MMP13, Birc2 (cIAP1) and Birc3 (cIAP2), Amplified on Chromosome 9, Collaborate with p53 Deficiency in Mouse Osteosarcoma Progression

Keywords: p53, osteosarcoma, MMP13, Birc2, Birc3

Array-based comparative genomic hybridization

The mouse BAC (bacterial artificial chromosome) arrays contained 19,000 BACs (printed in triplicate) covering the mouse genome with a resolution of less than 0.5 Mb. BAC array-CGH was performed on 41 p53+/− osteosarcomas and 10 p53+/− rhabdomyosarcomas as previously described (19). Normal genomic DNA from C57BL/6 mouse was used as diploid reference DNA, and mouse Cot-1 DNA was used to block repetitive sequences in sample probes and on BAC chips. Each sample/control pair was done twice by reciprocal labeling of sample or control with Cy5 or Cy3 dye to remove ratio artifacts. CGH data was analyzed as described in (19). Briefly, the fluorescent ratio representing the relative amounts of target sequences in the probe mix was analyzed by comparing the fluorescent intensity of corresponding individual spots after background subtraction. A control experiment comparing wild-type C57BL/6 and wild-type 129/SvEv genomic DNA copy number differences was performed in order to filter out strain-specific polymorphisms.

Isolation and culture of primary tumor cells

Tumor fragments were collected from p53+/− mice, washed twice with sterile PBS, minced, and incubated with 0.25% trypsin solution for two consecutive 15 minute incubations at 37°C with shaking. The cell solution was filtered through a 70 µm cell strainer (BD Falcon) to remove clumps, and the filtrate was centrifuged at 1500 rpm for 5 minutes at 4°C. Cells were collected and cultured in DMEM with 10% FBS, 1% penicillin-streptomycin at 37°C in a humidified incubator with 5% CO₂. Clonal populations of osteosarcoma cells were isolated by limiting dilution for both A+ (chromosome 9 amplicon positive) and A− (chromosome 9 amplicon negative) cell lines. The osteosarcoma origin of all lines was confirmed by alkaline phosphatase (ALP) staining.

Quantitative real-time PCR

Quantitative real-time PCR was performed with RT-3000 equipment (Corbett Research). Quantitation of genomic copy number was based on standard curves derived from sequential dilutions of genomic DNA from wild-type C57BL/6 mouse tail. For mRNA quantitation, total mRNA was extracted with RNeasy Mini Kit (QIAGEN) following the manufacturer’s instructions. Standard curves were performed by sequential dilutions of mRNA followed by reverse transcription with Superscript III First-strand Synthesis System (Invitrogen), and real-time PCR with iQ SYBR Green Super Mix (Bio-Rad) using gene-specific primers. After determination of the optimal reaction condition for the primer set, real-time PCR was done in triplicate for each sample, and expression of target genes was normalized to β-actin levels. Primer sequences used in the real-time PCR experiments are listed in the Supplementary Methods.

shRNA lentivirus construction, packaging and transduction

For MMP13, chemically synthesized siRNAs were purchased from Qiagen (#SI00177765 and #SI00177772), tested for efficient knockdown of siMMP13 compared to control siRNA (Ambion #4611). DNA oligonucleotides were synthesized based on the siRNA sequences with the best knockdown efficiency and were incorporated into the FG12 lentiviral vector, which contains GFP-expression sequences driven by a UbiC promoter (20). Viruses were packaged by co-transfecting 293T cells with FG12-shMMP13 vector plasmid, packaging constructs pRSVREV, pMDLg/pRRE, and the VSV-G expression plasmid pHCMVG. Viruses were harvested and transduced into osteosarcoma cells as described previously (20). Positively transduced cells were cell sorted for GFP expression. For Birc2, Birc3, and Yap1, virus packaging and transduction was done as previously described (12). Positively transduced clones were achieved by selection with puromycin at pre-determined concentrations (2ug/ml for A+ cell line and 4ug/ml for A− cell line). Real-time PCR confirmed the target gene (MMP13, Birc2, Birc3 or Yap1) knockdown in positively selected cells, and only those with knockdown efficiency of more than 75% were used in allograft studies.

