Reciprocal regulation of amino acid import and epigenetic state through Lat1 and EZH2.

Lat1 (SLC7A5) is an amino acid transporter often required for tumor cell import of essential amino acids (AA) including Methionine (Met). Met is the obligate precursor of S-adenosylmethionine (SAM), the methyl donor utilized by all methyltransferases including the polycomb repressor complex (PRC2)-specific EZH2. Cell populations sorted for surface Lat1 exhibit activated EZH2, enrichment for Met-cycle intermediates, and aggressive tumor growth in mice. In agreement, EZH2 and Lat1 expression are co-regulated in models of cancer cell differentiation and co-expression is observed at the invasive front of human lung tumors. EZH2 knockdown or small-molecule inhibition leads to de-repression of RXRα resulting in reduced Lat1 expression. Our results describe a Lat1-EZH2 positive feedback loop illustrated by AA depletion or Lat1 knockdown resulting in SAM reduction and concomitant reduction in EZH2 activity. shRNA-mediated knockdown of Lat1 results in tumor growth inhibition and points to Lat1 as a potential therapeutic target.

mTOR-targeted therapy: differential perturbation to mitochondrial membrane potential and permeability transition pore plays a role in therapeutic response.

While cancer cell mitochondria mediate actions of many successful chemotherapeutics, little is known about mitochondrial response in mTOR-targeted anticancer therapy. We have studied mitochondrial dynamics in relation to growth suppression employing an allosteric inhibitor rapalog, a highly selective mTOR kinase inhibitor (mTOR-KI) and mTOR-ShRNA. Global targeting of mTOR increased mitochondrial membrane potential (mΔψ) and inhibited mitochondrial permeability transition pore (mPTP). Importantly, these mTOR-KI-provoked anti-survival and pro-survival effects were differentially manifested in diverse cancer cells according to intrinsic susceptibility to mTOR-targeting. The most-sensitive cells including those possessing hyperactive PI3K/AKT/mTOR and/or growth factor-dependence (LNCap, MDA361 and MG63) all displayed a dramatic increase in mΔψ, whereas the mΔψ increase was not evident in majority of resistant cancer cells. Upon mTOR-KI treatment, the resistant cells including those harboring K-Ras- or B-Raf mutation (MDA231, HT29 and HCT116) all displayed a markedly reduced mPTP opening, which paralleled a sustained AKT-hexokinase 2 (HK2) survival signaling and persistent phosphorylation (inactivation) of GSK3ß. Further studies demonstrated that the mTOR-KI-provoked mPTP closure in resistant cells was mediated through an enhanced binding of HK2 to the mitochondrial voltage-dependent anion channel (VDAC), a molecular mechanism known to promote mPTP closure and cell survival. Detaching HK2 from VDAC by an HK2-displacing peptide or methyl jasmonate specifically blocked the mTOR-KI-provoked mPTP closure and potentiated growth suppression in resistant cells. Thus, mTOR-inhibition can exert complex and differential perturbation to mitochondrial dynamics in cancer cells, which likely influence therapeutic outcome of mTOR-targeted therapy.

Requirement of the mTOR kinase for the regulation of Maf1 phosphorylation and control of RNA polymerase III-dependent transcription in cancer cells.

The mammalian target of rapamycin (mTOR) regulates growth via promoting translation and transcription. Here, employing an mTOR active-site inhibitor WYE-125132 (WYE-132), we have performed quantitative phospho-proteomics and identified a Ser-75-containing phosphopeptide from Maf1, a known repressor of RNA polymerase III (Pol III) transcription. Treatment of cancer cells with WYE-132 or the rapamycin analog CCI-779 led to a rapid loss of the phosphorylation at Ser-75, whereas this effect was not seen in cells treated with cytotoxic agents or unrelated inhibitors. WYE-132-induced Maf1 dephosphorylation correlated with its accumulation in the nucleus and a marked decline in the cellular levels of pre-tRNAs. Depletion of cellular Maf1 via small interfering RNA increased basal pre-tRNA and rendered tRNA synthesis refractory to mTOR inhibitors. Maf1 mutant proteins carrying S75A alone or with S60A, T64A, and S68A (Maf1-S75A, Maf1-4A) progressively enhanced basal repression of tRNA in actively proliferating cells and attenuated amino acid-induced tRNA transcription. Gene alignment revealed conservation of all four Ser/Thr sites in high eukaryotes, further supporting a critical role of these residues in Maf1 function. Interestingly, mTOR inhibition led to an increase in the occupancy of Maf1 on a set of Pol III-dependent genes, with concomitant reduction in the binding of Pol III and Brf1. Unexpectedly, mTORC1 itself was also enriched at the same set of Pol III templates, but this association was not influenced by mTOR inhibitor treatment. Our results highlight a new and unique mode of regulation of Pol III transcription by mTOR and suggest that normalization of Pol III activity may contribute to the therapeutic efficacy of mTOR inhibitors.

