Biochemical and Structural Studies of the Carminomycin 4-O-Methyltransferase DnrK.

Structural and functional studies of the carminomycin 4-O-methyltransferase DnrK are described, with an emphasis on interrogating the acceptor substrate scope of DnrK. Specifically, the evaluation of 100 structurally and functionally diverse natural products and natural product mimetics revealed an array of pharmacophores as productive DnrK substrates. Representative newly identified DnrK substrates from this study included anthracyclines, angucyclines, anthraquinone-fused enediynes, flavonoids, pyranonaphthoquinones, and polyketides. The ligand-bound structure of DnrK bound to a non-native fluorescent hydroxycoumarin acceptor, 4-methylumbelliferone, along with corresponding DnrK kinetic parameters for 4-methylumbelliferone and native acceptor carminomycin are also reported for the first time. The demonstrated unique permissivity of DnrK highlights the potential for DnrK as a new tool in future biocatalytic and/or strain engineering applications. In addition, the comparative bioactivity assessment (cancer cell line cytotoxicity, 4E-BP1 phosphorylation, and axolotl embryo tail regeneration) of a select set of DnrK substrates/products highlights the ability of anthracycline 4-O-methylation to dictate diverse functional outcomes.

Himalaquinones A-G, Angucyclinone-Derived Metabolites Produced by the Himalayan Isolate Streptomyces sp. PU-MM59.

Himalaquinones A-G, seven new anthraquinone-derived metabolites, were obtained from the Himalayan-based Streptomyces sp. PU-MM59. The chemical structures of the new compounds were identified based on cumulative analyses of HRESIMS and NMR spectra. Himalaquinones A-F were determined to be unique anthraquinones that contained unusual C-4a 3-methylbut-3-enoic acid aromatic substitutions, while himalaquinone G was identified as a new 5,6-dihydrodiol-bearing angucyclinone. Comparative bioactivity assessment (antimicrobial, cancer cell line cytotoxicity, impact on 4E-BP1 phosphorylation, and effect on axolotl embryo tail regeneration) revealed cytotoxic landomycin and saquayamycin analogues to inhibit 4E-BP1p and inhibit regeneration. In contrast, himalaquinone G, while also cytotoxic and a regeneration inhibitor, did not affect 4E-BP1p status at the doses tested. As such, this work implicates a unique mechanism for himalaquinone G and possibly other 5,6-dihydrodiol-bearing angucyclinones.

Structure Determination, Functional Characterization, and Biosynthetic Implications of Nybomycin Metabolites from a Mining Reclamation Site-Associated Streptomyces.

We report the isolation and characterization of three new nybomycins (nybomycins B-D, 1-3) and six known compounds (nybomycin, 4; deoxynyboquinone, 5; α-rubromycin, 6; ß-rubromycin, 7; γ-rubromycin, 8; and [2α(1E,3E),4ß]-2-(1,3-pentadienyl)-4-piperidinol, 9) from the Rock Creek (McCreary County, KY) underground coal mine acid reclamation site isolate Streptomyces sp. AD-3-6. Nybomycin D (3) and deoxynyboquinone (5) displayed moderate (3) to potent (5) cancer cell line cytotoxicity and displayed weak to moderate anti-Gram-(+) bacterial activity, whereas rubromycins 6-8 displayed little to no cancer cell line cytotoxicity but moderate to potent anti-Gram-(+) bacterial and antifungal activity. Assessment of the impact of 3 or 5 cancer cell line treatment on 4E-BP1 phosphorylation, a predictive marker of ROS-mediated control of cap-dependent translation, also revealed deoxynyboquinone (5)-mediated downstream inhibition of 4E-BP1p. Evaluation of 1-9 in a recently established axolotl embryo tail regeneration assay also highlighted the prototypical telomerase inhibitor γ-rubromycin (8) as a new inhibitor of tail regeneration. Cumulatively, this work highlights an alternative nybomycin production strain, a small set of new nybomycin metabolites, and previously unknown functions of rubromycins (antifungal activity and inhibition of tail regeneration) and also provides a basis for revision of the previously proposed nybomycin biosynthetic pathway.

