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Supplementary Table 1: Patient characteristics; Supplementary Table 2: Primers for amplifying MET gene; Supplementary Figure 1: PC-9/LMC-GR did not express HGF or possess MET mutations; Supplementary Figure 2: Acquired gefitinib resistance of HCC827/luc cells in LMC model was associated with MET copy number gain; Supplementary Figure 3: Subcutaneous tumors with PC-9/LMC-GR cells are sensitive to gefitinib treatment.

ADAM28 (a disintegrin and metalloproteinase 28) is overexpressed by carcinoma cells in non–small cell lung carcinomas (NSCLC) and plays an important role in cancer cell proliferation and metastasis by reactivation of insulin-like growth factor-1 (IGF-1) and escaping from von Willebrand factor (VWF)–induced apoptosis through digestion of IGF-binding protein-3 and VWF, respectively. To aim for new target therapy of NSCLC patients, we developed human neutralizing antibodies 211-12 and 211-14 against ADAM28, which showed IC50 values of 62.4 and 37.5 nmol/L, respectively. Antibody 211-14 recognized the junctional region between cysteine-rich domain and secreted-specific domain and showed a KD value of 94.7 pmol/L for the epitope-containing peptide. This antibody detected monkey and human secreted-form ADAM28s, although it was not reactive with mouse membrane-anchored ADAM28m. Antibody 211-14 effectively inhibited IGF-1–stimulated cell proliferation of lung adenocarcinoma cell lines with ADAM28 expression, including PC-9 cells, and promoted VWF-induced cell death in these cell lines. In lung metastasis models, antibody 211-14 significantly reduced tumor growth and metastases of PC-9 cells and prolonged survivals in the antibody-treated mice compared with the control IgG-treated ones. Combination therapy of the antibody and docetaxel was more effective than that of bevacizumab and docetaxel and showed further elongation of survival time compared with monotherapy. No adverse effects were observed even after administration of 10-fold more than effective dose of anti-ADAM28 antibody to normal mice. Our data demonstrate that antibody 211-14 is a neutralizing antibody specific to ADAM28s and suggest that this antibody may be a useful treatment remedy for NSCLC patients. Mol Cancer Ther; 17(11); 2427–38. ©2018 AACR.

Supplementary Figure S3 shows results of Ki-67-positive cell index of the carcinoma cells within the lung tissue from mice treated with control mouse IgG or antibody 211-14.

Supplementary Table S2 shows hematological and biochemical results of mice treated with control mouse IgG or antibody 211-12 at 20 mg/kg body weight five times once a week.

Leptomeningeal carcinomatosis occurs frequently in EGFR-mutant lung cancer, and develops acquired resistance to EGFR tyrosine kinase inhibitors (EGFR-TKIs). This study aimed to clarify the mechanism of EGFR-TKI resistance in leptomeningeal carcinomatosis and seek for a novel therapeutic strategy. We examined EGFR mutations, including the T790M gatekeeper mutation, in 32 re-biopsy specimens from 12 leptomeningeal carcinomatosis and 20 extracranial lesions of EGFR-mutant lung cancer patients who became refractory to EGFR-TKI treatment. All the 32 specimens had the same baseline EGFR mutations, but the T790M mutation was less frequent in leptomeningeal carcinomatosis specimens than in extracranial specimens (8% vs. 55%, P < 0.01). To study molecular mechanisms of acquired EGFR-TKI resistance in leptomeningeal carcinomatosis, we utilized our previously developed mouse model of leptomeningeal carcinomatosis with the EGFR-mutant lung cancer cell line PC-9/ffluc cells, in which acquired resistance to gefitinib was induced by continuous oral treatment. Compared with subcutaneously inoculated gefitinib-resistant tumors, the T790M mutation was less frequent in leptomeningeal carcinomatosis that acquired resistance to gefitinib. PC-9/LMC-GR cells were established from the gefitinib-resistant leptomeningeal carcinomatosis model, and they were found to be intermediately resistant to gefitinib and osimertinib (third-generation EGFR-TKI). Although EGFR-T790M was negative, gefitinib resistance of PC-9/LMC-GR cells was related to MET copy number gain with MET activation. Moreover, combined use of EGFR-TKI and crizotinib, a MET inhibitor, dramatically regressed leptomeningeal carcinomatosis with acquired resistance to gefitinib or osimertinib. These findings suggest that combination therapy with MET inhibitors may be promising for controlling leptomeningeal carcinomatosis that acquires resistance to EGFR-TKIs. Mol Cancer Ther; 16(3); 506–15. ©2017 AACR.