mTOR complex-2 stimulates acetyl-CoA and de novo lipogenesis through ATP citrate lyase in HER2/PIK3CA-hyperactive breast cancer.

The mechanistic target of rapamycin (mTOR) is a major regulator of cell growth and is frequently dysregulated in cancer. While mTOR complex-1 (mTORC1) is a validated cancer target, the role of mTOR complex-2 (mTORC2) remains less defined. Here, we reveal mTORC2 as a critical regulator of breast cancer metabolism. We showed that hyperphosphorylation in ATP citrate lyase (ACL) occurs frequently in human breast tumors and correlates well with HER2+ and/or PIK3CA-mutant (HER2+/PIK3CAmut) status in breast tumor cell lines. In HER2+/PIK3CAmut cells, mTORC2 controls Ser-455 phosphorylation of ACL thereby promoting acetyl-CoA production, de novo lipogenesis and mitochondrial physiology, all of which were inhibited by an mTORC1/mTORC2 kinase inhibitor (mTOR-KI) or cellular depletion of mTORC2 or ACL. mTOR-KI but not rapamycin blocked the IGF-1-induced ACL phosphorylation and glucose to lipid conversion. Depletion of mTORC2 but not mTORC1 specifically inhibited the ACL-dependent acetyl-CoA production. In the HER2+/PIK3CAmut MDA361, MDA453, BT-474 and T47D cells, depletion of mTORC2 or ACL led to growth inhibition and mitochondrial hyperpolarization, which were partially rescued by an alternate source of acetyl-CoA. These same changes were not apparent in mTORC2- or ACL-depleted HER2-/PIK3CAwt MDA231 and HCC1806 cells, highlighting a differential dependence of mTORC2-ACL for survival in these two cell types. Moreover, ACL Ser-455 mutants S455E (phosphomimetic) and S455A (non-phosphorylatable) each increased or decreased, respectively, the acetyl-CoA production, mitochondrial homeostasis and survival in ACL-depleted MDA453 cells. These studies define a new and rapamycin-resistant mechanism of mTORC2-ACL in lipogenesis and acetyl-CoA biology and provide a rationale for targeting of mTORC1 and mTORC2 in HER2+/PIK3CAmut breast cancer.

Phosphorylation of extracellular signal-regulated kinase 1 and 2, protein kinase B, and signal transducer and activator of transcription 3 are differently inhibited by an epidermal growth factor receptor inhibitor, EKB-569, in tumor cells and normal human keratinocytes.

EKB-569 is an irreversible inhibitor of epidermal growth factor receptor (EGF-R) tyrosine kinase. It inhibits EGF-induced phosphorylation of EGF-R and the growth of tumors that overexpress EGF-R in animal models. In clinical trials, EKB-569 and all other EGF-R inhibitors cause skin rashes. To understand the latter phenomenon, the effect of EKB-569 on EGF-R as well as downstream signaling to phosphoinositide 3-kinase-protein kinase B (AKT), extracellular signal-regulated kinase 1 and 2 (ERK1/2), or signal transducer and activator of transcription 3 (STAT3) pathways were compared in tumor cell lines and normal human keratinocytes (NHEK) grown in tissue culture. Tumor cell lines that have high (A431 epidermoid and MDA-468 breast carcinomas) and low (MCF-7 breast carcinoma) expression of EGF-R were used. NHEK cells express at least 15-fold less EGF-R than A431 cells. EKB-569 was a potent inhibitor of proliferation in NHEK, A431, and MDA-468 cells (IC(50) = 61, 125, and 260 nM, respectively) but not MCF-7 cells (IC(50) = 3600 nM). EKB-569 was also a potent inhibitor of EGF-induced phosphorylated EGF-R (pEGF-R) in A431 and NHEK cells (IC(50) = 20-80 nM). The reduction in pEGF-R paralleled inhibition of phosphotyrosine-705 STAT3, while the inhibition of phosphorylated AKT and phosphorylated ERK1/2 occurred at higher concentrations of EKB-569 (75-500 nM) in both A431 and NHEK cells. The effects were specific because EKB-569 did not inhibit the nuclear factor-kappaB pathway. It is proposed that skin toxicity associated with EKB-569 is due to inhibition of EGF-R signaling. Downstream signal transduction markers, particularly the activation status of STAT3, may be useful surrogate markers to guide clinical development of EGF-R inhibitors.

