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. 2015 Sep;5(9):920-31.
doi: 10.1158/2159-8290.CD-15-0125. Epub 2015 Jun 17.

A Genome-Wide Scan Identifies Variants in NFIB Associated with Metastasis in Patients with Osteosarcoma

Lisa Mirabello  1 , Roelof Koster  2 , Branden S Moriarity  3 , Logan G Spector  4 , Paul S Meltzer  5 , Joy Gary  6 , Mitchell J Machiela  2 , Nathan Pankratz  7 , Orestis A Panagiotou  2 , David Largaespada  3 , Zhaoming Wang  8 , Julie M Gastier-Foster  9 , Richard Gorlick  10 , Chand Khanna  5 , Silvia Regina Caminada de Toledo  11 , Antonio S Petrilli  11 , Ana Patiño-Garcia  12 , Luis Sierrasesúmaga  12 , Fernando Lecanda  12 , Irene L Andrulis  13 , Jay S Wunder  13 , Nalan Gokgoz  13 , Massimo Serra  14 , Claudia Hattinger  14 , Piero Picci  14 , Katia Scotlandi  14 , Adrienne M Flanagan  15 , Roberto Tirabosco  16 , Maria Fernanda Amary  16 , Dina Halai  16 , Mandy L Ballinger  17 , David M Thomas  18 , Sean Davis  5 , Donald A Barkauskas  19 , Neyssa Marina  20 , Lee Helman  5 , George M Otto  21 , Kelsie L Becklin  22 , Natalie K Wolf  22 , Madison T Weg  21 , Margaret Tucker  2 , Sholom Wacholder  2 , Joseph F Fraumeni Jr  2 , Neil E Caporaso  2 , Joseph F Boland  8 , Belynda D Hicks  8 , Aurelie Vogt  8 , Laurie Burdett  8 , Meredith Yeager  8 , Robert N Hoover  2 , Stephen J Chanock  2 , Sharon A Savage  2
Affiliations

A Genome-Wide Scan Identifies Variants in NFIB Associated with Metastasis in Patients with Osteosarcoma

Lisa Mirabello et al. Cancer Discov. 2015 Sep.

Abstract

Metastasis is the leading cause of death in patients with osteosarcoma, the most common pediatric bone malignancy. We conducted a multistage genome-wide association study of osteosarcoma metastasis at diagnosis in 935 osteosarcoma patients to determine whether germline genetic variation contributes to risk of metastasis. We identified an SNP, rs7034162, in NFIB significantly associated with metastasis in European osteosarcoma cases, as well as in cases of African and Brazilian ancestry (meta-analysis of all cases: P = 1.2 × 10(-9); OR, 2.43; 95% confidence interval, 1.83-3.24). The risk allele was significantly associated with lowered NFIB expression, which led to increased osteosarcoma cell migration, proliferation, and colony formation. In addition, a transposon screen in mice identified a significant proportion of osteosarcomas harboring inactivating insertions in Nfib and with lowered NFIB expression. These data suggest that germline genetic variation at rs7034162 is important in osteosarcoma metastasis and that NFIB is an osteosarcoma metastasis susceptibility gene.

Significance: Metastasis at diagnosis in osteosarcoma is the leading cause of death in these patients. Here we show data that are supportive for the NFIB locus as associated with metastatic potential in osteosarcoma.

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Conflict of interest statement

