Search

Top genotyped SNP associations with metastasis at diagnosis in the discovery and all replication studies.

Top 30 loci associated with metastasis at diagnosis in the discovery set of 541 European osteosarcoma cases.

Relationship of variables to metastasis at diagnosis in the discovery set.

SNPs in the NFIB locus associated with metastasis at diagnosis with P{less than or equal to}1x10-5 in the discovery stage, and functionally annotated with information from the ENCODE and 1000 Genomes Project data.

Associations of the top GWAS and imputed SNP with metastasis at diagnosis in the discovery stage (European ancestry) by inheritance model.

Supplementary Figure 1. Manhattan plot of the Discovery stage. SNPs in the NFIB risk locus are highlighted in green. Supplementary Figure 2. LD structure of the NFIB risk locus. Plot was made using the CEU population of the 1000 Genomes Project data with LDlink (ref 54). Supplementary Figure 3. Significant relationship between the genotype of the metastasis associated SNP rs7034162, and expression of NFIB and IGFBP5 in osteosarcoma cell lines and tumors. Significance is based on linear regression comparing the distribution of expression of NFIBbetween the TT homozygous non-risk genotype (combined N=34) of rs7034162 genotypes to the heterozygous risk AT (combined N=10) and the homozygous risk AA genotypes (combined N=2). The green triangle represents the expression levels in the OSA cell line, the blue circle is expression levels in the HOS cell line, and the red square the U2OS cell line expression. These analyses were based on the publically available genotype and expression data from 17 osteosarcoma cell lines and 29 osteosarcoma tumors (ref 20). Supplementary Figure 4. No significant relationship between the genotype of the metastasis associated SNP, rs7034162, and expression of protein-encoding genes FREM1, ZDHHC1, and MPDZ in the neighborhood of NFIB in osteosarcoma cell lines and tumors. Significance is based on linear regression comparing the distribution of expression of FREM1, ZDHHC1, and MPDZ between the TT homozygous non-risk genotype (combined N=34) of rs7034162 genotypes to the heterozygous risk AT (combined N=10) and the homozygous risk AA genotypes (combined N=2). The green triangle represents the expression levels in the OSA cell line, the blue circle is expression levels in the HOS cell line, and the red square the U2OS cell line expression. These analyses were based on the publically available genotype and expression data from 17 osteosarcoma cell lines and 29 osteosarcoma tumors (ref 20). Supplementary Figure 5. The human osteosarcoma cell lines, U2OS and HOS, had higher NFIB protein levels then OSA cells. NFIB levels were determined using immunoblot analysis in the OSA, HOS and U2OS cells both basal (A) and after treatment with siRNA against NFIB (B). Supplementary Figure 6. NFIB expression significantly correlates with IGFBP5 expression in osteosarcoma cell lines and tumors. Significance is based on linear regression comparing the expression levels of NFIB with the corresponding IGFBP5 expression levels. Supplementary Figure 7. Expression levels of IGFBP5 decreases after NFIB suppression of human osteosarcoma cell lines. IGFBP5 expression of OSA, HOS and U2OS cells was determined 48 hours after transfection with control siRNA (si-NEG) or siRNA targeting NFIB (si-NFIB). Graph showing relative expression compared to control treated U2OS cells. Supplementary Figure 8. A model of how the NFIB risk locus leads to changes in NFIB expression and potentially may affect osteosarcoma metastatic potential. The risk allele (right panel) leads to lowered expression of NFIB and NFIB-mediated lower expression of IGFPB5, which then leads to less IGFBP5-mediated inhibition of IGF-1 (right panel, grey arrows), leading to an increase in proliferation, survival and metastasis of osteosarcoma cells (right panel, black arrows). The reference allele (left panel) leads to both higher expression of NFIB and higher expression of NFIB-mediated IGFPB5. In turn, higher IGFBP5 levels leads to increased inhibition of IGF-1 (left panel, black arrows), leading to a decrease in proliferation, survival and metastasis of osteosarcoma cells (left panel, grey arrows). Supplementary Figure 9. Plot of the PCA Eigenvectors of the Discovery stage. Supplementary Figure 10. QQ-plot of the Discovery stage.