Integrated molecular pathway analysis informs a synergistic combination therapy targeting PTEN/PI3K and EGFR pathways for basal-like breast cancer.

BACKGROUND: The basal-like breast cancer (BLBC) subtype is characterized by positive staining for basal mammary epithelial cytokeratin markers, lack of hormone receptor and HER2 expression, and poor prognosis with currently no approved molecularly-targeted therapies. The oncogenic signaling pathways driving basal-like tumorigenesis are not fully elucidated. METHODS: One hundred sixteen unselected breast tumors were subjected to integrated analysis of phosphoinositide 3-kinase (PI3K) pathway related molecular aberrations by immunohistochemistry, mutation analysis, and gene expression profiling. Incidence and relationships between molecular biomarkers were characterized. Findings for select biomarkers were validated in an independent series. Synergistic cell killing in vitro and in vivo tumor therapy was investigated in breast cancer cell lines and mouse xenograft models, respectively. RESULTS: Sixty-four % of cases had an oncogenic alteration to PIK3CA, PTEN, or INPP4B; when including upstream kinases HER2 and EGFR, 75 % of cases had one or more aberration including 97 % of estrogen receptor (ER)-negative tumors. PTEN-loss was significantly associated to stathmin and EGFR overexpression, positivity for the BLBC markers cytokeratin 5/14, and the BLBC molecular subtype by gene expression profiling, informing a potential therapeutic combination targeting these pathways in BLBC. Combination treatment of BLBC cell lines with the EGFR-inhibitor gefitinib plus the PI3K pathway inhibitor LY294002 was synergistic, and correspondingly, in an in vivo BLBC xenograft mouse model, gefitinib plus PI3K-inhibitor PWT-458 was more effective than either monotherapy and caused tumor regression. CONCLUSIONS: Our study emphasizes the importance of PI3K/PTEN pathway activity in ER-negative and basal-like breast cancer and supports the future clinical evaluation of combining EGFR and PI3K pathway inhibitors for the treatment of BLBC.

A divergent enantioselective strategy for the synthesis of griseusins.

The first enantioselective total synthesis of griseusin A, griseusin C, 4'-deacetyl-griseusin A, and two non-native counterparts in 11-14 steps is reported. This strategy highlights a key hydroxy-directed CH olefination of 1-methylene isochroman with an α,ß-unsaturated ketone followed by subsequent stereoselective epoxidation and regioselective cyclization to afford the signature tetrahydro-spiropyran ring. Colorectal cancer cell cytotoxicities of the final products highlight the impact of the griseusin tetrahydro-spiropyran ring on bioactivity. As the first divergent enantioselective synthesis, the strategy put forth sets the stage for further griseusin mechanism-of-action and SAR studies.

AKT and cancer--is it all mTOR?

AKT, a key regulator of cell proliferation and survival, is commonly dysregulated in human cancers. Activated AKT kinase is oncogenic and required for tumorigenesis in PTEN-deficient animals. However, the importance of AKT in mediating transformation by other oncogenes and which of its targets are necessary for this process are poorly understood. In this issue of Cancer Cell, Skeen et al. show that AKT is required for transformation by mutant H-Ras and for experimental skin carcinogenesis. Moreover, the effects of AKT are mediated predominantly or solely via mTORC1. This suggests that AKT or mTOR inhibitors will be useful treatments for many cancers.

The BAD protein integrates survival signaling by EGFR/MAPK and PI3K/Akt kinase pathways in PTEN-deficient tumor cells.

Tumor cells with mutated PTEN proliferate in an EGFR-independent manner. Induction of PTEN sensitizes cells to EGFR inhibition, and the combination causes synergistic apoptosis. Synergy is due to inhibition of two parallel pathways that phosphorylate the proapoptotic protein BAD at distinct sites. Serine 112 phosphorylation is EGFR/MEK/MAPK dependent, whereas serine 136 phosphorylation is PI3K/Akt dependent. Either phosphorylation is sufficient to sequester BAD to 14-3-3. BAD is released and apoptosis is induced only if both serines are dephosphorylated in response to inhibition of both pathways. Reduction of BAD expression by RNA interference prevents apoptosis in response to pathway inhibition. Thus, BAD integrates the antiapoptotic effects of both pathways. Combined inhibition of EGFR and PI3K signaling may be a useful therapeutic strategy.