Allograft study

Transduced cells were harvested, washed, counted and resuspended in HBSS (Invitrogen). 2×10⁶ of A+ or 0.5×10⁶ of A− cells were used for subcutaneous injection, by injecting dorsally above the scapula of 8–10 week Balb/c-nu/nu (Charles River Laboratories) mice. Tumor growth was monitored and measured every other day, and tumor volume (V) was determined by the formula V= (4/3)*π*[(L+W)/4]³, L being the longer cross-section and W being the shorter.

Western blotting

Tumor tissue or cell pellets were lysed in NP40 lysis buffer using PT100 homogenizer (Polytron). Equal amounts (25ug) of protein were separated on NuPAGE® Novex 4–12% Bis-Tris Gel (lnvitrogen) and transferred to PVDF membranes. The blots were probed with antibodies against MMP13 (Santa Cruz, #sc-30073, 1:500), Birc2 (12), Birc3 (Santa Cruz, #sc-7944, 1:100), Yap1 (12), or β-actin (Santa Cruz, #sc-1616, 1:2,000).

Immunohistochemistry

MMP13 staining was performed on the tumor sections with antibody from Santa Cruz (sc-30073). For cell proliferation experiments, BrdU solution (7mg/ml) was injected intraperitoneally into mice at 0.01 ml per gm of body weight. Mice were sacrificed 2 hours after injection and tumors were harvested, fixed with 4% paraformaldehyde, paraffin embedded and sectioned for imunohistochemistry. BrdU staining was performed with BrdU In-Situ Detection Kit (BD Biosciences #550803), and TUNEL staining was performed with In Situ Cell Death Detection Kit, Fluorescein (Roche #11 684 795 910), following manufacturer instructions. For the quantitation of BrdU staining and TUNEL staining, tumor sections from five mice were stained and counted for each group. Five fields from each tumor were randomly selected, both positively-strained cells and total cell number were counted, and the percentage was calculated by dividing positively-stained cell number by total cell number.

CT imaging

MicroCT imaging was performed on a Gamma Medica Xspect scanner at 50µm resolution at the BCM Mouse Phenotyping Core facility. Data were acquired with the system with X-ray tube voltage of 78kV(p), tube currents of 205 uA, and exposure matrix 512×512 views. A water phantom was included with each scan. Three-dimensional CT images of the tumors were reconstituted with Amira®.

Gene Expression Microarray Analysis

RNA was extracted from rhabdomyosarcoma or osteosarcomas (about 3mm cube) or mc3T3 cell (60mm dish) using the RNeasy Mini Kit (Qiagen). Following quality check of the RNA, microarray analysis was performed at the Baylor Microarray Core Facility (www.bcm.edu/mcfweb/) with Affymetrix Mouse Genome 430 2.0 Chips. Microarray data was analyzed with Genesifter software (www.genesifter.net).

Array-CGH identifies genome instability in p53+/− murine tumors

To identify copy number changes in the genomes of p53+/− sarcomas, we carried out microarray-based comparative genomic hybridization (array-CGH) analysis on genomic DNA isolated from 41 p53 +/− osteosarcoma and 10 rhabdomyosarcoma samples. For the 41 osteosarcomas, seven frequent regions of copy number gain or loss were observed (Table S1). Among these, three were regions of copy number gain and four were regions of copy number loss. Analysis of the mouse genome sequence within the regions of copy number losses revealed no obvious tumor suppressor candidates. However, two of the three regions of copy number gain had known candidate oncogenes and were on mouse chromosomes 9 and 15. Seven of 41 osteosarcomas had strong amplifications on chromosome 15 with the c-myc oncogene at the amplicon epicenter (Figure 1A). This was consistent with observations that human osteosarcomas also exhibit amplification and overexpression of c-myc (9, 21, 22). The chromosome 9 (band A1) amplicon was observed in 5 of 41 osteosarcomas and contains a cluster of 10 matrix metalloproteinase genes (Figure 1B,C,D). Moreover, the epicenter of this amplicon is near the Birc2 (cIAP1) and Birc3 (cIAP2) genes, which encode inhibitors of apoptosis (Figure 1D). As indicated earlier, DNA amplification centered on Birc2 and Birc3 has been observed in multiple human tumor types (12–17). Finally, the Yap1 gene, shown to be oncogenic in some contexts (12, 23), is also located in this amplicon.

