Novel chemical library screen identifies naturally occurring plant products that specifically disrupt glioblastoma-endothelial cell interactions

Tumor growth is not solely a consequence of autonomous tumor cell properties. Rather, tumor cells act upon and are acted upon by their microenvironment. It is tumor tissue biology that ultimately determines tumor growth. Thus, we developed a compound library screen for agents that could block essential tumor-promoting effects of the glioblastoma (GBM) perivascular stem cell niche (PVN). We modeled the PVN with three-dimensional primary cultures of human brain microvascular endothelial cells in Matrigel. We previously demonstrated stimulated growth of GBM cells in this PVN model and used this to assay PVN function. We screened the Microsource Spectrum Collection library for drugs that specifically blocked PVN function, without any direct effect on GBM cells themselves. Three candidate PVN-disrupting agents, Iridin, Tigogenin and Triacetylresveratrol (TAR), were identified and evaluated in secondary in vitro screens against a panel of primary GBM isolates as well as in two different in vivo intracranial models. Iridin and TAR significantly inhibited intracranial tumor growth and prolonged survival in these mouse models. Together these data identify Iridin and TAR as drugs with novel GBM tissue disrupting effects and validate the importance of preclinical screens designed to address tumor tissue function rather than the mechanisms of autonomous tumor cell growth

Conjugation to a SMAC mimetic potentiates sigma-2 ligand induced tumor cell death in ovarian cancer

BACKGROUND: Drug resistance is a significant problem in the treatment of ovarian cancer and can be caused by multiple mechanisms. Inhibition of apoptosis by the inhibitor of apoptosis proteins (IAPs) represents one such mechanism, and can be overcome by a mitochondrial protein called second mitochondria-derived activator of caspases (SMAC). We have previously shown that the ligands of sigma-2 receptors effectively induce tumor cell death. Additionally, because sigma-2 receptors are preferentially expressed in tumor cells, their ligands provide an effective mechanism for selective anti-cancer therapy. METHODS: In the current work, we have improved upon the previously described sigma-2 ligand SW43 by conjugating it to a pro-apoptotic small molecule SMAC mimetic SW IV-52, thus generating the novel cancer therapeutic SW IV-134. The new cancer drug was tested for receptor selectivity and tumor cell killing activity in vitro and in vivo. RESULTS: We have shown that SW IV-134 retained adequate sigma-2 receptor binding affinity in the context of the conjugate and potently induced cell death in ovarian cancer cells. The cell death induced by SW IV-134 was significantly greater than that observed with either SW43 or SW IV-52 alone and in combination. Furthermore, the intraperitoneal administration of SW IV-134 significantly reduced tumor burden and improved overall survival in a mouse xenograft model of ovarian cancer without causing significant adverse effects to normal tissues. Mechanistically, SW IV-134 induced degradation of cIAP-1 and cIAP-2 leading to NF-қB activation and TNFα-dependent cell death. CONCLUSIONS: Our findings suggest that coupling sigma-2 ligands to SMAC peptidomimetics enhances their effectiveness while maintaining the cancer selectivity. This encouraging proof-of-principle preclinical study supports further development of tumor-targeted small peptide mimetics via ligands to the sigma-2 receptor for future clinical applications

Synthetic Lethality of Chk1 Inhibition Combined with p53 and/or p21 Loss During a DNA Damage Response in Normal and Tumor Cells

Cell cycle checkpoints ensure genome integrity and are frequently compromised in human cancers. A therapeutic strategy being explored takes advantage of checkpoint defects in p53-deficient tumors in order to sensitize them to DNA-damaging agents by eliminating Chk1-mediated checkpoint responses. Using mouse models, we demonstrated that p21 is a key determinant of how cells respond to the combination of DNA damage and Chk1 inhibition (combination therapy) in normal cells as well as in tumors. Loss of p21 sensitized normal cells to the combination therapy much more than did p53 loss and the enhanced lethality was partially blocked by CDK inhibition. In addition, basal pools of p21 (p53 independent) provided p53 null cells with protection from the combination therapy. Our results uncover a novel p53-independent function for p21 in protecting cells from the lethal effects of DNA damage followed by Chk1 inhibition. As p21 levels are low in a significant fraction of colorectal tumors, they are predicted to be particularly sensitive to the combination therapy. Results reported in this study support this prediction