Enhanced bypass of PD-L1 translation reduces the therapeutic response to mTOR kinase inhibitors.

Increased PD-L1 expression in cancer cells is known to enhance immunosuppression, but the mechanism underlying PD-L1 upregulation is incompletely characterized. We show that PD-L1 expression is upregulated through internal ribosomal entry site (IRES)-mediated translation upon mTORC1 inhibition. We identify an IRES element in the PD-L1 5'-UTR that permits cap-independent translation and promotes continuous production of PD-L1 protein despite effective inhibition of mTORC1. eIF4A is found to be a key PD-L1 IRES-binding protein that enhances PD-L1 IRES activity and protein production in tumor cells treated with mTOR kinase inhibitors (mTORkis). Notably, treatment with mTORkis in vivo elevates PD-L1 levels and reduces the number of tumor-infiltrating lymphocytes in immunogenic tumors, but anti-PD-L1 immunotherapy restores antitumor immunity and enhances the therapeutic efficacy of mTORkis. These findings report a molecular mechanism for regulating PD-L1 expression through bypassing mTORC1-mediated cap-dependent translation and provide a rationale for targeting PD-L1 immune checkpoint to improve mTOR-targeted therapy.

Targeting Squalene Epoxidase Interrupts Homologous Recombination via the ER Stress Response and Promotes Radiotherapy Efficacy.

Over 50% of all patients with cancer are treated with radiotherapy. However, radiotherapy is often insufficient as a monotherapy and requires a nontoxic radiosensitizer. Squalene epoxidase (SQLE) controls cholesterol biosynthesis by converting squalene to 2,3-oxidosqualene. Given that SQLE is frequently overexpressed in human cancer, this study investigated the importance of SQLE in breast cancer and non-small cell lung cancer (NSCLC), two cancers often treated with radiotherapy. SQLE-positive IHC staining was observed in 68% of breast cancer and 56% of NSCLC specimens versus 15% and 25% in normal breast and lung tissue, respectively. Importantly, SQLE expression was an independent predictor of poor prognosis, and pharmacologic inhibition of SQLE enhanced breast and lung cancer cell radiosensitivity. In addition, SQLE inhibition enhanced sensitivity to PARP inhibition. Inhibition of SQLE interrupted homologous recombination by suppressing ataxia-telangiectasia mutated (ATM) activity via the translational upregulation of wild-type p53-induced phosphatase (WIP1), regardless of the p53 status. SQLE inhibition and subsequent squalene accumulation promoted this upregulation by triggering the endoplasmic reticulum (ER) stress response. Collectively, these results identify a novel tumor-specific radiosensitizer by revealing unrecognized cross-talk between squalene metabolites, ER stress, and the DNA damage response. Although SQLE inhibitors have been used as antifungal agents in the clinic, they have not yet been used as antitumor agents. Repurposing existing SQLE-inhibiting drugs may provide new cancer treatments. SIGNIFICANCE: Squalene epoxidase inhibitors are novel tumor-specific radiosensitizers that promote ER stress and suppress homologous recombination, providing a new potential therapeutic approach to enhance radiotherapy efficacy.

Spermine synthase and MYC cooperate to maintain colorectal cancer cell survival by repressing Bim expression.

Dysregulation of polyamine metabolism has been linked to the development of colorectal cancer (CRC), but the underlying mechanism is incompletely characterized. Here, we report that spermine synthase (SMS), a polyamine biosynthetic enzyme, is overexpressed in CRC. Targeted disruption of SMS in CRC cells results in spermidine accumulation, which inhibits FOXO3a acetylation and allows subsequent translocation to the nucleus to transcriptionally induce expression of the proapoptotic protein Bim. However, this induction is blunted by MYC-driven expression of miR-19a and miR-19b that repress Bim production. Pharmacological or genetic inhibition of MYC activity in SMS-depleted CRC cells dramatically induces Bim expression and apoptosis and causes tumor regression, but these effects are profoundly attenuated by silencing Bim. These findings uncover a key survival signal in CRC through convergent repression of Bim expression by distinct SMS- and MYC-mediated signaling pathways. Thus, combined inhibition of SMS and MYC signaling may be an effective therapy for CRC.

