Silencing of the novel p53 target gene Snk/Plk2 leads to mitotic catastrophe in paclitaxel (taxol)-exposed cells

Loss of p53 sensitizes to antimicrotubule agents in human tumor cells, but little is known about its role during mitosis. We have identified the Polo-like kinase family member serum inducible kinase (Snk/Plk2) as a novel p53 target gene. Snk/Plk2 mutagenesis demonstrated that its kinase activity is negatively regulated by its C terminus. Small interfering RNA (siRNA)-mediated Snk/Plk2 silencing in the presence of the mitotic poisons paclitaxel (Taxol) or nocodazole significantly increased apoptosis, similar to p53 mutations, which confer paclitaxel sensitivity. Furthermore, we have demonstrated that the apoptosis due to silencing of Snk/Plk2 in the face of spindle damage occurs in mitotic cells and not in cells that have progressed to a G(1)-like state without dividing. Since siRNA directed against Snk/Plk2 promoted death of paclitaxel-treated cells in mitosis, we envision a mitotic checkpoint wherein p53-dependent activation of Snk/Plk2 prevents mitotic catastrophe following spindle damage. Finally, these studies suggest that disruption of Snk/Plk2 may be of therapeutic value in sensitizing paclitaxel-resistant tumors.     

Phosphorylation of p21 in G2/M promotes cyclin B-Cdc2 kinase activity

Little is known about the posttranslational control of the cyclin-dependent protein kinase (CDK) inhibitor p21. We describe here a transient phosphorylation of p21 in the G2/M phase. G2/M-phosphorylated p21 is short-lived relative to hypophosphorylated p21. p21 becomes nuclear during S phase, prior to its phosphorylation by CDK2. S126-phosphorylated cyclin B1 binds to T57-phosphorylated p21. Cdc2 kinase activation is delayed in p21-deficient cells due to delayed association between Cdc2 and cyclin B1. Cyclin B1-Cdc2 kinase activity and G2/M progression in p21-/- cells are restored after reexpression of wild-type but not T57A mutant p21. The cyclin B1 S126A mutant exhibits reduced Cdc2 binding and has low kinase activity. Phosphorylated p21 binds to cyclin B1 when Cdc2 is phosphorylated on Y15 and associates poorly with the complex. Dephosphorylation on Y15 and phosphorylation on T161 promotes Cdc2 binding to the p21-cyclin B1 complex, which becomes activated as a kinase. Thus, hyperphosphorylated p21 activates the Cdc2 kinase in the G2/M transition.     

The Akt Inhibitor ISC-4 Synergizes with Cetuximab in 5-FU-Resistant Colon Cancer

Phenylbutyl isoselenocyanate (ISC-4) is an Akt inhibitor with demonstrated preclinical efficacy against melanoma and colon cancer. In this study, we sought to improve the clinical utility of ISC-4 by identifying a synergistic combination with FDA-approved anti-cancer therapies, a relevant and appropriate disease setting for testing, and biomarkers of response. We tested the activity of ISC-4 and 19 FDA-approved anticancer agents, alone or in combination, against the SW480 and RKO human colon cancer cell lines. A synergistic interaction with cetuximab was identified and validated in a panel of additional colon cancer cell lines, as well as the kinetics of synergy. ISC-4 in combination with cetuximab synergistically reduced the viability of human colon cancer cells with wild-type but not mutant KRAS genes. Further analysis revealed that the combination therapy cooperatively decreased cell cycle progression, increased caspase-dependent apoptosis, and decreased phospho-Akt in responsive tumor cells. The synergism between ISC-4 and cetuximab was retained independently of acquired resistance to 5-FU in human colon cancer cells. The combination demonstrated synergistic anti-tumor effects in vivo without toxicity and in the face of resistance to 5-FU. These results suggest that combining ISC-4 and cetuximab should be explored in patients with 5-FU-resistant colon cancer harboring wild-type KRAS.     

First-In-Class Small Molecule ONC201 Induces DR5 and Cell Death in Tumor but Not Normal Cells to Provide a Wide Therapeutic Index as an Anti-Cancer Agent

