Ubiquitin Ligase RNF146 Regulates Tankyrase and Axin to Promote Wnt Signaling

Canonical Wnt signaling is controlled intracellularly by the level of β-catenin protein, which is dependent on Axin scaffolding of a complex that phosphorylates β-catenin to target it for ubiquitylation and proteasomal degradation. This function of Axin is counteracted through relocalization of Axin protein to the Wnt receptor complex to allow for ligand-activated Wnt signaling. AXIN1 and AXIN2 protein levels are regulated by tankyrase-mediated poly(ADP-ribosyl)ation (PARsylation), which destabilizes Axin and promotes signaling. Mechanistically, how tankyrase limits Axin protein accumulation, and how tankyrase levels and activity are regulated for this function, are currently under investigation. By RNAi screening, we identified the RNF146 RING-type ubiquitin E3 ligase as a positive regulator of Wnt signaling that operates with tankyrase to maintain low steady-state levels of Axin proteins. RNF146 also destabilizes tankyrases TNKS1 and TNKS2 proteins and, in a reciprocal relationship, tankyrase activity reduces RNF146 protein levels. We show that RNF146, tankyrase, and Axin form a protein complex, and that RNF146 mediates ubiquitylation of all three proteins to target them for proteasomal degradation. RNF146 is a cytoplasmic protein that also prevents tankyrase protein aggregation at a centrosomal location. Tankyrase auto-PARsylation and PARsylation of Axin is known to lead to proteasome-mediated degradation of these proteins, and we demonstrate that, through ubiquitylation, RNF146 mediates this process to regulate Wnt signaling

Initiation and Characterization of Small Cell Lung Cancer Patient-Derived Xenografts from Ultrasound-Guided Transbronchial Needle Aspirates

<div><p>Small cell lung cancer (SCLC) is a devastating disease with limited treatment options. Due to its early metastatic nature and rapid growth, surgical resection is rare. Standard of care treatment regimens remain largely unchanged since the 1980’s, and five-year survival lingers near 5%. Patient-derived xenograft (PDX) models have been established for other tumor types, amplifying material for research and serving as models for preclinical experimentation; however, limited availability of primary tissue has curtailed development of these models for SCLC. The objective of this study was to establish PDX models from commonly collected fine needle aspirate biopsies of primary SCLC tumors, and to assess their utility as research models of primary SCLC tumors. These transbronchial needle aspirates efficiently engrafted as xenografts, and tumor histomorphology was similar to primary tumors. Resulting tumors were further characterized by H&E and immunohistochemistry, cryopreserved, and used to propagate tumor-bearing mice for the evaluation of standard of care chemotherapy regimens, to assess their utility as models for tumors in SCLC patients. When treated with Cisplatin and Etoposide, tumor-bearing mice responded similarly to patients from whom the tumors originated. Here, we demonstrate that PDX tumor models can be efficiently established from primary SCLC transbronchial needle aspirates, even after overnight shipping, and that resulting xenograft tumors are similar to matched primary tumors in cancer patients by both histology and chemo-sensitivity. This method enables physicians at non-research institutions to collaboratively contribute to the rapid establishment of extensive PDX collections of SCLC, enabling experimentation with clinically relevant tissues and development of improved therapies for SCLC patients.</p></div

PDX response to P/E <i>in vivo</i> generally reflects clinical response.

<p>Upon reaching a mean tumor volume of 150–200 mm³, mice bearing A) LU073p2 or B) LU086p3 PDX tumors were randomized, administered either vehicle (closed triangles) or P/E (open circles; 5 mg/kg Cisplatin on day 1 and 8 mg/kg Etoposide on days 1, 2 & 3 of treatment), and tumors were measured weekly. The bracket indicates the time to progression (TTP). C) The mean TTP of PDX tumors following one course of P/E treatment was plotted versus observed clinical TTP. Data is represented as Mean ± SEM and reflects cohorts of n = 5 mice per group.</p

Expression of SCLC antigens is maintained in PDX tumor models.

<p>FFPE sections were prepared from PDX tumors (LU086p1 is represented). (A) Tissue sections were stained by IHC for diagnostic SCLC markers Chromagranin A (CHGA), Synaptophysin (SYP), or CD56. Scale bars represent 10um. (B) Tissue sections were stained by IHC for diagnostic non-SCLC markers Keratin 5 (KRT5), Keratin 6 (KRT6A), Keratin 7 (KRT7), Keratin 14 (KRT14), Keratin 20 (KRT20), Napsin A (NAPSA), TP63, or TTF1. Scale bars represent 10um. (C) PDX tumor cells were dissociated into single-cell suspensions and analyzed by flow cytometry for expression of EpCAM (CD326) and NCAM1 (CD56). Histograms displaying expression levels are shown (dark black line), whereas background signal was determined using a matched isotype control antibody (filled gray).</p

Immunohistochemical Staining of Diagnostic Lung Cancer Markers.

<p>Immunohistochemical Staining of Diagnostic Lung Cancer Markers.</p

PDX Growth & P/E Responsiveness.

<p>PDX Growth & P/E Responsiveness.</p