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Supplementary Figure 2 from Coactivation of AKT and β-Catenin in Mice Rapidly Induces Formation of Lipogenic Liver Tumors

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Supplementary Figure 1 from Coactivation of AKT and β-Catenin in Mice Rapidly Induces Formation of Lipogenic Liver Tumors

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Supplementary Figure Legends, Supplementary Tables 1-2 from Coactivation of AKT and β-Catenin in Mice Rapidly Induces Formation of Lipogenic Liver Tumors

Supplementary Figure 6 from Coactivation of AKT and β-Catenin in Mice Rapidly Induces Formation of Lipogenic Liver Tumors

The primary cilium is a cellular sensor that detects light, chemicals, and movement and is important for morphogen and growth factor signaling. The small GTPase Rab11–Rab8 cascade is required for ciliogenesis. Rab11 traffics the guanine nucleotide exchange factor (GEF) Rabin8 to the centrosome to activate Rab8, needed for ciliary growth. Rabin8 also requires the transport particle protein complex (TRAPPC) proteins for centrosome recruitment during ciliogenesis. Here, using an MS-based approach for identifying Rabin8-interacting proteins, we identified C7orf43 (also known as microtubule-associated protein 11 (MAP11)) as being required for ciliation both in human cells and zebrafish embryos. We find that C7orf43 directly binds to Rabin8 and that C7orf43 knockdown diminishes Rabin8 preciliary centrosome accumulation. Interestingly, we found that C7orf43 co-sediments with TRAPPII complex subunits and directly interacts with TRAPPC proteins. Our findings establish that C7orf43 is a TRAPPII-specific complex component, referred to here as TRAPPC14. Additionally, we show that TRAPPC14 is dispensable for TRAPPII complex integrity but mediates Rabin8 association with the TRAPPII complex. Finally, we demonstrate that TRAPPC14 interacts with the distal appendage proteins Fas-binding factor 1 (FBF1) and centrosomal protein 83 (CEP83), which we show here are required for GFP-Rabin8 centrosomal accumulation, supporting a role for the TRAPPII complex in tethering preciliary vesicles to the mother centriole during ciliogenesis. In summary, our findings have revealed an uncharacterized TRAPPII-specific component, C7orf43/TRAPPC14, that regulates preciliary trafficking of Rabin8 and ciliogenesis and support previous findings that the TRAPPII complex functions as a membrane tether.

// Jian Wang 1 , Peijun He 1 , Matthias Gaida 2 , Shouhui Yang 1 Aaron J. Schetter 3 , Jochen Gaedcke 4 , B. Michael Ghadimi 4 , Thomas Ried 5 , Harris Yfantis 6 , Dong Lee 6 , Jonathan M. Weiss 7 , Jimmy Stauffer 8 , Nader Hanna 9 , H. Richard Alexander 9 , S. Perwez Hussain 1 1 Pancreatic Cancer Unit, Laboratory of Human Carcinogenesis, CCR, NCI, Bethesda, MD, USA 2 Institute of Pathology, University Hospital of Heidelberg, Heidelberg, Germany 3 US Food and Drug Administration, Silver spring, MD, USA 4 Department of General, Visceral and Pediatric Surgery, University Medicine, Gottingen, Germany 5 Genetics Branch, CCR, NCI, Baltimore Veterans Affairs Medical Center, Baltimore, MD, USA 6 Pathology and Laboratory Medicine, Baltimore Veterans Affairs Medical Center, Baltimore, MD, USA 7 Cancer and Inflammation Program, CCR, NCI Frederick, MD, USA 8 Laboratory of Cell and Developmental Signaling, NCI Frederick, MD, USA 9 Division of Surgical Oncology, University of Maryland School of Medicine, Baltimore, MD, USA Correspondence to: S. Perwez Hussain, email: hussainp@mail.nih.gov Keywords: NOS2, NO, PDAC, KPC mouse model Received: February 12, 2016 Accepted: June 12, 2016 Published: June 29, 2016 ABSTRACT Pancreatic cancer is one of the most lethal malignancies and is refractory to the available treatments. Pancreatic ductal adenocarcinoma (PDAC) expresses high level of inducible nitric oxide synthase (NOS2), which causes sustained production of nitric oxide (NO). We tested the hypothesis that an aberrantly increased NO-release enhances the development and progression of PDAC. Enhanced NOS2 expression in tumors significantly associated with poor survival in PDAC patients ( N = 107) with validation in independent cohorts. We then genetically targeted NOS2 in an autochthonous mouse model of PDAC to examine the effect of NOS2-deficiency on disease progression and survival. Genetic ablation of NOS2 significantly prolonged survival and reduced tumor severity in LSL-Kras G12D/+ ; LSL-Trp53 R172H/+ ; Pdx-1-Cre (KPC) mice. Primary tumor cells isolated from NOS2-deficient KPC (NKPC) mice showed decreased proliferation and invasiveness as compared to those from KPC mice. Furthermore, NKPC tumors showed reduced expression of pERK, a diminished inactivation of Forkhead box transcription factor O (FOXO3), a tumor suppressor, and a decrease in the expression of oncomir-21, when compared with tumors in KPC mice. Taken together, these findings showed that NOS2 is a predictor of prognosis in early stage, resected PDAC patients, and provide proof-of-principle that targeting NOS2 may have potential therapeutic value in this lethal malignancy.