Beyond rapalog therapy: preclinical pharmacology and antitumor activity of WYE-125132, an ATP-competitive and specific inhibitor of mTORC1 and mTORC2.

The mammalian target of rapamycin (mTOR) is a major component of the phosphoinositide 3-kinase (PI3K)/AKT signaling pathway that is dysregulated in 50% of all human malignancies. Rapamycin and its analogues (rapalogs) partially inhibit mTOR through allosteric binding to mTOR complex 1 (mTORC1) but not mTOR complex 2 (mTORC2), an emerging player in cancer. Here, we report WYE-125132 (WYE-132), a highly potent, ATP-competitive, and specific mTOR kinase inhibitor (IC(50): 0.19 +/- 0.07 nmol/L; >5,000-fold selective versus PI3Ks). WYE-132 inhibited mTORC1 and mTORC2 in diverse cancer models in vitro and in vivo. Importantly, consistent with genetic ablation of mTORC2, WYE-132 targeted P-AKT(S473) and AKT function without significantly reducing the steady-state level of the PI3K/PDK1 activity biomarker P-AKT(T308), highlighting a prominent and direct regulation of AKT by mTORC2 in cancer cells. Compared with the rapalog temsirolimus/CCI-779, WYE-132 elicited a substantially stronger inhibition of cancer cell growth and survival, protein synthesis, cell size, bioenergetic metabolism, and adaptation to hypoxia. Oral administration of WYE-132 to tumor-bearing mice showed potent single-agent antitumor activity against MDA361 breast, U87MG glioma, A549 and H1975 lung, as well as A498 and 786-O renal tumors. An optimal dose of WYE-132 achieved a substantial regression of MDA361 and A549 large tumors and caused complete regression of A498 large tumors when coadministered with bevacizumab. Our results further validate mTOR as a critical driver for tumor growth, establish WYE-132 as a potent and profound anticancer agent, and provide a strong rationale for clinical development of specific mTOR kinase inhibitors as new cancer therapy.

Biochemical, cellular, and in vivo activity of novel ATP-competitive and selective inhibitors of the mammalian target of rapamycin.

The mammalian target of rapamycin (mTOR) is centrally involved in cell growth, metabolism, and angiogenesis. While showing clinical efficacy in a subset of tumors, rapamycin and rapalogs are specific and allosteric inhibitors of mTOR complex 1 (mTORC1), but they do not directly inhibit mTOR complex 2 (mTORC2), an emerging player in cancer. Here, we report chemical structure and biological characterization of three pyrazolopyrimidine ATP-competitive mTOR inhibitors, WAY-600, WYE-687, and WYE-354 (IC(50), 5-9 nmol/L), with significant selectivity over phosphatidylinositol 3-kinase (PI3K) isofoms (>100-fold). Unlike the rapalogs, these inhibitors acutely blocked substrate phosphorylation by mTORC1 and mTORC2 in vitro and in cells in response to growth factor, amino acids, and hyperactive PI3K/AKT. Unlike the inhibitors of PI3K or dual-pan PI3K/mTOR, cellular inhibition of P-S6K1(T389) and P-AKT(S473) by the pyrazolopyrimidines occurred at significantly lower inhibitor concentrations than those of P-AKT(T308) (PI3K-PDK1 readout), showing mTOR selectivity in cellular setting. mTOR kinase inhibitors reduced AKT downstream function and inhibited proliferation of diverse cancer cell lines. These effects correlated with a strong G(1) cell cycle arrest in both the rapamycin-sensitive and rapamycin-resistant cells, selective induction of apoptosis, repression of global protein synthesis, and down-regulation of angiogenic factors. When injected into tumor-bearing mice, WYE-354 inhibited mTORC1 and mTORC2 and displayed robust antitumor activity in PTEN-null tumors. Together, our results highlight mechanistic differentiation between rapalogs and mTOR kinase inhibitors in targeting cancer cell growth and survival and provide support for clinical development of mTOR kinase inhibitors as new cancer therapy.

