Inhibition of Aurora Kinase A Induces Necroptosis in Pancreatic Carcinoma.

Induction of nonapoptotic cell death could be an approach to eliminate apoptosis-resistant tumors. We investigated necroptosis-based therapies in mouse models of pancreatic ductal adenocarcinoma cancer (PDAC). We screened 273 commercially available kinase inhibitors for cytotoxicity against a human PDAC cell line (PANC1). We evaluated the ability of the aurora kinase inhibitor CCT137690 to stimulate necroptosis in PDAC cell lines (PANC1, PANC2.03, CFPAC1, MiaPaCa2, BxPc3, and PANC02) and the HEK293 cell line, measuring loss of plasma membrane integrity, gain in cell volume, swollen organelles, and cytoplasmic vacuoles. We tested the effects of CCT137690 in colon formation assays, and the effects of the necroptosis (necrostatin-1 and necrosulfonamide), apoptosis, autophagy, and ferroptosis inhibitors. We derived cells from tumors that developed in Pdx1-Cre;K-RasG12D/+;p53R172H/+ (KPC) mice. Genes encoding proteins in cell death pathways were knocked out, knocked down, or expressed from transgenes in PDAC cell lines. Athymic nude or B6 mice were given subcutaneous injections of PDAC cells or tail-vein injections of KPC tumor cells. Mice were given CCT137690 (80 mg/kg) or vehicle and tumor growth was monitored; tumor tissues were collected and analyzed by immunohistochemistry. We compared gene expression levels between human pancreatic cancer tissues (n = 130) with patient survival times using the online R2 genomics analysis and visualization platform. CCT137690 induced necrosis-like death in PDAC cell lines and reduced colony formation; these effects required RIPK1, RIPK3, and MLKL, as well as inhibition of aurora kinase A (AURKA). AURKA interacted directly with RIPK1 and RIPK3 to reduce necrosome activation. AURKA-mediated phosphorylation of glycogen synthase kinase 3 beta (GSK3β) at serine 9 inhibited activation of the RIPK3 and MLKL necrosome. Mutations in AURKA (D274A) or GSK3β (S9A), or pharmacologic inhibitors of RIPK1 signaling via RIPK3 and MLKL, reduced the cytotoxic activity of CCT137690 in PDAC cells. Oral administration of CCT137690 induced necroptosis and immunogenic cell death in subcutaneous and orthotopic tumors in mice, and reduced tumor growth and tumor cell phosphorylation of AURKA and GSK3β. CCT137690 increased survival times of mice with orthotopic KPC PDACs and reduced tumor growth, stroma, and metastasis. Increased expression of AURKA and GSK3β mRNAs associated with shorter survival times of patients with pancreatic cancer. We identified the aurora kinase inhibitor CCT137690 as an agent that induces necrosis-like death in PDAC cells, via RIPK1, RIPK3, and MLKL. CCT137690 slowed growth of orthotopic tumors from PDAC cells in mice, and expression of AURKA and GSK3β associate with patient survival times. AURKA might be targeted for treatment of pancreatic cancer.

Xie Y ，Zhu S ，Zhong M ，Yang M ，Sun X ，Liu J ，Kroemer G ，Lotze M ，Zeh HJ 3rd ，Kang R ，Tang D ... -  《-》

JTC801 Induces pH-dependent Death Specifically in Cancer Cells and Slows Growth of Tumors in Mice.