PDF file - 33K, Correlation of clinicopathological parameters and MVD or Vasohibin-1 expression in the 175 study patients: Patients with high grade tumors and �pT2 had significantly higher levels of VASH1 density

PDF file - 169K, Supplementary Figure 1A. Kaplan-Meier recurrence-free survival of the 171 patients after surgery for UTUC stratified according to the cut-off level of MVD 70/mm2: High level of MVD was not an independent predictor of tumor recurrence (P = 0.216). Supplementary Figure 1B. Kaplan-Meier cancer-specific survival of the 171 patients after surgery for UTUC stratified according to the cut-off level of MVD 70/mm2: High level of MVD was not an independent predictor of cancer-specific survival (P = 0.473). Supplementary Figure 1C. Kaplan-Meier recurrence-free survival of the 171 patients after surgery for UTUC stratified according to the cut-off level of MVD 80/mm2: High level of MVD was not an independent predictor of tumor recurrence (P = 0.092). Supplementary Figure 1D. Kaplan-Meier cancer-specific survival of the 171 patients after surgery for UTUC stratified according to the cut-off level of MVD 80/mm2: High level of MVD was not an independent predictor of cancer-specific survival (P = 0.345). Supplementary Figure 1E. Kaplan-Meier recurrence-free survival of the 171 patients after surgery for UTUC stratified according to the cut-off level of VASH1 density 40/mm2: High level of VASH1 density was an independent predictor of tumor recurrence (P = 0.017). Supplementary Figure 1F. Kaplan-Meier cancer-specific survival of the 171 patients after surgery for UTUC stratified according to the cut-off level of VASH1 density 40/mm2: High level of VASH1 density was an independent predictor of cancer-specific survival (P = 0.042). Supplementary Figure 1G. Kaplan-Meier recurrence-free survival of the 171 patients after surgery for UTUC stratified according to the cut-off level of VASH1 density 50/mm2: High level of VASH1 density was an independent predictor of tumor recurrence (P = 0.005). Supplementary Figure 1H. Kaplan-Meier cancer-specific survival of the 171 patients after surgery for UTUC stratified according to the cut-off level of VASH1 density 50/mm2. High level of VASH1 density was an independent predictor of cancer-specific survival (P = 0.027).

Purpose: Vasohibin-1 (VASH1) is a novel angiogenic molecule that is specifically expressed in activated vascular endothelial cells, and the status of VASH1 expression has been documented in cancer angiogenesis. The aim of this study was to address the prognostic value of VASH1 expression in upper urinary tract urothelial carcinomas (UTUC).Experimental Design: We retrospectively analyzed the clinical records of 171 patients with locally advanced UTUC (Ta-3N0M0). The median follow-up period was 3.8 years. We immunohistochemically examined the accomplished microvessels with anti-CD34 as microvessel density (MVD) and the microvessels with activated endothelial cells as VASH1 density. Then, we analyzed the association between immunohistochemical expression and clinical outcomes.Results: Forty-two patients experienced tumor recurrence and of these 34 died of the disease during follow-up. VASH1 density was significantly associated with tumor grade, pathologic T stage, and MVD. The 5-year recurrence-free and cancer-specific survival rates were 66.1% and 72.8% in patients with VASH1 density (≥ 40/mm2) and 81.0% and 86.5% in their counterparts, respectively (P < 0.05). MVD was not an independent predictor of tumor recurrence or cancer-specific survival. Multivariate analyses revealed that high VASH1 density was an independent prognostic indicator of both tumor recurrence (P = 0.024, HR = 2.10) and cancer-specific survival (P = 0.031, HR = 2.23) as well as other standard prognostic factors including high tumor grade and lymphovascular invasion.Conclusions: VASH1 density represents a clinically relevant predictor of patient prognosis in UTUC. The results suggest that VASH1 density could become a new biomarker and provide additional prognostic information in patients with UTUC. Clin Cancer Res; 18(15); 4145–53. ©2012 AACR.