Background & Aims Liver inflammatory diseases associated with cancer promoting somatic oncogene mutations are increasing in frequency. Preclinical cancer models that allow for the study of early tumor progression are often protracted, which limits the experimental study parameters due to time and expense. Here we report a robust inexpensive approach using Sleeping Beauty transposition (SBT) delivery of oncogenes along with Gaussia Luciferase expression vector GLuc, to assess de novo liver tumor progression, as well as the detection of innate immune responses or responses induced by therapeutic intervention. Methods Tracking de novo liver tumor progression with GLuc was demonstrated in models of hepatocellular carcinoma (HCC) or adenoma (HCA) initiated by hydrodynamic delivery of SBT oncogenes. Results Rising serum luciferase levels correlated directly with increasing liver tumor burden and eventual morbidity. Early detection of hepatocyte apoptosis from mice with MET+CAT transfected hepatocytes was associated with a transient delay in HCC growth mediated by a CD8 + T-cell response against transformed hepatocytes. Furthermore, mice that lack B cells or macrophages had an increase in TUNEL + hepatocytes following liver MET transfection demonstrating that these cells provide protection from MET-induced hepatocyte apoptosis. Treatment with IL-18+IL-12 of mice displaying established HCC decreased tumor burden which was associated with decreased levels of serum luciferase. Conclusions Hydrodynamic delivery of the SBT vector GLuc to hepatocytes serves as a simple blood-based approach for real-time tracking of pathologically distinct types of liver cancer. This revealed tumor-induced immunologic responses and was beneficial in monitoring the efficacy of therapeutic interventions.

