LOSS OF CASPASE-8 FUNCTION IN COMBINATION WITH SMAC MIMETIC TREATMENT SENSITIZES HEAD AND NECK SQUAMOUS CARCINOMA TO RADIATION THROUGH INDUCTION OF NECROPTOSIS.

Caspase-8 (CASP8) is one of the most frequently mutated genes in Head and Neck Squamous Carcinomas (HNSCC), and mutations of CASP8 are associated with poor overall survival. The distribution of these mutations in HNSCC suggests that they are likely to be inactivating. Inhibition of CASP8 has been reported to sensitize cancer cells to necroptosis, a unique cell death mechanism. Here, we evaluated how CASP8 regulates necroptosis in HSNCC using cell line models and syngeneic mouse xenografts. In vitro, knockdown of CASP8 rendered HNSCCs susceptible to necroptosis induced by a second mitochondria-derived activator of caspase (SMAC) mimetic, Birinapant, when combined with pan-caspase inhibitors zVAD-FMK or emricasan. Strikingly, inhibition of CASP8 function via knockdown or emricasan treatment was associated with enhanced radiation killing by Birinapant through induction of necroptosis. In a syngeneic mouse model of oral cancer, Birinapant, particularly when combined with radiation delayed tumor growth and enhanced survival under CASP8 loss. Exploration of the molecular underpinnings of necroptosis sensitivity confirmed that the level of functional receptor-interacting serine/threonine-protein kinase-3 (RIP3), a key enzyme in the necroptosis pathway was crucial in determining susceptibility to this mode of death. Although an in vitro screen revealed that many HNSCC cell lines were resistant to necroptosis due to low levels of RIP3, patient tumors maintain RIP3 expression and should therefore remain sensitive. Collectively, these results suggest that targeting the necroptosis pathway with SMAC mimetics, especially in combination with radiation, may be a relevant therapeutic approach in HNSCC with compromised CASP8 status, provided that RIP3 function is maintained

Pancreatic cancer microenvironment: a current dilemma

Abstract Pancreatic cancer is one of the leading causes of cancer-related death in the United States and survival outcomes remain dismal despite significant advances in molecular diagnostics and therapeutics in clinical practice. The microenvironment of pancreatic cancer carries unique features with increased desmoplastic reaction and is infiltrated by regulatory T cells and myeloid-derived suppressor cells which negatively impact the effector immune cells. Current evidence suggests that stellate cell-induced hypovascular stroma may have direct effects on aggressive behavior of pancreatic cancer. Preclinical studies suggested improvement in drug delivery to cancer cells with stroma modifying agents. However these findings so far have not been confirmed in clinical trials. In this article, we elaborate current-state-of-the science of the pancreatic cancer microenvironment and its impact on molecular behavior of cancer cells, chemotherapy resistance and druggability of stroma elements in combination with other agents to enhance the efficacy of therapeutic approaches

Structure/function analysis of PARP-1 in oxidative and nitrosative stress-induced monomeric ADPR formation.

Poly adenosine diphosphate-ribose polymerase-1 (PARP-1) is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N) stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR), and formation of the soluble 2(nd) messenger monomeric adenosine diphosphate-ribose (mADPR). Previous studies have delineated specific roles for several of PARP-1's structural domains in the context of its involvement in a DNA damage response. However, little is known about the relationship between the mechanisms through which PARP-1 participates in DNA damage detection/response and those involved in the generation of monomeric ADPR. To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1's BRCT protein interaction domain. Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable. As the BRCT domain is required for PARP-dependent recruitment of XRCC1 to sites of DNA damage, these results suggest that DNA repair and monomeric ADPR 2(nd) messenger generation are parallel mechanisms through which PARP-1 modulates cellular responses to O/N stress

Attenuation of PARP-1 catalytic activity correlates with a reduction in O/N stress induced NAD degradation and TRPM2-dependent Ca²⁺ transients.