Models from experiments: combinatorial drug perturbations of cancer cells.

We present a novel method for deriving network models from molecular profiles of perturbed cellular systems. The network models aim to predict quantitative outcomes of combinatorial perturbations, such as drug pair treatments or multiple genetic alterations. Mathematically, we represent the system by a set of nodes, representing molecular concentrations or cellular processes, a perturbation vector and an interaction matrix. After perturbation, the system evolves in time according to differential equations with built-in nonlinearity, similar to Hopfield networks, capable of representing epistasis and saturation effects. For a particular set of experiments, we derive the interaction matrix by minimizing a composite error function, aiming at accuracy of prediction and simplicity of network structure. To evaluate the predictive potential of the method, we performed 21 drug pair treatment experiments in a human breast cancer cell line (MCF7) with observation of phospho-proteins and cell cycle markers. The best derived network model rediscovered known interactions and contained interesting predictions. Possible applications include the discovery of regulatory interactions, the design of targeted combination therapies and the engineering of molecular biological networks.

Total synthesis of griseusins and elucidation of the griseusin mechanism of action.

A divergent modular strategy for the enantioselective total synthesis of 12 naturally-occurring griseusin type pyranonaphthoquinones and 8 structurally-similar analogues is described. Key synthetic highlights include Cu-catalyzed enantioselective boration-hydroxylation and hydroxyl-directed C-H olefination to afford the central pharmacophore followed by epoxidation-cyclization and maturation via diastereoselective reduction and regioselective acetylation. Structural revision of griseusin D and absolute structural assignment of 2a,8a-epoxy-epi-4'-deacetyl griseusin B are also reported. Subsequent mechanistic studies establish, for the first time, griseusins as potent inhibitors of peroxiredoxin 1 (Prx1) and glutaredoxin 3 (Grx3). Biological evaluation, including comparative cancer cell line cytotoxicity and axolotl embryo tail inhibition studies, highlights the potential of griseusins as potent molecular probes and/or early stage leads in cancer and regenerative biology.

N-glycosylation-defective splice variants of neuropilin-1 promote metastasis by activating endosomal signals.

Neuropilin-1 (NRP1) is an essential transmembrane receptor with a variety of cellular functions. Here, we identify two human NRP1 splice variants resulting from the skipping of exon 4 and 5, respectively, in colorectal cancer (CRC). Both NRP1 variants exhibit increased endocytosis/recycling activity and decreased levels of degradation, leading to accumulation on endosomes. This increased endocytic trafficking of the two NRP1 variants, upon HGF stimulation, is due to loss of N-glycosylation at the Asn150 or Asn261 site, respectively. Moreover, these NRP1 variants enhance interactions with the Met and ß1-integrin receptors, resulting in Met/ß1-integrin co-internalization and co-accumulation on endosomes. This provides persistent signals to activate the FAK/p130Cas pathway, thereby promoting CRC cell migration, invasion and metastasis. Blocking endocytosis or endosomal Met/ß1-integrin/FAK signaling profoundly inhibits the oncogenic effects of both NRP1 variants. These findings reveal an important role for these NRP1 splice variants in the regulation of endocytic trafficking for cancer cell dissemination.

Frenolicin B Targets Peroxiredoxin 1 and Glutaredoxin 3 to Trigger ROS/4E-BP1-Mediated Antitumor Effects.

Peroxiredoxin 1 (Prx1) and glutaredoxin 3 (Grx3) are two major antioxidant proteins that play a critical role in maintaining redox homeostasis for tumor progression. Here, we identify the prototypical pyranonaphthoquinone natural product frenolicin B (FB) as a selective inhibitor of Prx1 and Grx3 through covalent modification of active-site cysteines. FB-targeted inhibition of Prx1 and Grx3 results in a decrease in cellular glutathione levels, an increase of reactive oxygen species (ROS), and concomitant inhibition of cancer cell growth, largely by activating the peroxisome-bound tuberous sclerosis complex to inhibit mTORC1/4E-BP1 signaling axis. FB structure-activity relationship studies reveal a positive correlation between inhibition of 4E-BP1 phosphorylation, ROS-mediated cancer cell cytotoxicity, and suppression of tumor growth in vivo. These findings establish FB as the most potent Prx1/Grx3 inhibitor reported to date and also notably highlight 4E-BP1 phosphorylation status as a potential predictive marker in response to ROS-based therapies in cancer.

mTOR inhibition induces upstream receptor tyrosine kinase signaling and activates Akt.