We previously identified ONC201 (TIC10) as a first-in-class orally active small molecule with robust antitumor activity that is currently in clinical trials in advanced cancers. Here, we further investigate the safety characteristics of ONC201 in preclinical models that reveal an excellent safety profile at doses that exceed efficacious doses by 10-fold. In vitro studies indicated a strikingly different dose-response relationship when comparing tumor and normal cells where maximal effects are much stronger in tumor cells than in normal cells. In further support of a wide therapeutic index, investigation of tumor and normal cell responses under identical conditions demonstrated large apoptotic effects in tumor cells and modest anti-proliferative effects in normal cells that were non-apoptotic and reversible. Probing the underlying mechanism of apoptosis indicated that ONC201 does not induce DR5 in normal cells under conditions that induce DR5 in tumor cells; DR5 is a pro-apoptotic TRAIL receptor previously linked to the anti-tumor mechanism of ONC201. GLP toxicology studies in Sprague-Dawley rats and beagle dogs at therapeutic and exaggerated doses revealed no dose-limiting toxicities. Observations in both species at the highest doses were mild and reversible at doses above 10-fold the expected therapeutic dose. The no observed adverse event level (NOAEL) was ≥42 mg/kg in dogs and ≥125 mg/kg in rats, which both correspond to a human dose of approximately 1.25 g assuming standard allometric scaling. These results provided the rationale for the 125 mg starting dose in dose escalation clinical trials that began in 2015 in patients with advanced cancer.     

Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

Background

SXT is an integrating conjugative element (ICE) originally isolated from Vibrio cholerae, the bacterial pathogen that causes cholera. It houses multiple antibiotic and heavy metal resistance genes on its ca. 100 kb circular double stranded DNA (dsDNA) genome, and functions as an effective vehicle for the horizontal transfer of resistance genes within susceptible bacterial populations. Here, we characterize the activities of an alkaline exonuclease (S066, SXT-Exo) and single strand annealing protein (S065, SXT-Bet) encoded on the SXT genetic element, which share significant sequence homology with Exo and Bet from bacteriophage lambda, respectively.

Results

SXT-Exo has the ability to degrade both linear dsDNA and single stranded DNA (ssDNA) molecules, but has no detectable endonuclease or nicking activities. Adopting a stable trimeric arrangement in solution, the exonuclease activities of SXT-Exo are optimal at pH 8.2 and essentially require Mn²⁺ or Mg²⁺ ions. Similar to lambda-Exo, SXT-Exo hydrolyzes dsDNA with 5'- to 3'-polarity in a highly processive manner, and digests DNA substrates with 5'-phosphorylated termini significantly more effectively than those lacking 5'-phosphate groups. Notably, the dsDNA exonuclease activities of both SXT-Exo and lambda-Exo are stimulated by the addition of lambda-Bet, SXT-Bet or a single strand DNA binding protein encoded on the SXT genetic element (S064, SXT-Ssb). When co-expressed in E. coli cells, SXT-Bet and SXT-Exo mediate homologous recombination between a PCR-generated dsDNA fragment and the chromosome, analogous to RecET and lambda-Bet/Exo.

Conclusions

The activities of the SXT-Exo protein are consistent with it having the ability to resect the ends of linearized dsDNA molecules, forming partially ssDNA substrates for the partnering SXT-Bet single strand annealing protein. As such, SXT-Exo and SXT-Bet may function together to repair or process SXT genetic elements within infected V. cholerae cells, through facilitating homologous DNA recombination events. The results presented here significantly extend our general understanding of the properties and activities of alkaline exonuclease and single strand annealing proteins of viral/bacteriophage origin, and will assist the rational development of bacterial recombineering systems.     

Human colon cancer stem cells are enriched by insulin-like growth factor-1 and are sensitive to figitumumab

Cancer stem cells (CSCs) are recognized as contributors to cancer progression and therapeutic resistance in liquid and solid malignancies. We analyzed a panel of human colon cancer cell lines for CSC populations by side population and aldehyde dehydrogenase activity. IGF-1 enriches these putative colon CSC populations in a β-catenin-dependent manner. Chemical inhibition of Akt depletes SP cells, and conversely, the overexpression of a constitutively active mutant version of Akt is sufficient to enrich CSC populations. CP-751,871, a fully human antibody with specificity to the IGF-1 receptor, is currently being tested in clinical trials for a variety of solid tumors. CP-751,871 reduces CSC populations in colon cancer cell lines in vitro and reduces tumor growth in vivo. We have identified a novel role for IGF-1 in the enrichment of chemoresistant CSC populations. Our results suggest that CP-751,871 has preferential activity against putative CSC populations and, therefore, may complement current standard chemotherapeutic regimens that target cycling cells.Key words: IGF-1, cancer stem cell, colon cancer, figitumumab     

The relative contribution of pro-apoptotic p53 target genes in the triggering of apoptosis following DNA damage in vitro and in vivo