<p><i>A) Left panel</i>: Calcium transients in DT40 cells are PARP and TRPM2 dependent. The indicated cell lines were stimulated with 100 mM MNNG, and cytosolic Ca²⁺ transients were monitored by ratiometric analysis of Indo-1 fluorescence by FACS. <i>Middle panel</i>: PARP clones with varying PARP expression levels by western blotting: PARP-deficient DT40 cells were reconstituted with WT human PARP-1, and the parent PARP-KO line and four clones with a range of expression levels are shown. 50 µg of cellular protein was loaded into each lane of an 8% SDS-PAGE gel and analyzed by western blotting. Rabbit anti-human PARP-1 polyclonal antibody was used as the primary antibody for immunoblotting of PARP-1 (1∶4000, Alexis Biochemicals), and IR680 conjugated goat anti-rabbit as secondary antibody (1∶3000, Licor Inc). Blots were analyzed on a Licor Odyssey. <i>Right panel</i>: Dependence of TRPM2-dependent Ca²⁺ transients on PARP expression level. The four PARP-WT expressing clones from the middle panel were compared for TRPM2-dependent Ca²⁺ transients in response to 100 µM MNNG, as measured by ratiometric analysis of Indo-1 fluorescence by FACS. Time to half maximum F400/F475 showed a standard deviation of±33 seconds: clone #2 with an intermediate level of PARP-1 expression was designated as <i>PARP-WT</i> and used as a positive control for subsequent experiments. <i>B) Left Panel</i>: <i>PARP-Y986H</i> protein expression is comparable to <i>PARP-WT</i>: 50 µg of cellular protein were loaded into each lane of an 8% SDS-PAGE gel and analyzed by western blotting. Antibodies were identical to <i>A.) Middle Panel</i>: Relative transcript abundance of <i>PARP-Y986H</i> normalized to <i>PARP-WT</i>, as determined by Q-PCR. <i>Right panel</i>: TRPM2-dependent whole cell currents are similar in <i>PARP-WT</i> and <i>PARP-Y986H</i> mutant clones. Average whole cell currents were not statistically different from one another across all cell types. Cells were patched in the whole cell configuration: the pipette solution contained 100 µM mADPR. The I-V relationship and current development across all cell types was characteristic of TRPM2 and identical to that previously shown by our lab (4). At least 3 whole cell recordings were taken for each cell type. <i>C) Left panel</i>: NAD turnover in <i>PARP-WT</i> and <i>PARP-Y986H</i> cells. Stars indicate a p-value of p≤.001 from baseline for all subsequent points. <i>Right panel</i>: TRPM2-dependent Ca²⁺ transients in <i>PARP-WT</i> and <i>PARP-Y986H</i> cells after stimulation with 100 µM MNNG, as measured by ratiometric analysis of Indo-1 fluorescence by FACS.</p

Loss of the AMD domain, but not the BRCT domain, is associated with reduced NAD degradation and TRPM2-dependent Ca²⁺ transients.

<p><i>A) Left Panel</i>: <i>PARP</i> AMD mutant expression levels relative to PARP-WT 50 µg of cellular protein from each cell line were loaded into each lane of an 8% SDS-PAGE gel and analyzed by western blotting. Rabbit anti-human PARP-1 polyclonal antibody was used as the primary antibody for immunoblotting of PARP-1 (1∶4000, Alexis Biochemicals), and IR680 conjugated goat anti-rabbit as secondary antibody (1∶3000, Licor Inc). Blots were analyzed on a Licor Odyssey. <i>Middle Panel</i>: Relative transcript abundance for PARP AMD mutants normalized to <i>PARP-WT</i>, as determined by Q-PCR. <i>Right Panel</i> TRPM2 dependent whole cell currents are similar in <i>PARP-WT</i> and PARP AMD mutant clones. Average whole cell currents were not statistically different from one another across all cell types. Cells were patched in the whole cell configuration: the pipette solution contained 100 µM mADPR. The I-V relationship and current development across all cell types was characteristic of TRPM2 and identical to that previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006339#pone.0006339-Buelow1" target="_blank">[5]</a>. At least 3 whole cell recordings were taken for each cell type. <i>B) Left Panel</i>: NAD turnover in <i>PARP-WT</i> and <i>PARP-dAMD</i> cells. Stars indicate a p-value of p≤.001 from baseline for all subsequent points. <i>Right Panel</i>: TRPM2-dependent Ca²⁺ transients in <i>PARP-WT</i> and <i>PARP-dAMD</i> cells after stimulation with 100 µM MNNG, as measured by ratiometric analysis of Indo-1 stained cells by FACS. <i>C) Left Panels</i>: NAD turnover in <i>PARP-WT</i>, <i>PARP-dBRCT</i>, and <i>PARP-nBD</i> cells. Stars indicate a p-value of p≤.001 from baseline for all subsequent points. <i>Right Panel</i>: TRPM2-dependent Ca²⁺ transients in <i>PARP-WT</i>, <i>PARP-dBRCT</i>, and <i>PARP-nBD</i> cells after stimulation with 100 µM MNNG, as measured by ratiometric analysis of Indo-1 stained cells by FACS.</p

