Tone Sandal

Axl is an essential epithelial-to-mesenchymal transition-induced regulator of breast cancer metastasis and patient survival

Metastasis underlies the majority of cancer-related deaths. Thus, furthering our understanding of the molecular mechanisms that enable tumor cell dissemination is a vital health issue. Epithelial-to-mesenchymal transitions (EMTs) endow carcinoma cells with enhanced migratory and survival attributes that facilitate malignant progression. Characterization of EMT effectors is likely to yield new insights into metastasis and novel avenues for treatment. We show that the presence of the receptor tyrosine kinase Axl in primary breast cancers independently predicts strongly reduced overall patient survival, and that matched patient metastatic lesions show enhanced Axl expression. We demonstrate that Axl is strongly induced by EMT in immortalized mammary epithelial cells that establishes an autocrine signaling loop with its ligand, Gas6. Epiallelic RNA interference analysis in metastatic breast cancer cells delineated a distinct threshold of Axl expression for mesenchymal-like in vitro cell invasiveness and formation of tumors in foreign and tissue-engineered microenvironments in vivo. Importantly, in two different optical imaging-based experimental breast cancer models, Axl knockdown completely prevented the spread of highly metastatic breast carcinoma cells from the mammary gland to lymph nodes and several major organs and increased overall survival. These findings suggest that Axl represents a downstream effector of the tumor cell EMT that is required for breast cancer metastasis. Thus, the detection and targeted treatment of Axl-expressing tumors represents an important new therapeutic strategy for breast cancer.

Activating glucokinase (GCK) Mutations as a cause of medically responsive congenital hyperinsulinism: prevalence in children and characterisation of a novel GCK mutation

OBJECTIVE Activating glucokinase (GCK) mutations are a rarely reported cause of congenital hyperinsulinism (CHI), but the prevalence of GCK mutations is not known. METHODS From a pooled cohort of 201 non-syndromic children with CHI from three European referral centres (Denmark, n=141; Norway, n=26; UK, n=34), 108 children had no K(ATP)-channel (ABCC8/KCNJ11) gene abnormalities and were screened for GCK mutations. Novel GCK mutations were kinetically characterised. RESULTS In five patients, four heterozygous GCK mutations (S64Y, T65I, W99R and A456V) were identified, out of which S64Y was novel. Two of the mutations arose de novo, three were dominantly inherited. All the five patients were medically responsive. In the combined Danish and Norwegian cohort, the prevalence of GCK-CHI was estimated to be 1.2% (2/167, 95% confidence interval (CI) 0-2.8%) of all the CHI patients. In the three centre combined cohort of 72 medically responsive children without K(ATP)-channel mutations, the prevalence estimate was 6.9% (5/72, 95% CI 1.1-12.8%). All activating GCK mutations mapped to the allosteric activator site. The novel S64Y mutation resulted in an increased affinity for the substrate glucose (S(0.5) 1.49+/-0.08 and 7.39+/-0.05 mmol/l in mutant and wild-type proteins respectively), extrapolating to a relative activity index of approximately 22 compared with the wild type. CONCLUSION In the largest study performed to date on GCK in children with CHI, GCK mutations were found only in medically responsive children who were negative for ABCC8 and KCNJ11 mutations. The estimated prevalence (approximately 7%) suggests that screening for activating GCK mutations is warranted in those patients.

A Novel, Extraneuronal Role for Cyclin-dependent Protein Kinase 5 (CDK5) MODULATION OF cAMP-INDUCED APOPTOSIS IN RAT LEUKEMIA CELLS

A number of cyclin-dependent protein kinase (CDK) inhibitors were tested for the ability to protect IPC-81 rat leukemic cells against cAMP-induced apoptosis. A near perfect proportionality was observed between inhibitor potency to protect against cAMP-induced apoptosis and to antagonize CDK5, and to a lesser extent, CDK2 and CDK1. Enforced expression of dominant negative CDK5 (but not CDK1-dn or CDK2-dn) protected against death, indicating that CDK5 activity was necessary for cAMP-induced apoptosis. The CDK inhibitors failed to protect the cells against daunorubicine-, staurosporine-, or okadaic acid-induced apoptosis. The inhibition of CDK5 prevented the cleavage of pro-caspase-3 in cAMP-treated cells. The cells could be saved closer to the moment of their onset of death by inhibitors of caspases than by inhibitors of CDK5. This suggested that the action of CDK5 was upstream of caspase activation. The cAMP treatment resulted in a moderate increase of the level of CDK5 mRNA and protein in IPC-81 wild-type cells. Such cAMP induction of CDK5 was not observed in cells expressing the inducible cAMP early repressor. The cAMP-induced increase of CDK5 contributed to apoptosis since cells overexpressing CDK5-wt were more sensitive for cAMP-induced death. These results demonstrate the first example of a proapoptotic CDK action upstream of caspase activation and of an extra-neuronal effect of CDK5.