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PDF file - 13K, Univariate and multivariate analysis for recurrence-free survival and cancer-specific survival of 175 UTUC patients: Multivariate analysis showed that high tumor grade, presence of LVI, positive pN and high VASH1 density were independent predictors of recurrence and cancer-specific survival

PDF file - 31K, Univariate and multivariate analysis for recurrence-free survival and cancer-specific survival in 171 UTUC patients: We used the cutoff value of MVD � 80/mm2 and VASH1 density � 50/mm2 according to the mean values. The levels of VASH1 density were independent predictors for a decrease in recurrence-free (HR = 2.08) and cancer-specific survival (HR = 2.06)

PDF file - 2.9MB, Supplementary Figure 2A. Immunostaining for CD34 in the case of pTa, low grade, and LVI negative UTUC with low VASH1 density: CD34 staining was observed in endothelial cells of microvascular vessels in the tumor lesion. Supplementary Figure 2B. Immunostaining for VASH1: the same case with 2A. VASH1 staining of vascular endothelial cells was negative or negligible. We observed low VASH1 density. Supplementary Figure 2C. Negative control slide: the same case with 2A-2B. The appropriate negative control slide was prepared. Supplementary Figure 2D. Immunostaining for CD34 in the case of pTa, low grade, and LVI negative UTUC with high VASH1 density: CD34 staining was observed in endothelial cells of microvascular vessels in the tumor lesion. Supplementary Figure 2E. Immunostaining for VASH1: the same case with 2D. VASH1 staining of vascular endothelial cells was strong. We observed high VASH1 density. Supplementary Figure 2F. Negative control slide: the same case with 2D-2E. The appropriate negative control slide was prepared. Supplementary Figure 2G. Immunostaining for CD34 in the case of pT3b, high grade, and LVI positive UTUC with low VASH1 density: CD34 staining was observed in endothelial cells of microvascular vessels in the tumor lesion. Supplementary Figure 2H. Immunostaining for VASH1: the same case with 2G. VASH1 staining of vascular endothelial cells was negative or negligible. We observed low VASH1 density. Supplementary Figure 2I. Negative control slide: the same case with 2G-2H. The appropriate negative control slide was prepared. Supplementary Figure 2J. Immunostaining for CD34 in the case of pT3b, high grade, and LVI positive UTUC with high VASH1 density: CD34 staining was observed in endothelial cells of microvascular vessels in the tumor lesion. Supplementary Figure 2K. Immunostaining for VASH1: the same case with 2J. VASH1 staining of vascular endothelial cells was strong. We observed high VASH1 density. Supplementary Figure 2L. Negative control slide: the same case with 2J-2K.