The effects of DBD mutations on PARP-dependent O/N STRESS induced NAD degradation and TRPM2-dependent Ca²⁺ transients.

<p><i>A) PARP-dZF</i> mutant expression levels relative to PARP-WT:<i>Left Panel</i>: 50 µg of cellular protein were loaded into each lane of an 8% SDS-PAGE gel and analyzed by western blotting. Rabbit anti-human PARP-1 polyclonal antibody was used as the primary antibody for immunoblotting of PARP-1 (1∶4000, Alexis Biochemicals), and IR680 conjugated goat anti-rabbit as secondary antibody (1∶3000, Licor Inc). Blots were analyzed on a Licor Odyssey. <i>Middle Panel</i>: Relative transcript abundance of <i>PARP-dZF</i> mutants normalized to <i>PARP-WT</i>, as determined by Q-PCR. <i>Right panel</i>: TRPM2-dependent whole cell currents are similar in <i>PARP-WT</i> and <i>PARP-dZF</i> mutant clones. Average whole cell currents were not statistically different from one another across all cell types. Cells were patched in the whole cell configuration: the pipette solution contained 100 µM mADPR. The I-V relationship and current development across all cell types was characteristic of TRPM2 and identical to that previously shown by our lab <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006339#pone.0006339-Buelow1" target="_blank">[5]</a>. At least 3 whole cell recordings were taken for each cell type. <i>B) Left panel</i>: NAD turnover in <i>PARP-WT and PARP-dZF1</i> DT40 cells. <i>PARP-dZF1</i> did not show NAD degradation over the course of 30 minutes following application of 100 µM MNNG. Stars indicate a p-value of p≤.001 from baseline for all subsequent points. <i>Right panel</i>: TRPM2-dependent Ca²⁺ transients in <i>PARP-WT and PARP-dZF1</i> DT40 cells after stimulation with 500 µM MNNG, as measured by ratiometric analysis of Indo-1 stained cells by FACS. No transients were seen in <i>PARP-KO</i> or <i>PARP-dZF1</i> at 100 µM MNNG (data not shown). <i>C) Left panel</i>: NAD turnover in <i>PARP-WT and PARP-dZF2</i> DT40 cells. <i>PARP-dZF2</i> cells show NAD degradation over the course of 240 minutes following application of 100 µM MNNG. Stars indicate a p-value of p≤.001 from baseline for all subsequent points. <i>Right panel</i>: TRPM2-dependent Ca²⁺ transients in <i>PARP-WT and PARP-dZF2</i> DT40 cells after stimulation with 100 µM MNNG, as measured by ratiometric analysis of Indo-1 stained cells by FACS. No transients were seen in <i>PARP-KO</i> or <i>PARP-dZF2</i> at 100 µM MNNG (data not shown).</p

human polyADP-ribose polymerase-1 (PARP-1) variants.

<p>PARP-1 variants with mutations or deletions of each of hPARP-1′s functional domains were made based on previous successful expression of the mutant construct either in bacteria or eukaryotic cells. Zn – zinc finger domain. Automod – automodification domain. BRCT – BRCA1 C-terminal homology domain. Catalytic – PARP catalytic domain.</p