Because of its previous association with various human tumors, we focused on the chromosome 9A1 amplicon, at chromosomal position 5–9 Mb (Figure 1D). The cluster of matrix metalloproteinase (MMP) genes within the amplicon is a family of extracellular endopeptidases that collectively cleave all the components of the extracellular matrix (ECM). MMP proteins may be important for osteosarcoma cell invasion if overexpressed (24, 25). The MMPs play important roles under both physiological and pathological conditions, including tumorigenesis, and are potential targets for cancer treatment (25–27).

Birc2, Birc3 and Yap1 are located in the chromosome 9 amplicon about one Mb downstream of the MMP cluster. Birc proteins, or the Baculoviral IAP Repeat-Containing proteins, are also called IAPs (Inhibitor of Apoptosis Proteins). This family is grouped by their characteristic BIR domains and is thought to be inhibitors of apoptosis (12, 28). Birc2 (cIAP1) and Birc3 (cIAP2) may regulate apoptotic pathways by multiple mechanisms. They bind caspases and Birc2 can affect the TNFα pathway by binding and ubiquitinating the TNFR-associated factor-2 (TRAF2) (29), whereas Birc3 preferentially binds and ubiquitinates TRAF1 (30). In addition, degradation of Birc2 has been shown to lead to NF-κB activation and TNFα secretion. The autocrine TNFα signaling in turn induces caspase 8 activation (31–33). Birc2 also affects the intrinsic apoptotic pathway by mediating the ubiquitination of Smac/DIABLO (34). Importantly, Birc2 acts as an oncogene in a mouse hepatocellular cancer model containing a similar chromosome 9A1 amplicon as described here (12).

Yap1, or Yes-Associated Protein 1, also resides on the chromosome 9A1 amplicon. Yap1 is a co-activator of p73-mediated apoptosis (35, 36), while others have shown that it is a potential oncoprotein (12, 23). Importantly, Yap1 has been shown to exhibit oncogenic activity in mouse hepatocellular cancers that contained similar chromosome 9A1 amplicons to those described here (12). Based on these findings, we decided to examine the roles of MMP13, Birc2, Birc3 and Yap1 in osteosarcomagenesis.

Southern blot hybridization of the osteosarcoma DNAs with MMP13 and MMP7 probes confirmed copy number gain in those tumors with chromosome 9A1 amplification measured by array CGH (Figure S1). Since these tumors were derived from p53+/− mice, we assessed p53 allele status, and found that the wildtype p53 allele was retained in some tumors, but was lost in cell lines derived from the same tumors (Figure S1). Historically, we have found that about half of all p53+/− tumors retain the wildtype p53 allele and about half undergo loss of the wildtype allele (37).

MMP13 is overexpressed in osteosarcomas

To determine whether a particular gene or set of genes in the chromosome 9A1 amplicon could be driving osteosarcomagenesis, we analyzed the RNA expression status of all genes within the chromosome 9A1 amplicon in both amplicon-positive and amplicon-negative osteosarcomas by microarray expression analysis and real-time PCR. MMP genes localized within and outside of this amplicon were first analyzed. A murine osteoblast cell line mc3T3, as well as normal bone, were used as controls for comparison. We found that only MMP13 and MMP12 (within the 9A1 amplicon), and MMP9 and MMP23 (outside the 9A1 amplicon) had high expression in osteosarcomas relative to the normal osteoblast control (Figure 2A, Figure S2, Figure S3). However, within the chromosome 9A1 amplicon, MMP1, MMP7, MMP8, MMP10 or MMP20 did not exhibit elevated RNA expression. This was confirmed by RNA microarray expression analysis on all of the MMP genes (Figure S2, Figure S3). Furthermore, MMP expression in the mouse osteosarcomas was consistent with corresponding gene expression in human osteosarcomas (except MMP19) (Figure 2A, Figure S3).