The spectrum of ABCC8 mutations in Norwegian patients with congenital hyperinsulinism of infancy.

Potassium channels in the plasma membrane of the pancreatic beta cells are critical in maintaining glucose homeostasis by responding to ATP and coupling metabolic changes to insulin secretion. These channels consist of subunits denoted the sulfonylurea receptor SUR1 and the inwardly rectifying ion channel KIR6.2, which are encoded by the genes ABCC8 and KCNJ11, respectively. Activating mutations in the subunit genes can result in monogenic diabetes, whereas inactivating mutations are the most common cause of congenital hyperinsulinism of infancy (CHI). Twenty‐six Norwegian probands with CHI were analyzed for alterations in ABCC8 and KCNJ11. Fifteen probands (58%) had mutations in the ABCC8 gene. Nine patients were homozygous or compound heterozygous for the mutations, indicating diffuse pancreatic disease. In five patients, heterozygous and paternally inherited mutations were found, suggesting focal disease. One patient had a de novo mutation likely to cause a milder, dominant form of CHI. Altogether, 16 different ABCC8 mutations (including the novel alterations W231R, C267X, IVS6‐3C>G, I462V, Q917X and T1531A) were identified. The mutations IVS10+1G>T, R1493W and V21D occurred in five, three and two families, respectively. KCNJ11 mutations were not found in any patients. Based on our mutation screening, we estimate the minimum birth prevalence of ABCC8‐CHI in Norway to 1:70,000 during the past decade. Our results considerably extend the knowledge of the molecular genetics behind CHI in Scandinavia.

Establishment of okadaic acid resistant cell clones using a cDNA expression library

The mechanism whereby the universal apoptogen and serine/threonine phosphatase inhibitor okadaic acid (OA) kills cells, is still unclear. To create a novel tool for probing of OA action, fibroblasts were selected for OA-resistance after infection with a retroviral Jurkat T-cell cDNA expression library. Twenty-one clones were selected. Two of these (OAR1, OAR2) were studied in detail. OAR1 and 2 had each a retrovirally introduced short cDNA, corresponding to a human gene (oar1 and oar2, respectively) with unknown function. Reintroduction of oar1 or oar2 cDNA into wild-type cells reproduced the OA-resistant phenotype. OAR1 and 2 were cross-resistant to other phosphatase inhibitors (calyculin A, cantharidin), but not to staurosporine or microinjected Cytochrome c, thus, indicating a disturbance in a limited number of death pathways, upstream or independent of apaf-1/caspases-3/9. The action of OA involved caspase-dependent and caspase-independent components. Both components were less efficient in OAR1 and 2, than in wild-type cells. Subtle differences existed between OA-induced phosphoprotein patterns in wild-type cells, OAR1, and OAR2, indicating that a narrow selection of protein phosphorylation events had been targeted. We propose that the clones have defects in a hitherto non-elucidated signal pathway linking OA-induced protein phosphorylation to initiation of a death execution pathway provided with a caspase-dependent amplification loop. The novel OA-resistant cell clones will be used to elucidate the significance for apoptosis of oar1 and 2, their link to altered protein phosphorylation, and the potential link of the latter to initiation of apoptosis. Cell Death and Differentiation (2001) 8, 754–766

Disease progression and search for monogenic diabetes among children with new onset type 1 diabetes negative for ICA, GAD- and IA-2 Antibodies.

