Supplementary Figure 6 from Sunitinib Acts Primarily on Tumor Endothelium rather than Tumor Cells to Inhibit the Growth of Renal Cell Carcinoma

<p>XLSX file - 613K, Genes significantly downregulated in samples with either CUL3 or NRF2 mutations</p>

Supplementary Figure 4 from Sunitinib Acts Primarily on Tumor Endothelium rather than Tumor Cells to Inhibit the Growth of Renal Cell Carcinoma

Supplementary Figure 7 from Sunitinib Acts Primarily on Tumor Endothelium rather than Tumor Cells to Inhibit the Growth of Renal Cell Carcinoma

Supplementary Figure Legends 1-7 from Sunitinib Acts Primarily on Tumor Endothelium rather than Tumor Cells to Inhibit the Growth of Renal Cell Carcinoma

Supplementary Figure 5 from Sunitinib Acts Primarily on Tumor Endothelium rather than Tumor Cells to Inhibit the Growth of Renal Cell Carcinoma

<p>PDF file - 197K, The pGL4luc2P-NRF2-ARE-Hygro construct (Promega)</p>

<p>PDF file - 727K, Dideoxynucleotide sequencing confirmed the presence of NRF2, CUL3, and SIRT1 mutations in case samples</p>

Supplementary Figure 3 from Sunitinib Acts Primarily on Tumor Endothelium rather than Tumor Cells to Inhibit the Growth of Renal Cell Carcinoma

PDF file - 772K, Knowledge-guided strategy of driver mutation discovery for NRF2 activation in sporadic PRCC2

Supplementary Figure 2 from Sunitinib Acts Primarily on Tumor Endothelium rather than Tumor Cells to Inhibit the Growth of Renal Cell Carcinoma

XLSX file - 186K, Somatic mutation in PRCC2

Supplementary Figure 1 from Sunitinib Acts Primarily on Tumor Endothelium rather than Tumor Cells to Inhibit the Growth of Renal Cell Carcinoma

<p>PDF file - 614K, Cycloheximide chase assay results indicate that the NRF2-E82G and the NRF2-V36del are more stable than the wild-type NRF2 (NRF2-WT)</p>

Fibronectin (FN) is a critical regulator of extracellular matrix (ECM) remodeling through its availability and stepwise polymerization for fibrillogenesis. Availability of FN is regulated by its synthesis and turnover, and fibrillogenesis is a multistep, integrin-dependent process essential for cell migration, proliferation, and tissue function. Transforming growth factor β (TGF-β) is an established regulator of ECM remodeling via transcriptional control of ECM proteins. Here we show that TGF-β, through increased FN trafficking in a transcription- and SMAD-independent manner, is a direct and rapid inducer of the fibrillogenesis required for TGF-β-induced cell migration. Whereas TGF-β signaling is dispensable for rapid fibrillogenesis, stable interactions between the cytoplasmic domain of the type II TGF-β receptor (TβRII) and the FN receptor (α5β1 integrin) are required. We find that, in response to TGF-β, cell surface-internalized FN is not degraded by the lysosome but instead undergoes recycling and incorporation into fibrils, a process dependent on TβRII. These findings are the first to show direct use of trafficked and recycled FN for fibrillogenesis, with a striking role for TGF-β in this process. Given the significant physiological consequences associated with FN availability and polymerization, our findings provide new insights into the regulation of fibrillogenesis for cellular homeostasis.

(99m)Tc-Methylene diphosphonate ((99m)Tc-MDP) is widely used in clinical settings to detect bone abnormalities. However, the mechanism of (99m)Tc-MDP uptake in bone is not well elucidated. In this study, we utilized a mouse tibia injury model, single-photon emission computed tomography (gamma scintigraphy or SPECT), ex vivo micro-computed tomography, and histology to monitor (99m)Tc-MDP uptake in injury sites during skeletal healing. In an ex vivo culture system, calvarial cells were differentiated into osteoblasts with osteogenic medium, pulsed with (99m)Tc-MDP at different time points, and quantitated for (99m)Tc-MDP uptake with a gamma counter. We demonstrated that (99m)Tc-MDP uptake in the injury sites corresponded to osteoblast generation in those sites throughout the healing process. The (99m)Tc-MDP uptake within the injury sites peaked on day 7 post-injury, while the injury sites were occupied by mature osteoblasts also starting from day 7. (99m)Tc-MDP uptake started to decrease 14 days post-surgery, when we observed the highest level of bony tissue in the injury sites. We also found that (99m)Tc-MDP uptake was associated with osteoblast maturation and mineralization in vitro. This study provides direct and biological evidence for (99m)Tc-MDP uptake in osteoblasts during bone healing in vivo and in vitro.