These results suggested that overexpression of the chromosome 9A1 amplicon-associated MMP13 gene might play a role in osteosarcoma progression. MMP13 (collagenase-3) expression is most abundant in adult bone compared to other tissues (38). MMP13-null mice show interstitial collagen accumulation and cartilaginous growth plate abnormalities. MMP13 knockdown increases apoptosis in squamous cell carcinomas (39). Real time RT-PCR analysis of MMP13 gene expression in 15 p53+/− osteosarcomas showed that virtually all of the osteosarcomas exhibited high RNA expression relative to osteoblast cells (Figure 2B). High MMP13 RNA expression was not dependent on gene amplification, as most tumors were diploid at the MMP13 locus (Figure 2B). High MMP13 RNA expression is also observed in many human osteosarcomas relative to normal human osteoblast cells, as measured by microarray expression analyses on 40 human osteosarcoma samples (Figure 2C). In contrast to high MMP13 expression in osteosarcomas, all six examined rhabdomyosarcomas exhibited low MMP13 expression. These expression patterns were confirmed at the protein level by immunohistochemistry staining of osteosarcoma and rhabdomyosarcoma sections (Figure 2D). No MMP13 staining was evident in rhabdomyosarcoma sections, while osteosarcoma sections showed both cytoplasmic and cell surface MMP13 staining. Other MMP genes did not show a pattern of elevated expression in human osteosarcomas (Figure S3).

Reduction of MMP13 expression decreases osteosarcoma growth rate

As a prelude to assessing the functional importance of high MMP13 expression in the murine osteosarcomas, we developed clonal cell lines from a primary osteosarcoma with chromosome 9A1 amplification (A+) and from primary osteosarcomas without this amplification event (A−). All clonal lines, when injected subcutaneously into immunodeficient Balb/c-nu/nu mice, formed osteosarcomas histopathologically indistinguishable from primary p53+/− osteosarcomas within several weeks following injection (Figure S4). Lentiviral vectors expressing two MMP13 shRNAs targeting different domains on MMP13 mRNA were constructed and transduced into 9A amplicon-positive and amplicon-negative osteosarcoma cells. Stably transduced cells were selected by flow cytometric cell sorting for GFP fluorescence (expressed only in vector-transduced cells). MMP13 mRNA levels in transduced osteosarcoma cells were reduced over 80% compared to empty vector-transduced cells (data not shown). Equal numbers of MMP13 shRNA-transduced or empty vector-transduced cells were subcutaneously injected into the dorsal flanks of nude mice, and tumor volume was monitored over time. Osteosarcomas formed by the shMMP13-transduced A+ and A− osteosarcoma cells grew at significantly reduced rates compared to their empty vector-transduced counterparts, suggesting that MMP13 plays an important role in osteosarcoma progression (Figure 3A,C, Figure S5). Western blots confirmed reduced MMP13 protein expression in the shRNA-expressing tumor allografts compared to empty vector-containing tumors (Figure 3B and 3D, upper panels).

The reductions in tumor growth rates caused by MMP13 knockdown could be a result of either reduced tumor cell proliferation or increased tumor cell apoptosis. To examine the effect of MMP13 shRNA on proliferation in vivo, we injected tumor bearing nude mice with bromodeoxyuridine (BrdU) for 3 hours prior to euthanasia and measured cell proliferation by staining tumor sections with an antibody directed against BrdU. Counting of stained cells provides an indicator of cells in S phase. Using this proliferation assay, no significant differences were observed between tumors with and without MMP13 shRNA expression, regardless of amplicon status (Figure 4A).

To compare rates of apoptosis in the transplanted tumors with and without MMP13 knockdown, we performed a TUNEL assay for apoptotic cells on tumor sections of the various tumors. Counting of stained apoptotic cells in microscopic fields from multiple tumor sections revealed that both A+ and A− transplanted MMP13 shRNA vector-transduced osteosarcomas displayed significantly higher levels of apoptosis than their empty vector-transduced counterparts (Figure 4B). Thus, reduction of MMP13 in the transplanted osteosarcomas increases rates of tumor cell apoptosis, leading to decreased overall tumor growth rates.