The p53 pathway displays a large degree of redundancy in the expression of a number of pro-apoptotic mechanisms following DNA damage that, among others, involves increased expression of several pro-apoptotic genes through transactivation. Spatial and temporal cellular contexts contribute to the complexity of the regulation of apoptosis, hence different genes may show a cell- and tissue-dependent specificity with regard to the regulation of cell death and act in concert or show redundancy with one and another. We used siRNA technology to assess the effect of multiple ablations of documented pro-apoptotic p53 target genes (PPG) in the colorectal cancer cell line HCT116 and generated mice deficient in both of the extrinsic and intrinsic PPGs genes Dr5 and Puma following treatment with chemotherapeutics and ionizing radiation. DR5, Fas, Bax, Bad, Puma and Bnip3L were induced by 5-FU and adriamycin (ADR) in HCT116 cells in a p53-dependent manner. The resulting caspase 3/7 activity in HCT116 cells following treatment were suppressed by ablated expression of the PPGs in the extrinsic as well as the intrinsic pathway. To our surprise, knocking-down any of the PPGs concomitantly with DR5 did not further inhibit caspase 3/7 activity whereas inhibiting DR5-expression in HCT116Bax knockdown (kd) and HCT116Fas kd did, suggesting that these genes act downstream or in synergy with DR5. This was supported by our in vivo observations, since Puma and Dr5 were equally efficient in protecting cells of the spleen from sub-lethal radiation-induced apoptosis but less effective compared with irradiated p53^−/− mice. To our surprise, Dr5^−/−; Puma^−/− mice did not show additive protection from radiation-induced apoptosis in any of the investigated organs. Our data indicates that the intrinsic pathway may rely on extrinsic signals to promote cell death in a cell- and tissue-dependent manner following DNA damage. Furthermore, p53 must rely on mechanisms independent of DR5 and PUMA to initiate apoptosis following γ-radiation in the spleen and thymus in vivo.Key words: p53, KILLER/DR5, PUMA, apoptosis, DNA damage     

CTGF-mediated autophagy-senescence transition in tumor stroma promotes anabolic tumor growth and metastasis

Comment on: Capparelli C, et al. Cell Cycle 2012; 11:2272-84 and Capparelli C, et al. Cell Cycle 2012; 11:2285-302.Otto Warburg first observed that cancer cells preferentially undergo glycolysis instead of the mitochondrial TCA cycle even under oxygen-rich conditions. This form of energy metabolism in cancer cells is called “aerobic glycolysis” or the “Warburg effect.”¹ Lisanti and colleagues have previously proposed an alternative model called the “the reverse Warburg effect,” in which aerobic glycolysis predominantly occurs in stromal fibroblasts.² During this process, cancer cells secrete oxidative stress factors, such as hydrogen peroxide, into the tumor microenvironment, which induces autophagy. This leads to degradation of mitochondria (mitophagy) and elevated glycolysis in cancer-associated fibroblasts.³ Aerobic glycolysis results in the elevated production of pyruvate, ketone bodies and L-lactate, which can be utilized by cancer cells for anabolic growth and metastasis. At the molecular level, stromal fibroblasts lose expression of caveolin-1 and activate HIF-1a (Fig. 1), TGFβ and NFκB signaling.⁴ Stromal caveolin-1 expression predicts clinical outcome in breast cancer patients.⁵Open in a separate windowFigure 1. CTGF-mediated autophagy-senescence transition in tumor stroma promotes anabolic tumor growth and metastasis. Cancer cells secrete oxidative stress factors (H₂O₂) that induce autophagy in cancer-associated fibroblasts. Additionally, caveolin-1 (cav-1) loss leads to activation of connective tissue growth factor (CTGF) and HIF-1α that mediate autophagy and senescence in these stromal cells. This is called the autophagy-senescence transition (AST). AST leads to mitophagy and elevated glycolysis in cancer-associated fibroblasts. Aerobic glycolysis results in the elevated production of several nutrients (pyruvate, ketone bodies and L-lactate), which can be utilized by cancer cells for tumor growth and metastasis.In the June 15, 2012 issue of Cell Cycle, two studies by Capparelli et al. further validate the “autophagic tumor stroma model of cancer” described above, as well as identify novel mechanisms involved in this process.^6,7 Autophagy and senescence are induced by the same stimuli and are known to occur simultaneously in cells. In the first study, the authors hypothesize that the onset of senescence in the tumor stroma in response to autophagy/mitophagy contributes to mitochondrial dysfunction and aerobic glycolysis. In order to genetically validate this process of autophagy-senescence transition (AST) (Fig. 1), Capparelli et al. overexpressed several autophagy-promoting factors (BNIP3, cathepsin B, Beclin-1 and ATG16L1) in hTERT fibroblasts to constitutively induce autophagy. Autophagic fibroblasts lost caveolin-1 expression and displayed enhanced tumor growth and metastasis when co-injected with breast cancer cells in mice, without an increase in angiogenesis. In contrast, constitutive activation of autophagy in breast cancer cells inhibited in vivo tumor growth. Autophagic fibroblasts also showed mitochondrial dysfunction, increased production of nutrients (L-lactate and ketone bodies) and features of senescence (β-galactosidase activity and p21 activation). AST was also demonstrated in human breast cancer patient samples.⁷ In the second study, using a similar experimental approach, the authors evaluated the role of the TGFβ target gene, connective tissue growth factor (CTGF), in the induction of AST and aerobic glycolysis in cancer-associated fibroblasts. CTGF would be activated in the tumor stroma upon loss of caveolin-1. CTGF overexpression in fibroblasts induced autophagy/mitophagy, glycolysis and L-lactate production in a HIF-1α-dependent manner along with features of senescence and oxidative stress. CTGF overexpression in fibroblasts also promoted tumor growth when co-injected with breast cancer cells in mice (Fig. 1), independent of angiogenesis. As expected, CTGF overexpression in breast cancer cells inhibited tumor growth. CTGF is known to be involved in extracellular matrix synthesis; however, the effects of CTGF overexpression in fibroblasts and tumor cells were found to be independent of this function.⁶Overall, the authors have identified a novel mechanism by which CTGF promotes AST and aerobic glycolysis in cancer-associated fibroblasts. In turn, the stromal cells stimulate anabolic tumor growth and metastasis. The authors also genetically validate the two-compartment model of cancer metabolism, whereby autophagy genes and CTGF have differential effects in stromal cells and tumor cells. The current studies have several implications for cancer therapy. The finding that HIF-1 activation is necessary for the induction of autophagy and senescence downstream of caveolin-1 loss and CTGF activation in stromal fibroblasts is intriguing. Activation of HIF-1 in the hypoxic tumor microenvironment is known to promote tumor cell growth, survival and therapeutic resistance.⁸ Therefore, targeting HIF-1 has the potential to block tumor progression through dual inhibitory effects on hypoxic cancer cell growth and survival as well as the induction of autophagy in stromal fibroblasts. CTGF and AST in the tumor stroma could serve as biomarkers for predicting clinical outcome, therapy response and metastasis. The two-compartment model of tumor metabolism raises further questions regarding the use of antioxidants and autophagy inhibitors/inducers for cancer therapy. The use of these agents in the clinic should be carefully evaluated considering their differential effects on stromal cells and cancer cells.     