Supplementary Figure 10 - PDF file 84K, HMGA2 mRNA expression in MCF7 and MDA-MB231 human breast cancer cells treated with TGFBeta 1 as detected by qRT-PCR

Supplementary Figure 6 - PDF file 132K, (A) HMGA2 was detected at the edge of the metastatic lung tumor (left, arrows) and from the vessel of the liver stroma to the parenchyma in the metastatic liver tumor (right, arrows). Scale bars represent 100 microm. (B) Liver metastases (arrows) were induced in 4TO7 cells stably over-expressing HMGA2 (4TO7-HMGA2)

Supplementary Figure 3 - PDF file 171K, Immunofluorescence analysis of ZEB1 (left), Fibronectin (right) expression in MCF7-Mock and MCF7-HMGA2 cells. Scale bars represent 50 microm. ZEB1 and fibronectin are expressed in the nucleus of MCF7-HMGA2 cells that have undergone the EMT as is consistent with the other EMT markers in Figure 1C

Supplementary Figure 2 - PDF file 77K, Agarose Colony Formation Assay. Anchorage-independent proliferation was performed by plating SW403, HT29, SW620, SW480 and HCT116 (1000 cells per well) in six-well plates, with a bottom layer of 0.6% agar and a top layer of 0.3% agar containing the cells. After 21 days incubation, colonies were counted

Supplementary Figure 7 - PDF file 97K, Schematic of liver metastasis. Metastatic cancer cells (4TO7 cells stably over-expressing HMGA2 and 4T1 cells) are located in Glisson's capsule (GC) and Liver parenchyma (LP)

Supplementary Figure 4 - PDF file 253K, beta-catenin was relocalized to the nucleus at the invasive front of Wnt1-mediated tumor on Hmga2+/+ genotypic backgrounds, but not on the Hmga2-/- genetic background. beta-catenin was detected in the nucleus of the cells at the invasive front consistent with the HMGA2 expressed cells in the Wnt1-mediated tumor on the Hmga2+/+ genetic background (arrows). On the other hand, no beta-catenin relocalization was detected in tumor on the Hmga2-/- genotypic backgrounds. Scale bars represent 100microm

PDF file - 80K, TGFBetaRII was up-regulated in 4T1 cells. TGFBetaRII mRNA expression was highly up-regulated in 4T1 cells compared to 4TO7 parental cells (## = P < 0.0001)

Supplementary Figure 8 - PDF file 82K, There is no statistically significant difference in Snail and Twist mRNA expression in ectopic HMGA2 transfected cells (MCF7-HMGA2 cells)

Supplementary Information - PDF file 81K, Supplementary Figure Legends and Supplementary Materials and Methods

The ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family includes nine members with aggrecan-degrading activity, i.e., ADAMTS1, 4, 5, 8, 9, 15, 16, 18, and 20. However, their systematic expression profile in knee osteoarthritis (OA) synovium and effects of cytokines and growth factors on the expression in OA synovial fibroblasts remain elusive. In this study, expression of all nine aggrecanolytic ADAMTS species was assessed by quantitative real-time PCR in OA and control normal synovial tissues. OA synovial fibroblasts were treated with interleukin-1α (IL-1α), IL-1β, tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), vascular endothelial growth factor165, and heparin-binding epidermal growth factor, and analyzed for the expression of the ADAMTS species. The signaling pathways and inhibition of ADAMTS4 expression by high-molecular-weight hyaluronan, adalimumab, tocilizumab, and signaling molecule inhibitors were studied. ADAMTS1, 4, 5, 9, and 16 were expressed in OA synovium, but only ADAMTS4 expression was significantly higher in OA as compared to normal synovium. IL-1α, TNF-α, and TGF-β markedly increased ADAMTS4 expression, while their effects were minimal for the other ADAMTS species. ADAMTS4 was synergistically upregulated by treatment with IL-1α and TNF-α, IL-1α and TGF-β, or IL-1α, TNF-α and TGF-β. The signaling molecules’ inhibitors demonstrated that IL-1α-induced ADAMTS4 expression is predominantly through TGF-β-associated kinase 1 (TAK1), and the TNF-α-stimulated expression is via TAK1 and nuclear factor-κB (NF-κB). The TGF-β-promoted expression was through the activin receptor-like kinase 5 (ALK5)/Smad2/3, TAK1, and non-TAK1 pathways. Adalimumab blocked TNF-α-stimulated expression. ADAMTS4 expression co-stimulated with IL-1α, TNF-α and TGF-β was abolished by treatment with adalimumab, TAK1 inhibitor, and ALK5/Smad2/3 inhibitor. These data demonstrate marked and synergistic upregulation of ADAMTS4 by IL-1α, TNF-α and TGF-β in OA synovial fibroblasts, and suggest that concurrent therapy with an anti-TNF-α drug and inhibitor(s) may be useful for prevention against aggrecan degradation in OA. Aggrecan degradation by ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) is essential to cartilage destruction in osteoarthritis, but its expression profile and regulation are unclear. The authors show overexpression of ADAMTS4 in osteoarthritis synovium and synergistic upregulation by interleukin-1α, tumor necrosis factor-α, and transforming growth factor-β in osteoarthritis synovial fibroblasts. These findings suggest that concurrent therapy with an anti-TNF-α drug and inhibitor(s) may be useful for prevention against aggrecan degradation in OA.