Stimulation of the insulin and insulin-like growth factor I (IGF-I) receptor activates the phosphoinositide-3-kinase/Akt/mTOR pathway causing pleiotropic cellular effects including an mTOR-dependent loss in insulin receptor substrate-1 expression leading to feedback down-regulation of signaling through the pathway. In model systems, tumors exhibiting mutational activation of phosphoinositide-3-kinase/Akt kinase, a common event in cancers, are hypersensitive to mTOR inhibitors, including rapamycin. Despite the activity in model systems, in patients, mTOR inhibitors exhibit more modest antitumor activity. We now show that mTOR inhibition induces insulin receptor substrate-1 expression and abrogates feedback inhibition of the pathway, resulting in Akt activation both in cancer cell lines and in patient tumors treated with the rapamycin derivative, RAD001. IGF-I receptor inhibition prevents rapamycin-induced Akt activation and sensitizes tumor cells to inhibition of mTOR. In contrast, IGF-I reverses the antiproliferative effects of rapamycin in serum-free medium. The data suggest that feedback down-regulation of receptor tyrosine kinase signaling is a frequent event in tumor cells with constitutive mTOR activation. Reversal of this feedback loop by rapamycin may attenuate its therapeutic effects, whereas combination therapy that ablates mTOR function and prevents Akt activation may have improved antitumor activity.

Beta-catenin cleavage enhances transcriptional activation.

Nuclear activation of Wnt/ß-catenin signaling is required for cell proliferation in inflammation and cancer. Studies from our group indicate that ß-catenin activation in colitis and colorectal cancer (CRC) correlates with increased nuclear levels of ß-catenin phosphorylated at serine 552 (pß-Cat552). Biochemical analysis of nuclear extracts from cancer biopsies revealed the existence of low molecular weight (LMW) pß-Cat552, increased to the exclusion of full size (FS) forms of ß-catenin. LMW ß-catenin lacks both termini, leaving residues in the armadillo repeat intact. Further experiments showed that TCF4 predominantly binds LMW pß-Cat552 in the nucleus of inflamed and cancerous cells. Nuclear chromatin bound localization of LMW pß-Cat552 was blocked in cells by inhibition of proteasomal chymotrypsin-like activity but not by other protease inhibitors. K48 polyubiquitinated FS and LMW ß-catenin were increased by treatment with bortezomib. Overexpressed in vitro double truncated ß-catenin increased transcriptional activity, cell proliferation and growth of tumor xenografts compared to FS ß-catenin. Serine 552-> alanin substitution abrogated K48 polyubiquitination,  ß-catenin nuclear translocation and tumor xenograft growth. These data suggest that a novel proteasome-dependent posttranslational modification of ß-catenin enhances transcriptional activation. Discovery of this pathway may be helpful in the development of diagnostic and therapeutic tools in colitis and cancer.

Snail determines the therapeutic response to mTOR kinase inhibitors by transcriptional repression of 4E-BP1.

Loss of 4E-BP1 expression has been linked to cancer progression and resistance to mTOR inhibitors, but the mechanism underlying 4E-BP1 downregulation in tumors remains unclear. Here we identify Snail as a strong transcriptional repressor of 4E-BP1. We find that 4E-BP1 expression inversely correlates with Snail level in cancer cell lines and clinical specimens. Snail binds to three E-boxes present in the human 4E-BP1 promoter to repress transcription of 4E-BP1. Ectopic expression of Snail in cancer cell lines lacking Snail profoundly represses 4E-BP1 expression, promotes cap-dependent translation in polysomes, and reduces the anti-proliferative effect of mTOR kinase inhibitors. Conversely, genetic and pharmacological inhibition of Snail function restores 4E-BP1 expression and sensitizes cancer cells to mTOR kinase inhibitors by enhancing 4E-BP1-mediated translation-repressive effect on cell proliferation and tumor growth. Our study reveals a critical Snail-4E-BP1 signaling axis in tumorigenesis, and provides a rationale for targeting Snail to improve mTOR-targeted therapies.