BackgroundTo investigate disease progression the first 12 months after diagnosis in children with type 1 diabetes negative (AAB negative) for pancreatic autoantibodies [islet cell autoantibodies(ICA), glutamic acid decarboxylase antibodies (GADA) and insulinoma-associated antigen-2 antibodies (IA-2A)]. Furthermore the study aimed at determining whether mutations in KCNJ11, ABCC8, HNF1A, HNF4A or INS are common in AAB negative diabetes.Materials and methodsIn 261 newly diagnosed children with type 1 diabetes, we measured residual β-cell function, ICA, GADA, and IA-2A at 1, 6 and 12 months after diagnosis. The genes KCNJ11, ABCC8, HNF1A, HNF4A and INS were sequenced in subjects AAB negative at diagnosis. We expressed recombinant K-ATP channels in Xenopus oocytes to analyse the functional effects of an ABCC8 mutation.ResultsTwenty-four patients (9.1%) tested AAB negative after one month. Patients, who were AAB-negative throughout the 12-month period, had higher residual β-cell function (P = 0.002), lower blood glucose (P = 0.004), received less insulin (P = 0.05) and had lower HbA1c (P = 0.02) 12 months after diagnosis. One patient had a heterozygous mutation leading to the substitution of arginine at residue 1530 of SUR1 (ABCC8) by cysteine. Functional analyses of recombinant K-ATP channels showed that R1530C markedly reduced the sensitivity of the K-ATP channel to inhibition by MgATP. Morover, the channel was highly sensitive to sulphonylureas. However, there was no effect of sulfonylurea treatment after four weeks on 1.0-1.2 mg/kg/24 h glibenclamide.ConclusionGAD, IA-2A, and ICA negative children with new onset type 1 diabetes have slower disease progression as assessed by residual beta-cell function and improved glycemic control 12 months after diagnosis. One out of 24 had a mutation in ABCC8, suggesting that screening of ABCC8 should be considered in patients with AAB negative type 1 diabetes.

Abstract 1747: BGB324, a selective small molecule Axl kinase inhibitor to overcome EMT-associated drug resistance in carcinomas: Therapeutic rationale and early clinical studies

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Axl is a member of the TAM (Tyro3, Axl and Mer) family of receptor tyrosine kinases that regulate multiple cellular responses including cell survival, proliferation, and migration. Axl expression is predictive of poor patient overall survival in a variety of human cancers including triple negative breast (TNBC), pancreatic ductal adenocarcinoma (PDA) and non-small cell lung cancer (NSCLC). Axl expression is induced by the epithelial-to-mesenchymal transition (EMT) gene program in cancer cells and Axl signaling is required to maintain EMT-associated features including invasiveness, metastasis, stem cell-like traits and resistance to targeted inhibitors and other chemotherapeutic agents. BGB324/R428 is an oral, selective small molecule inhibitor of Axl that recently was evaluated in early clinical safety studies in healthy volunteers. Treatment with BGB324 up to and including 1.5 gms daily (per os) was established as being safe and well tolerated. The endpoints of the study included pharmacokinetics and safety. Bioavailability was increased in the presence of food and systemic exposure increased dose proportionately. At the highest dose the apparent elimination half-life approached four days presenting a range of dosing options. We evaluated the effects of BGB324 in preclinical models of TNBC, PDA and NSCLC, including 2D/3D cell culture and mouse xenograft models, in combination with targeted and chemotherapeutic agents. BGB324 treatment of mesenchymal carcinoma cells blocked invasiveness and enhanced chemotherapeutic efficacy. BGB324 abrogated the tumor initiation capacity of TNBC cells, an activity associated with cancer stem cells. BGB324 treatment blocked the emergence of EMT-associated acquired resistance to erlotinib in human NSCLC xenografts. Furthermore, combination treatment of BGB324 with chemotherapy inhibited the growth of human NSCLC xenografts and significantly prolonged survival in orthotopic and genetically engineered mouse models of PDA. Collectively, these data suggest that the first-in-class selective Axl inhibitor BGB324 can overcome EMT-related acquired therapeutic resistance and enhance the efficacy of multiple anti-cancer strategies. Together with the results of results of the early clinical safety studies, this provides a rationale for further clinical studies. Citation Format: Katarzyna Wnuk-Lipinska, Crina Tiron, Gro Gausdal, Tone Sandal, Robin Frink, Stefan Hinz, Monica Hellesoy, Lavina Ahmed, Hallvard Haugen, Xiao Liang, Magnus Blo, David Micklem, Murray Yule, John Minna, Longen Zhou, Rolf Brekken, James Lorens. BGB324, a selective small molecule Axl kinase inhibitor to overcome EMT-associated drug resistance in carcinomas: Therapeutic rationale and early clinical studies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1747. doi:10.1158/1538-7445.AM2014-1747