Description of participating case studies.

5' UTR structures of BONCAT identified proteins

Legends for Supplementary Tables and Figures

Purpose: Thrombospondin-I (TSP-I) is a natural antiangiogenic protein that enhances apoptosis of activated endothelial cells. A modified nonapeptide from TSP-I, ABT-526, has been found to be active in mouse cancer models and in dogs with naturally occurring cancers. To further assist in the development of ABT-526, we report herein on its evaluation in combination with cytotoxic chemotherapy in pet dogs with relapsed non-Hodgkin's lymphoma (NHL).Experimental Design: Ninety-four pet dogs with naturally occurring first-relapse NHL were entered into a prospective randomized placebo controlled double-blinded trial of ABT-526 plus CeeNu (Bristol-Myers Squibb, New York, NY) versus CeeNu alone. Endpoints included response rate, duration of response, time to progression, and incidence of toxicoses.Results: No significant ABT-526-specific toxicities were seen. CeeNu-associated toxicities, including neutropenia, thrombocytopenia, gastroenteritis, and elevated alanine transaminase, were similar. No significant difference in objective response rate was seen (ABT-526 + CeeNu versus placebo + CeeNu, 23/49 versus 23/37; P > 0.25). Cooperative activity between ABT-526 and CeeNu chemotherapy was evident based on a significant increase in the median response duration of dogs receiving ABT-526 plus CeeNu compared with placebo plus CeeNu (35 versus 15 days; P < 0.05). The time to progression for responding cases was also significantly greater in dogs receiving ABT-526 plus CeeNu compared with placebo plus CeeNu (41 versus 21 days; P < 0.05).Conclusions: Results of this preclinical trial suggest that the activity of ABT-526 is sustained when combined with cytotoxic chemotherapy; furthermore, the activity seems to be associated with the maintenance of CeeNu-induced treatment responses. Further studies of TSP-I peptide antiangiogenic therapy in pet dogs and humans with NHL are warranted.

Supplementary Table 3 contains a summary of the outcomes, with statistical comparisons, of the dogs enrolled in the SOC clinical trial arm. This includes the DFI and outcomes of the groups of dogs stratified by tumor location and ALP status.

Supplementary Fig. 1 depicts the chronology of clinical trial visits and associated procedures for dogs enrolled in this clinical trial.

All supplementary files together

Supplementary Table 2 contains the summary comparison of both the Intent-to-treat and Per-protocol analyses of clinical outcomes for dogs enrolled in Standard of Care (SOC) and Standard of Care + sirolimus

Purpose: To successfully metastasize, tumor cells must respond appropriately to biological stressors encountered during metastatic progression. We sought to test the hypothesis that enhanced efficiency of mRNA translation during periods of metastatic stress is required for metastatic competence of osteosarcoma and that this metastasis-specific adaptation is amenable to therapeutic intervention.Experimental Design: We employ novel reporter and proteomic systems that enable tracking of mRNA translation efficiency and output in metastatic osteosarcoma cells as they colonize the lungs. We test the potential to target mRNA translation as an antimetastatic therapeutic strategy through pharmacokinetic studies and preclinical assessment of the prototypic mTOR inhibitor, rapamycin, across multiple models of metastasis.Results: Metastatic osteosarcoma cells translate mRNA more efficiently than nonmetastatic cells during critical stressful periods of metastatic colonization of the lung. Rapamycin inhibits translational output during periods of metastatic stress, mitigates lung colonization, and prolongs survival. mTOR-inhibiting exposures of rapamycin are achievable in mice using treatment schedules that correspond to human doses well below the MTDs defined in human patients, and as such are very likely to be tolerated over long exposures alone and in combination with other agents.Conclusions: Metastatic competence of osteosarcoma cells is dependent on efficient mRNA translation during stressful periods of metastatic progression, and the mTOR inhibitor, rapamycin, can mitigate this translation and inhibit metastasis in vivo. Our data suggest that mTOR pathway inhibitors should be reconsidered in the clinic using rationally designed dosing schedules and clinical metrics related to metastatic progression. Clin Cancer Res; 22(24); 6129–41. ©2016 AACR.