Differential gene expression profiles of Jnk1- and Jnk2-deficient murine fibroblast cells.

c-Jun NH(2)-terminal kinase (JNK) 1 and JNK2 have been assumed to complement each other and mediate the same or similar biological functions. However, our recent reports indicated that 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate-induced tumor development is suppressed in Jnk2 knockout mice but enhanced in Jnk1 knockout mice. In the present work, primary embryo cells were isolated from wild-type, Jnk1(-/-) and Jnk2(-/-) mice and used for cDNA microarray analysis. The patterns of gene expression in Jnk1(-/-), Jnk2(-/-), and wild-type cells are different. After 12-O-tetradecanoylphorbol-13-acetate treatment, the changes in the gene expression profiles in three different kinds of cells appear to agree with the differences in susceptibility to tumorigenesis of each respective animal model. These results suggest that JNK1 and JNK2 proteins have different roles in modulating cell function.

Deficiency of c-Jun-NH(2)-terminal kinase-1 in mice enhances skin tumor development by 12-O-tetradecanoylphorbol-13-acetate.

The c-Jun NH(2)-terminal kinase (JNK) has been implicated in regulating cell survival, apoptosis, and transformation. However, the distinct role of JNK isoforms in regulating tumor development is not yet clear. We have found previously that skin tumor formation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), is suppressed in JNK2-deficient (Jnk2(-/-)) mice. Here, we show that JNK1-deficient (Jnk1(-/-)) mice are more susceptible to TPA-induced skin tumor development than wild-type mice. The rate of tumor development in Jnk1(-/-) mice was significantly more rapid than that observed in wild-type mice (P < 0.0001). At the end of 33 weeks of TPA promotion, the number of skin tumors and tumors >1.5 mm in diameter per mouse in Jnk1(-/-) mice was significantly increased by 71% (P < 0.03) and 82% (P < 0.03), respectively, relative to the wild-type mice. Furthermore, the carcinoma incidence and the number of carcinomas per mouse were also higher in Jnk1(-/-) mice. Strikingly, Jnk1(-/-) mouse skin was more sensitive to TPA-induced AP-1 DNA binding activity and phosphorylation of extracellular signal-regulated kinases and Akt, which are two important survival signaling components. These results suggest that JNK1 is a crucial suppressor of skin tumor development.

Role of MAP kinases in UVB-induced phosphorylation of p53 at serine 20.

Phosphorylation of the p53 tumor suppressor protein is one of the key regulatory steps in its activation process. Serine 20 phosphorylation of p53 has been shown to be required for the activation of p53 following UV radiation, but the signaling pathway mediating UV-induced phosphorylation is unknown. Here, we determined the role of MAP kinases in UVB-induced phosphorylation and found that JNKs are directly involved in the phosphorylation of p53 at serine 20. In a mouse JB6 epidermal cell line, dominant negative JNK1 abrogated UVB-induced phosphorylation of p53 at serine 20, whereas dominant negative p38 kinase or its inhibitor, SB202190, partially attenuated the phosphorylation. In contrast, dominant negative ERK2 or the MEK1 inhibitor, PD98059, had no effect on p53 phosphorylation at serine 20. Importantly, UVB-activated or active recombinant JNK1/2, or the p38 kinase downstream target, MAPKAPK-2, but not ERKs or p38 kinase, phosphorylated p53 at serine 20 in vitro. Furthermore, phosphorylation of p53 at serine 20 by UVB-activated JNKs and UVB-induced p53-dependent transcriptional activity were suppressed in Jnk1 or Jnk2 knockout (Jnk1(-/-) or Jnk2(-/-)) cells. Additionally, Jnk1(-/-), Jnk2(-/-), and p53-deficient (p53(-/-)) cells, as well as re-introduction of a p53 mutant with substitution of serine 20 to alanine into p53(-/-) cells, were defective for UVB-induced apoptosis. These findings strongly suggest that JNKs are the major direct signaling mediators of UVB-induced p53 phosphorylation at serine 20.