Hyaluronan (HA)-binding protein involved in HA depolymerization (HYBID) is involved in cartilage destruction via HA depolymerization in human knee osteoarthritis. However, roles of HYBID in the progression of osteoarthritis remain elusive. This study sought to examine whether genetic depletion of Hybid could suppress surgically induced osteoarthritis of mouse knee joints. In osteoarthritis induced by medial collateral ligament transection with meniscus removal, articular cartilage destruction and osteophyte formation at the medial femoral-tibial joint were significantly inhibited in Hybid-deficient (Hybid-/-) mouse group compared to wild-type group. Hybid was highly produced by synovial cells and articular chondrocytes in wild-type mouse osteoarthritis joint. Interleukin-1β, interleukin-6 and tumor necrosis factor-α were up-regulated in the osteoarthritis joint tissues of both wild-type and Hybid-/- mice. Vascular density at the synovial and periosteal junction was significantly reduced in Hybid-/- mice compared to wild-type mice. High-molecular-weight HA was accumulated in osteoarthritis joint tissues of Hybid-/- mice. Injections of high-molecular-weight HA to knee joints attenuated the cartilage destruction and osteophyte formation in wild-type mouse osteoarthritis group. Inhibition of cartilage destruction and osteophyte formation in Hybid-/- mice was also observed in destabilization of the medial meniscus model. These data are the first to demonstrate that cartilage destruction and osteophyte formation are suppressed in Hybid-/- mice and suggest that Hybid-mediated HA depolymerization is implicated for the progression of mechanically induced knee osteoarthritis.

Increased vascularity in the synovium of patients with rheumatoid arthritis (RA) is thought to result from angiogenesis. However, other possibilities to develop vasculatures by mechanism(s) like vasculogenic mimicry remain elusive. In this study, expression of vascular endothelial (VE)-cadherin was immunohistochemically detected in both microvessel lining cells and fibroblastic cells in RA synovium. VE-cadherin-positive lining cells surrounded by periodic acid-Schiff staining-positive substance were occasionally negative for endothelial cell markers CD31/CD34 but positive for osteoblast (OB)-cadherin that is specific to fibroblastic cells. RA synovial fibroblast-like cells (RSFLs) in culture expressed VE-cadherin and OB-cadherin, but not CD31/CD34. Tube formation was evaluated by real-time adhesion and tube formation assays. RSFLs specifically bound to VE-cadherin-chimera-coated plates. They developed tube-like structures solely, which were hindered by anti-VE-cadherin antibody or VE-cadherin-chimera, and with endothelial cells. VE-cadherin was up-regulated by vascular endothelial growth factor-A165 (VEGF165) treatment, and shed from cell surface by treatment with tumor necrosis factor-a. RSFLs expressed VEGF and VEGF receptors (VEGFRs), and VEGF165 induced up-regulation of VE-cadherin was suppressed by inhibiting VEGFR2 activity. These data demonstrate that fibroblastic cells in RA synovium express VE-cadherin, and suggest that the fibroblastic cells are involved in formation of vasculogenic mimicry-like tubes in RA synovium through the VEGF/VEGFR2/VE-cadherin pathway.

