Background: We examined the potential of metformin (MET) to enhance non-small cell lung cancer (NSCLC) responses to ionising radiation (IR).
Maternal obesity results in a number of obstetrical and fetal complications with both immediate and long-term consequences. The increased prevalence of obesity has resulted in increasing numbers of women of reproductive age in this high-risk group. Since many of these obese women have been subjected to hypercaloric diets from early childhood we have developed a rodent model of life-long maternal obesity to more clearly understand the mechanisms that contribute to adverse pregnancy outcomes in obese women. Female Sprague Dawley rats were fed a control diet (CON - 16% of calories from fat) or high fat diet (HF - 45% of calories from fat) from 3 to 19 weeks of age. Prior to pregnancy HF-fed dams exhibited significant increases in body fat, serum leptin and triglycerides. A subset of dams was sacrificed at gestational day 15 to evaluate fetal and placental development. The remaining animals were allowed to deliver normally. HF-fed dams exhibited a more than 3-fold increase in fetal death and decreased neonatal survival. These outcomes were associated with altered vascular development in the placenta, as well as increased hypoxia in the labyrinth. We propose that the altered placental vasculature may result in reduced oxygenation of the fetal tissues contributing to premature demise and poor neonatal survival.
Figure 2. Active ABL assembles the RUNX2-TAZ transcription factor complex required for osteoblast differentiation. (A) Primary murine osteoblasts were cultured in osteogenic medium for 14 days, and phosphotyrosine immune complexes were probed with an anti-ABL antibody. WCLs were probed with the indicated antibodies for Western blot analysis. (B) HEK293T cells were cotransfected with Flag-TAZ and RUNX2, with or without ABL (PP or KD). Flag-TAZ immune complexes were probed with an anti-RUNX2 antibody. (C) Luciferase activity from a BGLAP reporter assay in HEK293T cells cotransfected with the indicated constructs. n = 3. (D and E) HEK293T cells were cotransfected with the indicated constructs, and RUNX2 (D) or ABL (E) immune complexes were probed with an anti-ABL (D) or anti-Flag (E) antibody. (F) HEK293T cells infected with shGFP or shABL were cotransfected with RUNX2 and Flag-TAZ. The nuclear compartment was extracted from the cells, and Flag-TAZ immune complexes were probed with an anti-RUNX2 antibody. (G and H) HEK293T cells were cotransfected with WT or YF RUNX2 (G) or Flag-TAZ (H), with or without ABL (PP). RUNX2 (G) or Flag-TAZ (H) immune complexes were probed with an anti-p-Tyr antibody. (I) Primary murine osteoblasts infected with shGFP or shABL were cultured in osteogenic medium. p-Tyr or RUNX2 immune complexes were probed with an anti-RUNX2 or anti-TAZ antibody. (J) Luciferase activity from a Bglap reporter assay in primary murine osteoblasts in the presence of shGFP or shABL. n = 3. (K and L) Saos-2 cells infected with an empty vector control or an FKBP-ABL-expressing retroviral vector in the presence of shGFP (K and L), shRUNX2 (K), or shTAZ (L) were cultured in growth medium containing 50 nM FK1012. Cells were stained with alizarin red S solution. n = 3. *P < 0.05, by ANOVA with a Tukey-Kramer post-hoc test. Data represent the mean ± SEM. YAP has previously been reported to be stabilized following tyrosine phosphorylation by ABL (25). In distinction, we observed that all-tyrosine-to-phenylalanine-mutant TAZ (YF) was also stabilized by active ABL (PP) (Supplemental Figure 4F), suggesting that TAZ stabilization by ABL is not mediated by tyrosine phosphorylation. TAZ is degraded following the phosphorylation of Ser311 by LATS, which primes for the phosphorylation of Ser314 by CK1ε (26). Phosphorylated Ser314 lies in the TAZ phosphodegron and triggers binding to and subsequent ubiquitylation of TAZ by the E3-ubiquitin ligase β-TrCP (26). We hypothesized that ABL suppressed TAZ ubiquitylation through disruption of the interaction between TAZ and β-TrCP and observed that ABL (PP), but not ABL (KD), diminished the interaction between these proteins ( Figure 4F). These data demonstrate that ABL stabilizes TAZ through the suppression of its ubiquitin-mediated degradation pathway initiated by β-TrCP. The Journal of Clinical Investigation R E S E A R C H A R T I C L EABL stabilizes the TAZ-TEAD complex required for osteoblast expansion. We have shown that ABL mediated stabilization of TAZ protein and asked whet...