Mitomycin C potentiates TRAIL-induced apoptosis through p53-independent upregulation of death receptors: Evidence for the role of c-Jun N-terminal kinase activation

The discovery of the molecular targets of chemotherapeutic medicines and their chemical footprints can validate and improve the use of such medicines. In the present report, we investigated the effect of mitomycin C (MMC), a classical chemotherapeutic agent on cancer cell apoptosis induced by TRAIL. We found that MMC not only potentiated TRAIL-induced apoptosis in HCT116 (p53−/−) colon cancer cells but also sensitized TRAIL-resistant colon cancer cells HT-29 to the cytokine both in vitro and in vivo. MMC also augmented the pro-apoptotic effects of two TRAIL receptor agonist antibodies, mapatumumab and lexatumumab. At a mechanistic level, MMC downregulated cell survival proteins, including Bcl2, Mcl-1 and Bcl-XL, and upregulated pro-apoptotic proteins including Bax, Bim and the cell surface expression of TRAIL death receptors DR4 and DR5. Gene silencing of DR5 by short hairpin RNA reduced the apoptosis induced by combination treatment of MMC and TRAIL. Induction of DR4 and DR5 was independent of p53, Bax and Bim but was dependent on c-Jun N terminal kinase (JNK) as JNK pharmacological inhibition and siRNA abolished the induction of the TRAIL receptors by MMC.     

Noninvasive vascular imaging in fluorescent tumors using multispectral unmixing

Noninvasive imaging of tumor vascularization in animal models provides an important tool for studying the biology of tumor angiogenesis as well as monitoring the effects of antiangiogenic therapies. Through the use of in vivo multispectral fluorescent imaging, we have discovered a distinct spectral signature associated with blood vessels present in fluorescent tumors in mice. This unique spectral signature allows for the tumor vasculature to be imaged and quantified without the use of vascular imaging probes. This noninvasive vascular imaging technique allows for real-time analysis of tumor vascularization, which provides a powerful and efficient tool for monitoring the effect of antiangiogenic therapies in preclinical animal models.