We reported that full-length width of medial tibial osteophyte composed of cartilage and bone parts is directly correlated with medial meniscus extrusion (MME) in early-stage knee osteoarthritis (OA). However, no data are available for MME prevalence and its relationship to osteophyte in elderlies. 1,191 elderlies (females 57%; 72.9 years old on average) in the Bunkyo Health Study underwent standing plain radiograph and proton density-weighted MRI on knee joints. MRI-detected OA changes were evaluated according to the Whole Organ Magnetic Resonance Imaging Score. A new method to assess cartilage and bone parts of osteophyte was developed by pseudo-coloring images of proton density-weighted fat-suppressed MRI. Most of the subjects showed the Kellgren-Lawrence grade 1 or 2 of radiographic medial knee OA (88.1%), MME (98.7%, 3.90 ± 2.01 mm) and medial tibial osteophyte (99.3%, 3.27 ± 1.50 mm). Among the OA changes, MME was most closely associated with full-length width of medial tibial osteophyte (β = 1.114; 95%CI, 1.069–1.159; p

Herein, we describe two counterexamples of the previously reported β/α-selectivity of 96/4 for glycosylation using ethyl 2-O-[2,3,4-tris-O-tert-butyldimethylsilyl (TBS)-α-L-rhamnopyranosyl]-3,4,6-tris-O-TBS-thio-β-D-glucopyranoside as the glycosyl donor. Furthermore, we investigated the effects of protecting group on the rhamnose moieties in the glycosylation with cholestanol and revealed that β-selectivity originated from the two TBS groups at the 3-O and 4-O positions of rhamnose. In contrast, the TBS group at the 2-O position of rhamnose hampered the β-selectivity. Finally, the β/α-selectivity during the glycosylation was enhanced to ≥99/1. The results obtained herein suggest that the protecting groups on the sugar connected to the 2-O of a glycosyl donor with axial-rich conformation can control the stereoselectivity of glycosylation.

The development of systemic therapies, including vascular endothelial growth factor-tyrosine kinase inhibitors (VEGF-TKI) and mammalian target of rapamycin (mTOR) inhibitors, represents a major breakthrough in the treatment of patients with renal cell carcinoma (RCC). However, inherent resistance is observed in some patients and acquired resistance commonly develops in many patients within several months of the initiation of systemic therapies. Since these treatments rarely cure patients, their aim is to suppress tumor progression and prolong survival. Therefore, the establishment of dependable criteria that predict responses and resistance to systemic therapies is clinically important, and the underlying molecular mechanisms also need to be elucidated for the future development of more effective therapies. We herein review recent advances in research on the molecular mechanisms underlying resistance, with a focus on morphological characteristics, tumor angiogenesis, and the tumor immune microenvironment in RCC and their relationships with VEGF-TKI treatments. Recent therapies using immune checkpoint inhibitors (ICI) and newly developed VEGF-TKI also appear to be effective for advanced RCC, with stable and durable responses to ICI being observed in some RCC patients. These new drugs and their outcomes have been briefly described.

The aim of this paper is to characterize continuous endomorphisms in the space of entire functions of exponential type of order $p>0$. Let $A_p$ denote the space of entire functions of $n$ complex variables $z\in{\mathbb C}^n$ of order $p$ of normal type. We consider an endomorphism $F$ in the space, which is considered to be a DFS-space. We show that there is a unique linear differential operator $P$ of infinite order with coefficients in the space which realizes $F$, that is, $Ff=Pf$ holds for any $f\in A_p$. The coefficients satisfy certain growth conditions and conversely, if a formal differential operator of infinite order with coefficients in $A_p$ satisfy these conditions, then it induces a continuous endomorphism., 13 pages