Related Article from Catching Cancer by the Tail: New Perspectives on the Use of Kinase Inhibitors

Supplementary Materials and Methods

Purpose:The mTOR pathway has been identified as a key nutrient signaling hub that participates in metastatic progression of high-grade osteosarcoma. Inhibition of mTOR signaling is biologically achievable with sirolimus, and might slow the outgrowth of distant metastases. In this study, pet dogs with appendicular osteosarcoma were leveraged as high-value biologic models for pediatric osteosarcoma, to assess mTOR inhibition as a therapeutic strategy for attenuating metastatic disease progression.Patients and Methods:A total of 324 pet dogs diagnosed with treatment-naïve appendicular osteosarcoma were randomized into a two-arm, multicenter, parallel superiority trial whereby dogs received amputation of the affected limb, followed by adjuvant carboplatin chemotherapy ± oral sirolimus therapy. The primary outcome measure was disease-free interval (DFI), as assessed by serial physical and radiologic detection of emergent macroscopic metastases; secondary outcomes included overall 1- and 2-year survival rates, and sirolimus pharmacokinetic variables and their correlative relationship to adverse events and clinical outcomes.Results:There was no significant difference in the median DFI or overall survival between the two arms of this trial; the median DFI and survival for standard-of-care (SOC; defined as amputation and carboplatin therapy) dogs was 180 days [95% confidence interval (CI), 144–237] and 282 days (95% CI, 224–383) and for SOC + sirolimus dogs, it was 204 days (95% CI, 157–217) and 280 days (95% CI, 252–332), respectively.Conclusions:In a population of pet dogs nongenomically segmented for predicted mTOR inhibition response, sequentially administered adjuvant sirolimus, although well tolerated when added to a backbone of therapy, did not extend DFI or survival in dogs with appendicular osteosarcoma.

Summary of BONCAT proteomic analysis of mRNA translation during metastasis

Supplementary Table 4 contains a summary of the outcomes, with statistical comparisons, of the dogs enrolled in the SOC + sirolimus clinical trial arm. This includes the DFI and outcomes of the groups of dogs stratified by tumor location and ALP status.

In this issue of Clinical Cancer Research, London and colleagues evaluate a small molecule multiple-targeted tyrosine kinase inhibitor in dogs with c-kit driven skin cancer. The study represents another example of opportunities to include pet dogs in studies that improve our understanding of human cancer biology and therapy.

Supplementary Figure 1 from Cooperative Activity of Cytotoxic Chemotherapy with Antiangiogenic Thrombospondin-I Peptides, ABT-526 in Pet Dogs with Relapsed Lymphoma

Highly metastatic osteosarcoma cells activate the mTOR pathway during metastatic progression

Supplementary Figure Legend from Cooperative Activity of Cytotoxic Chemotherapy with Antiangiogenic Thrombospondin-I Peptides, ABT-526 in Pet Dogs with Relapsed Lymphoma

Supplementary Table 1 contains the inclusion and exclusion criteria for pet dogs considered for enrollment into the SOC and SOC + S clinical trial arms.

Supplementary Fig. 2 shows the walk-in pharmacokinetics, both measured results and simulated values, for dogs treated with oral sirolimus in preparation for the definitive adjuvant trial.