Maintenance of acid-base homeostasis is required for normal physiology, metabolism, and development. It is not clear how cell death is activated in response to changes in pH. We performed a screen to identify agents that induce cell death in a pH-dependent manner (we call this alkaliptosis) in pancreatic ductal adenocarcinoma cancer (PDAC) cells and tested their effects in mice. We screened a library of 254 compounds that interact with G-protein-coupled receptors (GPCRs) to identify those with cytotoxic activity against a human PDAC cell line (PANC1). We evaluated the ability of JTC801, which binds the opiod receptor and has analgesic effects, to stimulate cell death in human PDAC cell lines (PANC1, MiaPaCa2, CFPAC1, PANC2.03, BxPc3, and CAPAN2), mouse pancreatic cancer-associated stellate cell lines, primary human pancreatic ductal epithelial cells, and 60 cancer cell lines (the NCI-60 panel). Genes encoding proteins in cell death and GPCR signaling pathways, as well as those that regulate nuclear factor-κB (NF-κB) activity, were knocked out, knocked down, or expressed from transgenes in cancer cell lines. JTC801 was administered by gavage to mice with xenograft tumors, C57BL/6 mice with orthographic pancreatic tumors grown from Pdx1-Cre;KRasG12D/+;Tp53R172H/+ (KPC) cells, mice with metastases following tail-vein injection of KPC cells, and Pdx-1-Cre;KrasG12D/+ mice crossed with Hmgb1flox/flox mice (KCH mice). Pancreata were collected from mice and analyzed for tumor growth and by histology and immunohistochemistry. We compared gene and protein expression levels between human pancreatic cancer tissues and patient survival times using online R2 genomic or immunohistochemistry analyses. Exposure of human PDAC cell lines (PANC1 and MiaPaCa2) to JTC801 did not induce molecular markers of apoptosis (cleavage of caspase 3 or poly [ADP ribose] polymerase [PARP]), necroptosis (interaction between receptor-interacting serine-threonine kinase 3 [RIPK3] and mixed lineage kinase domain like pseudokinase [MLKL]), or ferroptosis (degradation of glutathione peroxidase 4 [GPX4]). Inhibitors of apoptosis (Z-VAD-FMK), necroptosis (necrosulfonamide), ferroptosis (ferrostatin-1), or autophagy (hydroxychloroquine) did not prevent JTC801-induced death of PANC1 or MiaPaCa2 cells. The cytotoxic effects of JTC801 in immortalized fibroblast cell lines was not affected by disruption of genes that promote apoptosis (Bax-/-/Bak-/- cells), necroptosis (Ripk1-/-, Ripk3-/-, or Mlkl-/- cells), ferroptosis (Gpx4-/- cells), or autophagy (Atg3-/-, Atg5-/-, Atg7-/-, or Sqstm1-/- cells). We found JTC801 to induce a pH-dependent form cell death (alkaliptosis) in cancer cells but not normal cells (hepatocytes, bone marrow CD34+ progenitor cells, peripheral blood mononuclear cells, or dermal fibroblasts) or healthy tissues of C57BL/6 mice. JTC801 induced alkaliptosis in cancer cells by activating NF-κB, which repressed expression of the carbonic anhydrase 9 gene (CA9), whose product regulates pH balance in cells. In analyses of Cancer Genome Atlas data and tissue microarrays, we associated increased tumor level of CA9 mRNA or protein with shorter survival times of patients with pancreatic, kidney, or lung cancers. Knockdown of CA9 reduced the protective effects of NF-κB inhibition on JTC801-induced cell death and intracellular alkalinization in PANC1 and MiaPaCa2 cell lines. Oral administration of JTC801 inhibited growth of xenograft tumors (from PANC1, MiaPaCa2, SK-MEL-28, PC-3, 786-0, SF-295, HCT116, OV-CAR3, and HuH7 cells), orthotropic tumors (from KPC cells), lung metastases (from KPC cells) of mice, and slowed growth of tumors in KCH mice. In a screen of agents that interact with GPCR pathways, we found JTC801 to induce pH-dependent cell death (alkaliptosis) specifically in cancer cells such as PDAC cells, by reducing expression of CA9. Levels of CA9 are increased in human cancer tissues. JTC801 might be developed for treatment of pancreatic cancer.

Song X ，Zhu S ，Xie Y ，Liu J ，Sun L ，Zeng D ，Wang P ，Ma X ，Kroemer G ，Bartlett DL ，Billiar TR ，Lotze MT ，Zeh HJ ，Kang R ，Tang D ... -  《-》

Regulation of pH by Carbonic Anhydrase 9 Mediates Survival of Pancreatic Cancer Cells With Activated KRAS in Response to Hypoxia.

Most pancreatic ductal adenocarcinomas (PDACs) express an activated form of KRAS, become hypoxic and dysplastic, and are refractory to chemo and radiation therapies. To survive in the hypoxic environment, PDAC cells upregulate enzymes and transporters involved in pH regulation, including the extracellular facing carbonic anhydrase 9 (CA9). We evaluated the effect of blocking CA9, in combination with administration of gemcitabine, in mouse models of pancreatic cancer. We knocked down expression of KRAS in human (PK-8 and PK-1) PDAC cells with small hairpin RNAs. Human and mouse (KrasG12D/Pdx1-Cre/Tp53/RosaYFP) PDAC cells were incubated with inhibitors of MEK (trametinib) or extracellular signal-regulated kinase (ERK), and some cells were cultured under hypoxic conditions. We measured levels and stability of the hypoxia-inducible factor 1 subunit alpha (HIF1A), endothelial PAS domain 1 protein (EPAS1, also called HIF2A), CA9, solute carrier family 16 member 4 (SLC16A4, also called MCT4), and SLC2A1 (also called GLUT1) by immunoblot analyses. We analyzed intracellular pH (pHi) and extracellular metabolic flux. We knocked down expression of CA9 in PDAC cells, or inhibited CA9 with SLC-0111, incubated them with gemcitabine, and assessed pHi, metabolic flux, and cytotoxicity under normoxic and hypoxic conditions. Cells were also injected into either immune-compromised or immune-competent mice and growth of xenograft tumors was assessed. Tumor fragments derived from patients with PDAC were surgically ligated to the pancreas of mice and the growth of tumors was assessed. We performed tissue microarray analyses of 205 human PDAC samples to measure levels of CA9 and associated expression of genes that regulate hypoxia with outcomes of patients using the Cancer Genome Atlas database. Under hypoxic conditions, PDAC cells had increased levels of HIF1A and HIF2A, upregulated expression of CA9, and activated glycolysis. Knockdown of KRAS in PDAC cells, or incubation with trametinib, reduced the posttranscriptional stabilization of HIF1A and HIF2A, upregulation of CA9, pHi, and glycolysis in response to hypoxia. CA9 was expressed by 66% of PDAC samples analyzed; high expression of genes associated with metabolic adaptation to hypoxia, including CA9, correlated with significantly reduced survival times of patients. Knockdown or pharmacologic inhibition of CA9 in PDAC cells significantly reduced pHi in cells under hypoxic conditions, decreased gemcitabine-induced glycolysis, and increased their sensitivity to gemcitabine. PDAC cells with knockdown of CA9 formed smaller xenograft tumors in mice, and injection of gemcitabine inhibited tumor growth and significantly increased survival times of mice. In mice with xenograft tumors grown from human PDAC cells, oral administration of SLC-0111 and injection of gemcitabine increased intratumor acidosis and increased cell death. These tumors, and tumors grown from PDAC patient-derived tumor fragments, grew more slowly than xenograft tumors in mice given control agents, resulting in longer survival times. In KrasG12D/Pdx1-Cre/Tp53/RosaYFP genetically modified mice, oral administration of SLC-0111 and injection of gemcitabine reduced numbers of B cells in tumors. In response to hypoxia, PDAC cells that express activated KRAS increase expression of CA9, via stabilization of HIF1A and HIF2A, to regulate pH and glycolysis. Disruption of this pathway slows growth of PDAC xenograft tumors in mice and might be developed for treatment of pancreatic cancer.