IntroductionEarlier, we showed that in cancer cells, AMP-activated kinase (AMPK) participates in a signal transduction pathway involving ATM-AMPK-p53/p21cip1 which is activated by ionizing radiation (IR) to mediate G2-M arrest and enhanced cytotoxicity. We also observed that AMPK modulates ATM expression and activity and the IR response of the Akt-mTOR pathway. Since the ATM, AMPK and Akt pathways are key targets of novel radio-sensitizing therapeutics, we examined the chronic modultion of expression and activity of those pathways by IR alone in xenograft models of lung cancer.MethodsImmuno-compromised mice were grafted with human lung A549 and H1299 cells, were treated with a single fraction of 0 or 10 Gy, and left to grow for 8 weeks. Extracted tumors were subjected to lysis and immunoblotting or fixation and immunohistochemical analysis.ResultsIR inhibited significantly xenograft growth and was associated with increased expression of Ataxia Telengiectasia Mutated (ATM) and enhanced phosphorylation of two ATM targets, H2Ax and checkpoint kinase Chk2. Irradiated tumours showed increased total AMPK levels and phosphorylation of AMPK and its substrate Acetyl-CoA Carboxylase (ACC). IR led to enhanced expression and phosphorylation of p53 and cyclin dependent kinase inhibitors p21cip1 and p27kip1. However, irradiated tumours had reduced phosphorylation of Akt, mTOR and it‘s target translation initiation inhibitor 4EBP1. Irradiated xenografts showed reduced microvessel density, reduced expression of CD31 but increased expression of hypoxia-induced factor 1A (HIF1a) compared to controls.ConclusionIR inhibits epithelial cancer tumour growth and results in sustained expression and activation of ATM-Chk2, and AMPK-p53/p21cip1/p27kip1 but partial inhibition of the Akt-mTOR signaling pathways. Future studies should examine causality between those events and explore whether further modulation of the AMPK and Akt-mTOR pathways by novel therapeutics can sensitize lung tumours to radiation.
Objectives: FPI-1434 is a targeted alpha-particle therapeutic consisting of an IGF-1R targeting antibody radiolabeled with Actinium-225. The primary mechanism of action for FPI-1434 is induction of double strand DNA breaks (DSB) in targeted tumors resulting in cell death. Poly (ADP-ribose) polymerase (PARP) is part of the cellular mechanism that repairs DSB. In cancer patients with genetic defects in DSB repair (eg. BRCA1/2), the PARP pathway becomes a primary repair system and its inhibition results in cell death. This mechanism has been leveraged as a therapy against DNA-repair deficient tumors leading to FDA-approval of PARP inhibitors (PARPi) including Olaparib. Treatment with PARPi to block repair of DNA damage driven by FPI-1434 may act synergistically to increase the lethal DNA damage load. To that end, Fusion has performed studies to combine FPI-1434 and Olaparib against tumor models with no pre-disposing defects in DNA repair. Methods: Colorectal (Colo-205) or lung (A549) cancer xenografts were established in Balb/c nude mice. For efficacy studies, a dose combination matrix for FPI-1434 and Olaparib was tested. FPI-1434 was dosed (i.v.) once at 20-200 nCi followed with Olaparib (i.p.) at doses of 0-50 mg/kg. Olaparib was dosed 24h after FPI-1434 using a 5 day on/2 day off schedule (5 mice/group). DSB formation (γH2AX) and apoptosis (cleaved caspase 3) were evaluated in treated tumors by IHC staining. Results: DSB formation and induction of apoptosis were detected in FPI-1434-treated tumors in a time and dose dependent manner. DSB formation was observed in all areas of tumor containing intact or apoptotic cancer cells confirming FPI-1434 mechanism. In tumor efficacy studies, Olaparib had no single-agent efficacy in the Colo-205 or A549 models. Single doses of FPI-1434 at 20 nCi had no effect on Colo-205 tumors, suppressed growth at 50 nCi and caused regression at 100 nCi. Combination efficacy was seen in the 20 and 50 nCi dose groups, including Olaparib. Doses of 20 nCi FPI-1434 and 25 mg/kg Olaparib had the strongest combined effect in this model. In the A549 model, single doses of FPI-1434 had no effect at 20 or 50 nCi, caused growth suppression at 100 nCi and regression at 200 nCi. Combination efficacy was seen in the 20, 50 and 100 nCi dose groups, including Olaparib. Doses of 50 nCi FPI-1434 and 25 mg/kg Olaparib had the strongest combined effect in this model. In general, the strongest efficacy was observed by combining ineffective single-agent doses of the two compounds. Strong efficacy of high dose FPI-1434 masked observable combination effects suggesting that FPI-1434 was the therapeutic driver. Conclusions: Olaparib co-dosing enhanced FPI-1434 efficacy which supports consideration of this combination for clinical use. Ineffective single-agent doses, when combined, resulted in synergistic efficacy in both Colo-205 and A549 models suggesting that the mechanism can be applied to multiple cancer types where predisposing mutations in DNA repair are lacking. The strongest combination effect appeared to occur at the low single-agent doses suggesting that PARP inhibition may potentiate efficacy at lower clinical doses of FPI-1434. Citation Format: Meiduo Hu, John Forbes, Ryan Simms, Yaryna Storozhuk, Eric Burak, John Valliant. Combination of IGF-1R targeted alpha therapy with Olaparib results in synergistic efficacy against colorectal and lung cancer xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB130.
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