Purpose:Only one chemical class of topoisomerase I (TOP1) inhibitors is FDA approved, the camptothecins with irinotecan and topotecan widely used. Because of their limitations (chemical instability, drug efflux-mediated resistance, and diarrhea), novel TOP1 inhibitors are warranted. Indenoisoquinoline non-camptothecin topoisomerase I (TOP1) inhibitors overcome chemical instability and drug resistance that limit camptothecin use. Three indenoisoquinolines, LMP400 (indotecan), LMP776 (indimitecan), and LMP744, were examined in a phase I study for lymphoma-bearing dogs to evaluate differential efficacy, pharmacodynamics, toxicology, and pharmacokinetics.Experimental Design:Eighty-four client-owned dogs with lymphomas were enrolled in dose-escalation cohorts for each indenoisoquinoline, with an expansion phase for LMP744. Efficacy, tolerability, pharmacokinetics, and target engagement were determined.Results:The MTDs were 17.5 mg/m2 for LMP 776 and 100 mg/m2 for LMP744; bone marrow toxicity was dose-limiting; up to 65 mg/m2 LMP400 was well-tolerated and MTD was not reached. None of the drugs induced notable diarrhea. Sustained tumor accumulation was observed for LMP744; γH2AX induction was demonstrated in tumors 2 and 6 hours after treatment; a decrease in TOP1 protein was observed in most lymphoma samples across all compounds and dose levels, which is consistent with the fact that tumor response was also observed at low doses LMP744. Objective responses were documented for all indenoisoquinolines; efficacy (13/19 dogs) was greatest for LMP744.Conclusions:These results demonstrate proof-of-mechanism for indenoisoquinoline TOP1 inhibitors supporting their further clinical development. They also highlight the value of the NCI Comparative Oncology Program (https://ccr.cancer.gov/Comparative-Oncology-Program) for evaluating novel therapies in immunocompetent pets with cancers.

Figure 4, Tables 4-8, supplementary text

Supplementary Figure 3 from Insulin-Like Growth Factor-I Regulates the Liver Microenvironment in Obese Mice and Promotes Liver Metastasis

PDF file - 848K

Supplementary Table S5A from Cancers as Wounds that Do Not Heal: Differences and Similarities between Renal Regeneration/Repair and Renal Cell Carcinoma

Metastatic breast cancer may emerge from latent tumor cells that remain dormant at disseminated sites for many years. Identifying mechanisms regulating the switch from dormancy to proliferative metastatic growth has been elusive due to the lack of experimental models of tumor cell dormancy. We characterized the in vitro growth characteristics of cells that exhibit either dormant (D2.0R, MCF-7, and K7M2AS1.46) or proliferative (D2A1, MDA-MB-231, and K7M2) metastatic behavior in vivo. Although these cells proliferate readily in two-dimensional culture, we show that when grown in three-dimensional matrix, distinct growth properties of the cells were revealed that correlate to their dormant or proliferative behavior at metastatic sites in vivo. In three-dimensional culture, cells with dormant behavior in vivo remained cell cycle arrested with elevated nuclear expression of p16 and p27. The transition from quiescence to proliferation of D2A1 cells was dependent on fibronectin production and signaling through integrin β1, leading to cytoskeletal reorganization with filamentous actin (F-actin) stress fiber formation. We show that phosphorylation of myosin light chain (MLC) by MLC kinase (MLCK) through integrin β1 is required for actin stress fiber formation and proliferative growth. Inhibition of integrin β1 or MLCK prevents transition from a quiescent to proliferative state in vitro. Inhibition of MLCK significantly reduces metastatic outgrowth in vivo. These studies show that the switch from dormancy to metastatic growth may be regulated, in part, through epigenetic signaling from the microenvironment, leading to changes in the cytoskeletal architecture of dormant cells. Targeting this process may provide therapeutic strategies for inhibition of the dormant-to-proliferative metastatic switch. [Cancer Res 2008;68(15):6241–50]

Supplementary Table S8 from Cancers as Wounds that Do Not Heal: Differences and Similarities between Renal Regeneration/Repair and Renal Cell Carcinoma

Ezrin links the plasma membrane to the actin cytoskeleton where it plays a pivotal role in the metastatic progression of several human cancers; however, the precise mechanistic basis for its role remains unknown. Here, we define transitions between active (phosphorylated open) and inactive (dephosphorylated closed) forms of Ezrin that occur during metastatic progression in osteosarcoma. In our evaluation of these conformations we expressed C-terminal mutant forms of Ezrin that are open (phosphomimetic T567D) or closed (phosphodeficient T567A) and compared their biologic characteristics to full-length wild-type Ezrin in osteosarcoma cells. Unexpectedly, cells expressing open, active Ezrin could form neither primary orthotopic tumors nor lung metastases. In contrast, cells expressing closed, inactive Ezrin were also deficient in metastasis but were unaffected in their capacity for primary tumor growth. By imaging single metastatic cells in the lung, we found that cells expressing either open or closed Ezrin displayed increased levels of apoptosis early after their arrival in the lung. Gene expression analysis suggested dysregulation of genes that are functionally linked to carbohydrate and amino acid metabolism. In particular, cells expressing closed, inactive Ezrin exhibited reduced lactate production and basal or ATP-dependent oxygen consumption. Collectively, our results suggest that dynamic regulation of Ezrin phosphorylation at amino acid T567 that controls structural transitions of this protein plays a pivotal role in tumor progression and metastasis, possibly in part by altering cellular metabolism. Cancer Res; 72(4); 1001–12. ©2011 AACR.

