with the iron chelator deferoxamine mesylate salt (DFO) abrogated induction of GRP78 and GADD153 upon DHA treatment, indicating iron is required for DHA-induced ER stress. This result is consistent with the fact that the anti-proliferative activity of DHA is also mediated by iron. We thus suggest the unbalance of redox may result in DHA-induced ER stress, which may contribute, at least in part, to its anti-cancer activity.

The insulin-like growth factor-1 receptor (IGF1R) is over-expressed in a wide variety of tumors and contributes to tumor cell proliferation, metastasis and drug resistance. The aim of this study was to establish a sensitive screening platform to identify novel IGF1R inhibitors. The catalytic domain of IGF1R was expressed using the Bac-to-Bac baculovirus expression system. The screening platform for IGF1R inhibitors was established based on ELISA. The binding profile of IGF1R with the inhibitors was predicted with molecular docking and then subjected to the surface plasmon resonance (SPR) approach. The growth inhibition of cancer cells by the inhibitors was assessed with MTT assay. Apoptosis was analyzed using flow cytometry and Western blotting. A naturally occurring small molecule compound hematoxylin was identified as the most potent inhibitor (IC50 value=1.8±0.1 μmol/L) within a library of more than 200 compounds tested. Molecular simulation predicted the possible binding mode of hematoxylin with IGF1R. An SPR assay further confirmed that hematoxylin bound directly to IGF1R with high binding affinity (Kd=4.2×10-6 mol/L). In HL-60 cancer cells, hematoxylin inactivated the phosphorylation of IGF1R and downstream signaling and therefore suppressed cell proliferation. Mechanistic studies revealed that hematoxylin induced apoptosis in HL-60 cells via both extrinsic and intrinsic pathways. A simple, sensitive ELISA-based screening platform for identifying IGF1R inhibitors was established. Hematoxylin was identified as a promising IGF1R inhibitor with effective antitumor activity that deserves further investigation.

PI3K is a promising therapeutic target for cancer. With PI-103 as the lead compound, we designed and synthesized 4-(2-arylpyrido[3′,2′:3,4]pyrrolo[1,2-f][1,2,4]triazin-4-yl)morpholine derivatives. 9, 10a, 10d, 10e had the IC50 against PI3Kα comparable with PI-103. All of the compounds showed selectivity over 15 tested protein kinases and anti-proliferative activity at micromolar concentration against several cancer cell lines.

The phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt)-mammalian target of rapamycin (mTOR) signaling pathway is often constitutively activated in various human cancers, providing validated targets for cancer therapy. Among a series of 5-cyano-6-morpholino-4-substituted-pyrimidine analogs designed and synthesized based on PI3K target, 4-(2-(dimethylamino)vinyl)-2-(3-hydroxyphenyl)-6-morpholinopyrimidine-5-carbonitrile (WJD008) was selected for further pharmacological characterization because of its potent activity against PI3K signaling. WJD008 inhibited kinase activity of PI3Kα and mTOR with less activity against PIKK family members. In cellular settings, WJD008 abrogated insulin-like growth factor-I-activated PI3K-Akt-mTOR signaling cascade and blocked the membrane translocation of a pleckstrin homology domain containing enhanced green fluorescent protein-general receptor for phosphoinositides, isoform 1-pleckstrin homology fusion protein, suggesting down-regulation of phosphatidylinositol (3,4,5)-trisphosphate output induced by WJD008 resulted in inactivation of PI3K pathway. Consequently, WJD008 arrested cells in G1 phase without induction of apoptosis. Furthermore, WJD008 reversed the hyperactivation of the PI3K pathway caused by the oncogenic mutation of p110α H1047R and suppressed the proliferation and clonogenesis of transformed RK3E cells harboring this mutant. WJD008 was superior to the pan-PI3K inhibitor wortmannin against proliferation of a panel of cancer cells independently of their status of PI3K pathway or tissue originations. In summary, WJD008 is a potent dual PI3K/mTOR modulator with antiproliferative and anticlonogenic activity in tumor cells and transformed cells with PIK3CA mutant, which provides new clues for the design and development of this chemical scaffold as an anticancer drug.