McDonald PC ，Chafe SC ，Brown WS ，Saberi S ，Swayampakula M ，Venkateswaran G ，Nemirovsky O ，Gillespie JA ，Karasinska JM ，Kalloger SE ，Supuran CT ，Schaeffer DF ，Bashashati A ，Shah SP ，Topham JT ，Yapp DT ，Li J ，Renouf DJ ，Stanger BZ ，Dedhar S ... -  《-》

The Evaluation of Effect of Aurora Kinase Inhibitor CCT137690 in Melanoma and Melanoma Cancer Stem Cell.

Dysregulation of the cell cycle is one of the main causes of melanomagenesis. Genomewide studies showed that the expression of Aurora -A and -B significantly has been upregulated in melanoma. However, there is no FDA approved drug targeting aurora kinases in the treatment of melanoma. In addition, the development of resistance to chemotherapeutic agents in the treatment of melanoma and, as a result, the relapse due to heterogeneous cell groups in patients is a second phenomenon that causes treatment failure. Therefore, there is an urgent need for therapeutic alternatives targeting both melanoma and Melanoma Cancer Stem Cells (MCSCs) in treatments. At this stage, cell cycle regulators become promising targets. In this study, we aimed to identify the effects of Aurora kinase inhibitor CCT137690 on the cytotoxicity, apoptosis, cell cycle, migration, and colony formation and expression changes of genes related to proliferation, cell death and cell cycle in melanoma and melanoma cancer stem cell. In addition, we investigated the apoptotic and cytostatic effects of CCT137690 in normal fibroblast cells. We evaluated the cytotoxic effect of CCT137690 in MCSCs, NM2C5 referring as melanoma model cells and WI-38 cells by using the WST-1 test. The effect of CCT137690 on apoptosis was detected via Annexin V and JC-1 method; on cell cycle progression by cell cycle test; on gene expression by using RT-PCR, on migration activity by wound healing assay and clonal growth by clonogenic assay in NM2C5 cells and MCSCs. The effects of CCT137690 in WI-38, referring as healthy fibroblast cell, were assessed through Annexin V and cell cycle method. CCT137690 was determined to have a cytotoxic and apoptotic effect in MCSCs and melanoma. It caused polyploidy and cell cycle arrest at the G2/M phase in MCSCs and melanoma cells. The significant decrease in the expression of MMP2, MMP7, MMP10, CCNB1, IRAK1, PLK2 genes, and the increase in the expression of PTEN, CASP7, p53 genes were detected. Aurora kinases inhibitor CCT137690 displays promising anticancer activity in melanoma and especially melanoma cancer stem cells. The effect of CCT137690 on melanoma and MCSC may provide a new approach to treatment protocols.

Sogutlu F ，Kayabasi C ，Yelken BO ，Asik A ，Gasimli R ，Kipcak S ，Susluer SY ，Avci CB ，Gunduz C ... -  《-》

Aurora A kinase and its activator TPX2 are potential therapeutic targets in KRAS-induced pancreatic cancer.

Gomes-Filho SM ，Dos Santos EO ，Bertoldi ERM ，Scalabrini LC ，Heidrich V ，Dazzani B ，Levantini E ，Reis EM ，Bassères DS ... -  《-》