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Regional plots of the discovery and combined association results, recombination hotspots and linkage disequilibrium (LD) for the 9p24.1:14,081,842-14,398,982 osteosarcoma metastasis susceptibility locus
Y-axes represent the statistical significance (−log10 transformed P values) of SNP association results from a trend test (left) and the recombination rate (right). SNPs are color-coded based on pairwise linkage disequilibrium (r2) with the most statistically significant SNP. The most statistically significant SNP is labeled and shown in purple. Allelic P values generated by SNPTEST using score method (two-sided). Physical locations of the SNPs are based on NCBI human genome build 36, and gene annotation was based on the NCBI RefSeq genes from the UCSC Genome Browser. (A.) European discovery set, (B.) meta-analyzed European discovery and replication sets (imputed), (C.) meta-analyzed European discovery/replication sets, plus African and Brazilian ancestry cases (imputed). (D.) Kaplan-Meier curve of cumulative (cum) survival for osteosarcoma patients by rs7034162 genotype.
Figure 2
Figure 2. Relationship between the genotype of the metastasis-associated SNP, rs7034162, and expression of NFIB and other nearby protein-encoding genes FREM1, ZDHHC1, and MPDZ in osteosarcoma cell lines and tumors
Significance is based on linear regression comparing the distribution of NFIB, FREM1, ZDHHC1, and MPDZ expression between the TT homozygous non-risk genotype (N=34) of rs7034162 genotypes to the heterozygous risk AT (N=10) and the homozygous risk AA genotypes (N=2). These analyses were based on the publically available genotype and expression data from 17 osteosarcoma cell lines and 29 osteosarcoma tumors (20).
Figure 3
Figure 3. NFIB expression correlates with invasion and migration potential of human osteosarcoma cells
(A.) NFIB expression in OSA, HOS and U2OS cells was determined 48 hours after transfection with control siRNA (si-NEG) or siRNA targeting NFIB (si-NFIB). Graphs show relative expression compared with control treated U2OS cells. The rs7034162 genotype is shown for the osteosarcoma cell lines: U2OS and HOS cells carry the homozygous non-risk allele (TT), and OSA cells carry the homozygous risk allele (AA). (B.) 48 hours after NFIB suppression, cells were evaluated for migratory potential. Cells that invaded through the matrigel-coated membrane inserts were stained with crystal violet, air-dried and bright field images were taken (inset photographs depict staining intensity differences). To quantify cell invasion/migration, the inserts were treated with 200μl of methanol and the absorbance measured in triplicate at 560 nm. Invading cells are depicted relative to control treated U2OS cell. Results of at least three experiments in triplicate are expressed as mean ± SD. The invasion and migration rate of each cell line was inversely correlated with NFIB expression levels.
Figure 4
Figure 4. Increased migration and podia formation in NFIB suppressed human osteosarcoma cells
Subconfluent osteosarcoma cells were infected with the indicated siRNAs, and 48 hours post-infection wounds were made on the monolayers. Bright field images were taken at time 0 and 16, 24, and/or 30 hours (h). The wound healing cell migration and filamentous actin staining assays were done in triplicate for three cell lines: OSA (A.), HOS (B.) and U2OS (C.). The rs7034162 genotype is shown for the osteosarcoma cell lines: U2OS and HOS cells carry the homozygous non-risk allele (TT), and OSA cells carry the homozygous risk allele (AA). A representative example of the three independent experiments performed is shown. The cells showed increased migration after treatment with siRNA against NFIB (si-NFIB) compared with the si-NEG control, and the filamentous actin staining (far right) shows increased podia formation in the cells treated with si-NFIB. (D.) Soft agar colony formation assay in OSA, HOS, and U2OS cell lines. The HOS and OSA cells showed a significant reduction in colony formation after over-expressed of NFIB compared with the Luciferase (Luc) control.
Figure 5
Figure 5. Nfib insertions from a Sleeping Beauty (SB) transposon screen for osteosarcoma in mice, and expression in mouse tumors
(A.) Diagram depicts T2/Onc insertion sites in Nfib from the SB transposon screen (23). Black and red arrows represent T2/Onc insertions identified in tumors from Trp53-SB and SB animals, respectively. Gray and light red arrows represent T2/Onc insertions identified in metastases from Trp53-SB and SB animals, respectively. The direction of the arrows represents the direction of the mouse stem cell virus (MSCV) LTR and splice donor (SD) sequence of T2/Onc. RT-PCR analysis of Nfib (B.) and Igfbp5 (C.) expression in tumors with and without transposon insertions in Nfib. (D.) Semi-quantitative immunohistochemical scores for nuclear NFIB labeling in sections of mouse osteosarcomas with Nfib insertions and in normal adjacent osteoblasts. (E.) Immunohistochemical staining (brown) for NFIB in sections containing mouse osteosarcomas with or without an Nfib insertion and adjacent, normal osteoblasts to evaluate tissue-specific changes in NFIB expression. A two-tailed ttest was used to estimate the P-values for the differences in Nfib and/or Igfbp5 expression.
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