The ADP receptor P2RY12 regulates osteoclast function and pathologic bone remodeling

The adenosine diphosphate (ADP) receptor P2RY12 (purinergic receptor P2Y, G protein coupled, 12) plays a critical role in platelet aggregation, and P2RY12 inhibitors are used clinically to prevent cardiac and cerebral thrombotic events. Extracellular ADP has also been shown to increase osteoclast (OC) activity, but the role of P2RY12 in OC biology is unknown. Here, we examined the role of mouse P2RY12 in OC function. Mice lacking P2ry12 had decreased OC activity and were partially protected from age-associated bone loss. P2ry12(–/–) OCs exhibited intact differentiation markers, but diminished resorptive function. Extracellular ADP enhanced OC adhesion and resorptive activity of WT, but not P2ry12(–/–), OCs. In platelets, ADP stimulation of P2RY12 resulted in GTPase Ras-related protein (RAP1) activation and subsequent α(IIb)β(3) integrin activation. Likewise, we found that ADP stimulation induced RAP1 activation in WT and integrin β(3) gene knockout (Itgb3(–/–)) OCs, but its effects were substantially blunted in P2ry12(–/–) OCs. In vivo, P2ry12(–/–) mice were partially protected from pathologic bone loss associated with serum transfer arthritis, tumor growth in bone, and ovariectomy-induced osteoporosis: all conditions associated with increased extracellular ADP. Finally, mice treated with the clinical inhibitor of P2RY12, clopidogrel, were protected from pathologic osteolysis. These results demonstrate that P2RY12 is the primary ADP receptor in OCs and suggest that P2RY12 inhibition is a potential therapeutic target for pathologic bone loss

Cerenkov Radiation Energy Transfer (CRET) Imaging: A Novel Method for Optical Imaging of PET Isotopes in Biological Systems

Positron emission tomography (PET) allows sensitive, non-invasive analysis of the distribution of radiopharmaceutical tracers labeled with positron (β(+))-emitting radionuclides in small animals and humans. Upon β(+) decay, the initial velocity of high-energy β(+) particles can momentarily exceed the speed of light in tissue, producing Cerenkov radiation that is detectable by optical imaging, but is highly absorbed in living organisms.To improve optical imaging of Cerenkov radiation in biological systems, we demonstrate that Cerenkov radiation from decay of the PET isotopes (64)Cu and (18)F can be spectrally coupled by energy transfer to high Stokes-shift quantum nanoparticles (Qtracker705) to produce highly red-shifted photonic emissions. Efficient energy transfer was not detected with (99m)Tc, a predominantly γ-emitting isotope. Similar to bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET), herein we define the Cerenkov radiation energy transfer (CRET) ratio as the normalized quotient of light detected within a spectral window centered on the fluorophore emission divided by light detected within a spectral window of the Cerenkov radiation emission to quantify imaging signals. Optical images of solutions containing Qtracker705 nanoparticles and [(18)F]FDG showed CRET ratios in vitro as high as 8.8±1.1, while images of mice with subcutaneous pseudotumors impregnated with Qtracker705 following intravenous injection of [(18)F]FDG showed CRET ratios in vivo as high as 3.5±0.3.Quantitative CRET imaging may afford a variety of novel optical imaging applications and activation strategies for PET radiopharmaceuticals and other isotopes in biomaterials, tissues and live animals

Valosin-containing protein (VCP) is required for autophagy and is disrupted in VCP disease

Accumulation of autophagosomes because of impaired autophagy during valosin-containing protein (VCP)–linked dementia is explained by the absence or reduced activity of VCP

The clinicopathological and prognostic impact of 14-3-3 sigma expression on vulvar squamous cell carcinomas