Response and determinants of cancer cell susceptibility to PI3K inhibitors: combined targeting of PI3K and Mek1 as an effective anticancer strategy.

While small molecule inhibitors of the phosphatidylinositide-3-kinase (PI3K) are expected to impact the development of new cancer therapy, the tumor types and underlying cellular pathways determining inhibitor response remain poorly defined. In this report, we have studied anti-proliferative effects of the PI3K inhibitors WAY-266176 and WAY-266175 in a panel of histologically diverse cancer cells. Inactivation of PI3K caused potent growth suppression in some cells (MDA468, BT549, MDA361, MCF7, LNCap, PC3MM2) but minimal suppression in others (MDA231, MDA435, DU145, HCT116, A549), which correlated with a differential down-regulation of cyclin D1, c-Myc, and induction of apoptosis. A heightened PI3K/AKT/mTOR signaling was linked to the sensitive phenotype but did not generally predict inhibitor response. Interestingly, the resistant cells all displayed an elevated phospho-ERK that remained elevated after serum deprivation. In HCT116 cells, activation mutations in the PI3K catalytic subunit PIK3CA and Ki-Ras correlated with a resistant phenotype, which was partially sensitized by homologous replacement with the wild-type Ki-Ras but not by deletion of cellular PTEN. Depletion of Mek1 via siRNA in resistant cells enhanced PI3K inhibitor-induced growth suppression. Moreover, a profoundly augmented growth suppression and apoptosis were achieved in resistant cells by combination treatment with WAY-266176/WAY-266175 and Mek1 kinase inhibitor CI-1040 or UO126. The combination therapy efficiently inhibited mitogenic signaling and reduced expression of cyclin D1 and c-Myc. Our results identify deregulation of the Ras/Raf/Mek/ERK pathway as a dominant determinant in cancer cell resistance to PI3K inhibitors and highlight combined targeting of PI3K and Mek1 as an effective anticancer strategy.

Two photoaffinity analogues of the tripeptide, hemiasterlin, exclusively label alpha-tubulin.

A synthetic analogue of the tripeptide hemiasterlin, designated HTI-286, depolymerizes microtubules, is a poor substrate for P-glycoprotein, and inhibits the growth of paclitaxel-resistant tumors in xenograft models. Two radiolabeled photoaffinity analogues of HTI-286, designated 4-benzoyl-N,beta,beta-trimethyl-l-phenylalanyl-N(1)-[(1S,2E)-3-carboxy-1-isopropylbut-2-enyl]-N(1),3-dimethyl-l-valinamide (probe 1) and N,beta,beta-trimethyl-l-phenylalanyl-4-benzoyl-N-[(1S,2E)-3-carboxy-1-isopropyl-2-butenyl]-N,beta,beta-trimethyl-l-phenylalaninamide (probe 2), were made to help identify HTI-286 binding sites in tubulin. HTI-286, probe 1, and probe 2 had similar affinities for purified tubulin [apparent K(D(app)) = 0.2-1.1 microM], inhibited polymerization of purified tubulin approximately 80%, and were potent inhibitors of cell growth (IC(50) = 1.0-22 nM). Both radiolabeled probes labeled exclusively alpha-tubulin. Labeling by [(3)H]probe 1 was inhibited by probe 1, HTI-286, vinblastine, or dolastatin 10 (another peptide antimitotic agent that depolymerizes microtubules) but was either unaffected or enhanced (at certain temperatures) by colchicine or paclitaxel. [(3)H]Probe 1 also labeled exclusively tubulin in cytosolic extracts of whole cells. The major, if not exclusive, contact site for probe 1 was mapped to residues 314-339 of alpha-tubulin and corresponds to the sheet 8 and helix 10 region. This region is known to (1) have longitudinal interactions with beta-tubulin across the interdimer interface, (2) have lateral interactions with adjacent protofilaments, and (3) contact the N-terminal region of stathmin, a protein that induces depolymerization of tubulin. Binding of probe 1 to this region may alter the conformation of tubulin outside the labeling domain, since enzymatic removal of the C-terminus of only alpha-tubulin by subtilisin after, but not before, photolabeling is blocked by probe 1. These results suggest that hemiasterlin is in close contact with alpha-tubulin and may span the interdimer interface so that it contacts the vinblastine- and dolastatin 10-binding sites believed to be in beta-tubulin. In addition, we speculate that antimitotic peptides mimic the interaction of stathmin with tubulin.