Supplementary Video 1 from Inhibition of Metastatic Outgrowth from Single Dormant Tumor Cells by Targeting the Cytoskeleton

Fold increase in myeloid cell subsets in the lungs.

CCR1 expression in tumor cells.

Supplementary Figure 4C-D from Insulin-Like Growth Factor-I Regulates the Liver Microenvironment in Obese Mice and Promotes Liver Metastasis

Supplementary Figure 1 from Insulin-Like Growth Factor-I Regulates the Liver Microenvironment in Obese Mice and Promotes Liver Metastasis

Supplementary Figure 2 from Insulin-Like Growth Factor-I Regulates the Liver Microenvironment in Obese Mice and Promotes Liver Metastasis

Nm23-H1 transcriptionally down-regulates expression of the lysophosphatidic acid receptor EDG2 and this down-regulation is critical for Nm23-H1–mediated motility suppression in vitro. We investigated the effect of altered EDG2 expression on Nm23-H1–mediated metastasis suppression in vivo. Clonal MDA-MB-435–derived tumor cell lines transfected with Nm23-H1 together with either a vector control or EDG2 had similar anchorage-dependent and anchorage-independent growth rates in vitro. However, a 45- and 300-fold inhibition of motility and invasion (P < 0.0001), respectively, was observed in Nm23-H1/vector lines, whereas coexpression of EDG2 restored activity to levels observed in the parental line. Using fluorescently labeled cells and ex vivo microscopy, the capacity of these cells to adhere, arrest, extravasate, and survive in the murine lung over a 24-h time course was measured. Only 5% of Nm23-H1/vector–transfected cells were retained in the murine lung 6 h following tail vein injection; coexpression of EDG2 enhanced retention 8- to 13-fold (P < 0.01). In a spontaneous metastasis assay, the primary tumor size of Nm23-H1/vector and Nm23-H1/EDG2 clones was not significantly different. However, restoration of EDG2 expression augmented the incidence of pulmonary metastasis from 51.9% to 90.4% (P = 2.4 × 10−5), comparable with parental MDA-MB-435 cells. To determine the relevance of this model system to human breast cancer, a cohort of breast carcinomas was stained for Nm23-H1 and EDG2 and a statistically significant inverse correlation between these two proteins was revealed (r = −0.73; P = 0.004). The data indicate that Nm23-H1 down-regulation of EDG2 is functionally important to suppression of tumor metastasis. [Cancer Res 2007;67(24):11751–9]

Supplementary Table S7 from Cancers as Wounds that Do Not Heal: Differences and Similarities between Renal Regeneration/Repair and Renal Cell Carcinoma

CCL23, the human CCL9 orthologous.

Supplementary Methods, Tables 1-4, Figure 1 from Credentialing Preclinical Pediatric Xenograft Models Using Gene Expression and Tissue Microarray Analysis

Supplementary Figure 2 from Alterations in Gemin5 Expression Contribute to Alternative mRNA Splicing Patterns and Tumor Cell Motility

Supplementary Table 3.1 from Alterations in Gemin5 Expression Contribute to Alternative mRNA Splicing Patterns and Tumor Cell Motility

Supplementary Table 1 from Alterations in Gemin5 Expression Contribute to Alternative mRNA Splicing Patterns and Tumor Cell Motility

Supplementary Table S4 from Cancers as Wounds that Do Not Heal: Differences and Similarities between Renal Regeneration/Repair and Renal Cell Carcinoma

Myeloid cells, CCL9 and its effect on tumor phenotype.