<p>Abstract</p> <p>Background</p> <p>14-3-3 sigma (σ) promotes G2/M cell cycle arrest by sequestering cyclin B1-CDC2 complex in cytoplasm. Down-regulation of 14-3-3σ, which has been demonstrated in various carcinomas, may contribute to malignant transformation. However, the exact role of 14-3-3σ in the pathogenesis of vulvar carcinoma is not fully characterized, and the prognostic impact of 14-3-3σ protein expression is still unknown.</p> <p>Methods</p> <p>We investigated the 14-3-3σ expression in a series of 302 vulvar squamous cell carcinomas using immunohistochemistry and its associations with clinicopathological factors and clinical outcome.</p> <p>Results</p> <p>In cytoplasm, nucleus and cytoplasm/nucleus of vulvar carcinomas high 14-3-3σ protein expression was found in 72%, 59% and 75% of the carcinomas, respectively, and low levels in 28%, 41% and 25% of the cases, respectively. High level of 14-3-3σ in cytoplasm, nucleus and cytoplasm/nucleus was significantly correlated to large tumor diameter (<it>p </it>= 0.001, <it>p </it>= 0.002 and <it>p </it>= 0.001, respectively) and deep invasion (<it>p </it>= 0.01, <it>p </it>= 0.001 and <it>p </it>= 0.007, respectively). Variations of 14-3-3σ protein expression were not associated to disease-specific survival.</p> <p>Conclusion</p> <p>Our results indicate that 14-3-3σ may be involved in the development of a subset of vulvar squamous cell carcinomas by down-regulation of 14-3-3σ protein. Neither cytoplasmic nor nuclear level of 14-3-3σ expression was associated with prognosis.</p

Metformin promotes antitumor immunity via endoplasmic-reticulum-associated degradation of PD-L1

Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformin’s role in cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy

KAI1 suppresses HIF-1α and VEGF expression by blocking CDCP1-enhanced Src activation in prostate cancer

<p>Abstract</p> <p>Background</p> <p>KAI1 was initially identified as a metastasis-suppressor gene in prostate cancer. It is a member of the tetraspan transmembrane superfamily (TM4SF) of membrane glycoproteins. As part of a tetraspanin-enriched microdomain (TEM), KAI1 inhibits tumor metastasis by negative regulation of Src. However, the underlying regulatory mechanism has not yet been fully elucidated. CUB-domain-containing protein 1 (CDCP1), which was previously known as tetraspanin-interacting protein in TEM, promoted metastasis via enhancement of Src activity. To better understand how KAI1 is involved in the negative regulation of Src, we here examined the function of KAI1 in CDCP1-mediated Src kinase activation and the consequences of this process, focusing on HIF-1 α and VEGF expression.</p> <p>Methods</p> <p>We used the human prostate cancer cell line PC3 which was devoid of KAI1 expression. Vector-transfected cells (PC3-GFP clone #8) and KAI1-expressing PC3 clones (PC3-KAI1 clone #5 and #6) were picked after stable transfection with KAI1 cDNA and selection in 800 <it>μ</it>g/ml G418. Protein levels were assessed by immunoblotting and VEGF reporter gene activity was measured by assaying luciferase activitiy. We followed tumor growth <it>in vivo </it>and immunohistochemistry was performed for detection of HIF-1, CDCP1, and VHL protein level.</p> <p>Results</p> <p>We demonstrated that Hypoxia-inducible factor 1α (HIF-1α) and VEGF expression were significantly inhibited by restoration of KAI1 in PC3 cells. In response to KAI1 expression, CDCP1-enhanced Src activation was down-regulated and the level of von Hippel-Lindau (VHL) protein was significantly increased. In an <it>in vivo </it>xenograft model, KAI1 inhibited the expression of CDCP1 and HIF-1α.</p> <p>Conclusions</p> <p>These novel observations may indicate that KAI1 exerts profound metastasis-suppressor activity in the tumor malignancy process via inhibition of CDCP1-mediated Src activation, followed by VHL-induced HIF-1α degradation and, ultimately, decreased VEGF expression.</p