Objectives The relative contributions of inflammatory signalling and sequential oncogenic dysregulation driving liver cancer pathogenesis remain incompletely understood. Lymphotoxin-β receptor (LTβR) signalling is critically involved in hepatitis and liver tumorigenesis. Therefore, we explored the interdependence of inflammatory lymphotoxin signalling and specific oncogenic pathways in the progression of hepatic cancer. Design Pathologically distinct liver tumours were initiated by hydrodynamic transfection of oncogenic V-Akt Murine Thymoma Viral Oncogene Homolog 1 (AKT)/β-catenin or AKT/Notch expressing plasmids. To investigate the relationship of LTβR signalling and specific oncogenic pathways, LTβR antagonist (LTβR-Fc) or agonist (anti-LTβR) were administered post oncogene transfection. Initiated livers/tumours were investigated for changes in oncogene expression, tumour proliferation, progression, latency and pathology. Moreover, specific LTβR-mediated molecular events were investigated in human liver cancer cell lines and through transcriptional analyses of samples from patients with intrahepatic cholangiocarcinoma (ICC). Results AKT/β-catenin-transfected livers displayed increased expression of LTβ and LTβR, with antagonism of LTβR signalling reducing tumour progression and enhancing survival. Conversely, enforced LTβR-activation of AKT/β-catenin-initiated tumours induced robust increases in proliferation and progression of hepatic tumour phenotypes in an AKT-dependent manner. LTβR-activation also rapidly accelerated ICC progression initiated by AKT/Notch, but not Notch alone. Moreover, LTβR-accelerated development coincides with increases of Notch, Hes1, c-MYC, pAKT and β-catenin. We further demonstrate LTβR signalling in human liver cancer cell lines to be a regulator of Notch, pAKTser473 and β-catenin. Transcriptome analysis of samples from patients with ICC links increased LTβR network expression with poor patient survival, increased Notch1 expression and Notch and AKT/PI3K signalling. Conclusions Our findings link LTβR and oncogenic AKT signalling in the development of ICC.

Serum starvation stimulates cilia growth in cultured cells, yet serum factors associated with ciliogenesis are unknown. Previously, we showed that starvation induces rapid Rab11-dependent vesicular trafficking of Rabin8, a Rab8 guanine-nucleotide exchange factor (GEF), to the mother centriole, leading to Rab8 activation and cilium growth. Here, we demonstrate that through the LPA receptor 1 (LPAR1), serum lysophosphatidic acid (LPA) inhibits Rab11a-Rabin8 interaction and ciliogenesis. LPA/LPAR1 regulates ciliogenesis initiation via downstream PI3K/Akt activation, independent of effects on cell cycle. Akt stabilizes Rab11a binding to its effector, WDR44, and a WDR44-pAkt-phosphomimetic mutant blocks ciliogenesis. WDR44 depletion promotes Rabin8 preciliary trafficking and ciliogenesis-initiating events at the mother centriole. Our work suggests disruption of Akt signaling causes a switch from Rab11-WDR44 to the ciliogenic Rab11-FIP3-Rabin8 complex. Finally, we demonstrate that Akt regulates downstream ciliogenesis processes associated with Rab8-dependent cilia growth. Together, this study uncovers a mechanism whereby serum mitogen signaling regulates Rabin8 preciliary trafficking and ciliogenesis initiation.

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and the third most common cause of cancer related deaths worldwide. We set out to perform immune studies in HCC. In a recent published mouse model of HCC, the hydrodynamic injection of two plasmids, pT3-EF5-hMET (pMET) and pT3CAT led to prompt tumor growth. We modified the pT3CAT plasmid with conventional PCR cloning methods to create pCAT_LucOS, in which luciferase including T cell epitopes is expressed in addition to the onogene. Whereas co-delivery of pMET and pT3CAT in C57BL/6 mice led to rapid tumor development, that required euthanasia within 9 weeks, co-delivery of pMET and pCAT_LucOS did not lead to any signs of tumor burden over a course of 7 months. In contrast, survival studies with immunocompromised Rag1KO mice showed similar survival between mice that were injected with the same plasmid combinations. CAT gene expression was elevated over normal liver tissue level in pMET and pT3CAT injected C57BL/6 mice over the course of the 9 week experiment. In pMET and pCAT_LucOS injected mice the CAT expression level was elevated only in the first 2 weeks after the injection. The tumor bearing pMET and pT3CAT injected mice showed increased frequencies of CD11b+Gr-1+ MDSC and CD11b+F4/80+ Macrophages and decreased frequencies of CD4+ and CD8+ T cells in the liver. In pMET and pCAT_LucOS injected mice an increase in CD8+ T cells in the first two weeks was seen together with strong CD8 response against one of the tumor specific T cell epitopes.