Background : Canonical and noncanonical Wnt signaling pathways both play pivotal roles in bone development. Wntless/GPR177 is a chaperone protein that is required for secretion of all Wnt ligands. We previously showed that deletion of Wntless within mature osteoblasts severely impaired postnatal bone homeostasis. Results : In this study, we systemically evaluated how deletion of Wntless in different stages of osteochondral differentiation affected embryonic bone development, by crossing Wntless ( Wls )‐flox/flox mice with strains expressing cre recombinase behind the following promoters: Osteocalcin, Collagen 2a1 , or Dermo1 . Ex vivo µCT and whole‐mount skeletal staining were performed to examine skeletal mineralization. Histology and immunohistochemistry were used to evaluate cellular differentiation and alterations in Wnt signaling. In this work, we found that Wntless regulated chondrogenesis and osteogenesis through both canonical and noncanonical Wnt signaling. Conclusions : These findings provide more insight into the requirements of different Wnt‐secretion cell types critical for skeletal development. Developmental Dynamics 244:1347–1355, 2015 . © 2015 Wiley Periodicals, Inc.

Abstract Sustained activation of the stress-regulated transcription factor NRF2 (NFE2L2) is a prominent feature of many types of cancer, implying that mutations driving NRF2 may be important to tumor progression. In hereditary type 2 papillary renal cell carcinoma (PRCC2, also known as hereditary leiomyomatosis and renal cell cancer), NRF2 activation is a direct consequence of the accumulation of intracellular fumarate, a result of fumarate hydratase (FH) inactivation, but it is not clear how NRF2 may be activated in sporadic forms of PRCC2. Here we show that somatic mutations in NRF2, CUL3, and SIRT1 are responsible for driving the NRF2 activation phenotype in sporadic PRCC2. Transcriptome sequencing revealed the expression pattern of mutant alleles of NRF2, CUL3, and SIRT1 and also confirmed NRF2 activation in clinical specimens. Our results show a convergence in somatic mutations in sporadic PRCC2 with FH mutation in hereditary PRCC2. Cancer Res; 73(7); 2044–51. ©2013 AACR.

Abstract Sunitinib is a broad-spectrum small-molecule inhibitor of receptor tyrosine kinases (RTK) that serves as the present standard of care for first-line therapy of advanced clear cell renal cell carcinoma (ccRCC). A full understanding of the targets and mechanism of action of sunitinib in ccRCC treatment remains incomplete. In this study, we evaluated several tumor cell and endothelial targets of sunitinib and investigated which RTK(s) may specifically contribute to its therapeutic effects. Microarray expression profiling and Western blot analysis revealed that among known sunitinib targets, only platelet-derived growth factor receptor-β and vascular endothelial growth factor receptor-2 (VEGFR-2) were overexpressed in ccRCCs relative to normal tissues. Sunitinib was unable to inhibit survival or proliferation of ccRCC cells at pharmacologically relevant concentrations (∼0.1 μmol/L) that inhibit RTK targets. In contrast, sunitinib inhibited endothelial cell proliferation and motility at the same concentrations by suppressing VEGFR-2 signaling. Moreover, whereas sunitinib inhibited the growth of ccRCC xenograft tumors and decreased tumor microvessel density as soon as 12 hours after treatment, sunitinib showed no significant effects on tumor cell proliferation or apoptosis up to 72 hours after treatment. Our findings indicate that sunitinib inhibits ccRCC growth primarily through an antiangiogenic mechanism and not through direct targeting of ccRCC tumor cells. Cancer Res; 70(3); 1053–62

Abstract Sunitinib, a small-molecule inhibitor of multiple receptor tyrosine kinases (RTKs), is considered the standard of care for first line therapy of advanced clear cell renal cell carcinoma (ccRCC). However, a complete understanding of its targets and mechanism of action in the treatment of ccRCC remains incomplete. We aimed to evaluate the primary targets of sunitinib in the treatment of ccRCC (i.e. tumor cells versus endothelial cells) and to determine which RTK(s) specifically contribute to the therapeutic effect of sunitinib. Microarray gene expression profiling of human ccRCC samples showed that, of the known RTK targets of sunitinib, only PDGFR-β and VEGFR-2 were overexpressed in ccRCC relative to normal tissues. Western blotting of ccRCC and endothelial cell lines confirmed that PDGFR and VEGFR, but not other sunitinib targets, were expressed in these cells. In vitro studies found that sunitinib was unable to inhibit survival or proliferation of ccRCC cells at pharmacologically relevant concentrations (∼0.1 μM) which inhibit RTK targets. In contrast, sunitinib inhibited endothelial cell proliferation and invasion at these concentrations through suppression of VEGFR-2 signaling. Sunitinib inhibited growth of ccRCC xenografts and decreased tumor microvessel density as soon as 12 hours post-treatment; however, sunitinib showed no significant effects on tumor cell proliferation or apoptosis after up to 72 hours post-treatment. Our studies indicate that sunitinib inhibits ccRCC growth primarily through an anti-angiogenic mechanism and not through direct targeting of ccRCC tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1644.