The Molecular Genetics and Pathophysiology of Congenital Hyperinsulinism Caused by Short-Chain 3-Hydroxyacyl-CoA Dehydrogenase Deficiency

Deficiency of the metabolic enzyme short-chain 3-hydroxyacyl-CoA (SCHAD) is a rare autosomal recessive form of congenital hyperinsulinism of infancy caused by mutations in the HADH

Abstract B78: Warfarin blocks Gas6-mediated Axl activation required for pancreatic tumor plasticity and metastasis

Warfarin, a vitamin K antagonist anti-coagulant in clinical use for over 50 years, is reported to exert anti-cancer effects. We hypothesized that the molecular mechanism underlying the observed anti-tumor effects of warfarin is unrelated to generalized anti-coagulation, but rather due to inhibition of the Axl receptor tyrosine kinase. Activation of Axl by its ligand Gas6, a vitamin K-dependent protein, is inhibited at doses of warfarin that do not affect coagulation. Here we document that inhibiting Gas6-mediated Axl activation with low dose warfarin blocks pancreatic cancer progression and spread. Warfarin and other Axl-targeting agents inhibit tumor progression and block spontaneous metastasis in multiple murine models of pancreatic cancer. Warfarin inhibited Axl-dependent tumor cell migration, invasiveness and proliferation while increasing apoptosis and sensitivity to chemotherapy. We demonstrate that Axl signaling is necessary for pancreatic tumor cell epithelial plasticity which is potently reversed by warfarin or selective Axl inhibition in vitro and in vivo. We anticipate Axl is a critical driver of pancreatic cancer progression and its inhibition with low dose warfarin or Axl-selective targeting agents may significantly improve outcome in patients. Citation Format: Amanda Kirane, Kathleen W. Ludwig, Gry Haaland, Tone Sandal, Renata Ranaweera, Jason E. Toombs, Laura A. Sullivan, Miao Wang, Noah Sorrelle, Sean P. Dineen, Michael T. Dellinger, James B. Lorens, Rolf A. Brekken. Warfarin blocks Gas6-mediated Axl activation required for pancreatic tumor plasticity and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B78.

Abstract 4888: Axl receptor signaling in required for stem cell traits and metastasis in breast cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Epithelial-to-mesenchymal transition (EMT) endows carcinoma cells with migratory, survival and stem cell-like attributes that facilitate therapeutic resistance and metastasis. Expression of the Axl receptor tyrosine kinase in aggressive breast cancer correlates with EMT, poor survival and is prevalent in patient metastases. Induction of EMT in immortalized mammary epithelial cells by EMT-transcription factors, TGFbeta or hypoxia upregulates Axl and establishes an autocrine-signaling loop with its ligand, Gas6. Axl receptor signaling is required to maintain EMT-related invasive, drug resistance and cancer stem cell (CSC) traits of malignant breast cancer cells. Targeting Axl signaling with RNAi or pharmacological agents blocks the EMT/CSC gene program and inhibits malignant functions including invasiveness, drug resistance, mammosphere formation, in vivo tumor initiation, and spontaneous metastasis in orthotopic breast cancer models. These results suggest that Axl expression is requisite for the maintenance of EMT and stem cell-like traits during malignant progression. The EMT program is characteristic of normal adult mammary epithelial stem/progenitor cells. Congruently, we show that the Axl receptor expressed on mammary epithelial stem/progenitor cells, and Axl signaling is necessary for mammary epithelial multipotent progenitor activity. Thus Axl receptor signaling represents a novel regulatory pathway linking normal mammary stem/progenitor cells and breast cancer stem cells. Hence, clinical Axl inhibitors represent a novel therapeutic avenue to target EMT/CSC traits of aggressive breast cancer. Citation Format: Crina Tiron, Fanny Pelissier, Katarzyna Wnuk-Lipinska, Ingunn Stefansson, Reeta Virtakoivu, Masaru Miyano, Tone Sandal, David Micklem, James Garbe, Martha Stampfer, Johanna Ivaska, Lars Akslen, Mark LaBarge, James Lorens. Axl receptor signaling in required for stem cell traits and metastasis in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4888. doi:10.1158/1538-7445.AM2013-4888