Histopathological examination of the transplanted tumors revealed an additional effect of MMP13 knockdown. The stained sections of both A+ and A− tumors transduced with MMP13 shRNA vectors showed reduced cellular density and increased osteoid component compared to control vector transduced tumors, suggesting a higher level of differentiation (Figure 4C). In osteosarcomas, differentiation has been considered as antithetical to the oncogenic process, and loss of differentiation has powerful prognostic significance (40). To quantitatively determine whether bone matrix and tumor density were increased when MMP13 expression was reduced, computed tomography (CT) scans were performed, and the tumor density was measured three-dimensionally by Amira® and converted into Hounsfield units. Both A+ and A− shMMP13-expressing tumors were denser than their vector-expressing controls (p<0.05) (Figure 4D, Figure S5). Since differentiated osteosarcoma cells secrete more osteoid than proliferating cells, and mineralized osteoid is the major component of the bone matrix, these results indicate that reduction of MMP13 induces a more differentiated tumor cell state.

Reduction of Birc2 and Birc3 enhances apoptosis and reduces tumor growth rates in amplicon positive osteosarcomas

The anti-apoptotic genes Birc2 (cIAP1) and Birc3 (cIAP2) are located near the epicenter of the chromosome 9A1 amplicon. Real-time PCR and RNA microarray analysis of the p53+/− osteosarcomas and rhabdomyosarcomas showed high expression of Birc2 and Birc3 mRNA expression in virtually all tumors (Figure 5A, Figure S6). Moreover, high Birc3 expression was observed in most human osteosarcomas (Figure 5A, lower right panel). To determine whether Birc2 and/or Birc3 influences osteosarcoma growth, lentiviral vectors expressing shRNAs against Birc2 or Birc3 (or empty vectors) were transduced into clonal A+ and A− osteosarcoma cell lines and stably transduced lines were selected in puromycin (lentiviral vectors in these experiments encoded puromycin resistance genes). After allografting into nude mice, tumor growth was monitored. Birc2 or Birc3 shRNA-expressing A+ tumors grew significantly slower than empty vector-transduced A+ tumors (Figure 5B, upper panels). In contrast, Birc2 and Birc3 shRNA did not significantly affect the growth rate of the A− osteosarcoma cells (Figure 5B, lower panels). TUNEL experiments showed that Birc2 or Birc3 shRNA-expressing A+ osteosarcoma sections contained significantly increased apoptotic cell percentages (Figure 5C), while no significant differences in apoptotic cell percentages were noted in shRNA vector transduced A− osteosarcoma sections. These results indicated that high Birc2 and high Birc3 expression contributed to tumor growth and reduced apoptosis only in osteosarcomas with the chromosome 9A1 amplicon.

Yap1 knockdown does not affect osteosarcoma growth rate

The Yap1 gene is directly adjacent to Birc2 and Birc3 and is co-amplified with these anti-apoptotic genes in the chromosome 9A1 amplicon containing osteosarcomas. Because Yap1 has been shown to be oncogenic in some contexts (12, 23), we analyzed Yap1 RNA expression by real time RT-PCR and microarray analyses. We found that Yap1 mRNA was highly expressed only in the mouse p53+/− osteosarcomas that contained the chromosome 9A amplicon (Figure 6A, Figure S7). Osteosarcomas without this amplification event and rhabdomyosarcomas generally did not show elevated expression of this gene. In human osteosarcomas, Yap1 expression was consistently downregulated relative to osteoblasts (Figure 6B). We then transduced A+ and A− osteosarcoma cells with a Yap1 shRNA lentiviral vector (or control vector), selected stable transductants and performed allograft studies. No significant changes in tumor growth rate between Yap1 shRNA and empty vector transduced tumors, either A+ or A−, were observed (Figure 6C,D). However, despite reduction of Yap1 RNA levels by the shRNA vectors in the osteosarcoma cells (Figure S8), western blot analyses of the tumors indicated that Yap1 protein in all tumors was undetectable (data not shown). These results argue that Yap1 expression is not significant in altering osteosarcoma cell growth in either the A+ or A− transplanted osteosarcomas.

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