MicroRNAs as Driver Genes in the Pathogenesis of Osteosarcoma
Osteosarcoma (OS) is the most common type of bone cancer diagnosed in children and adolescents. Significant improvement in overall survival of OS patients was achieved after implementation of relatively effective chemotherapy in 1970s, but a third of patients still die during 5 years after diagnosis. Overall 5 years survival drops even further to about 20% for OS patients with metastases at diagnosis. Therefore, improvements in therapy of OS patients, particularly, those with the metastatic disease are needed. Advances in OS treatment are inconceivable without better understanding of molecular mechanisms of osteosarcomagenesis and, especially, metastatic processes in this kind of cancer.
MicroRNAs (miRNAs) are small non-coding RNAs that have been widely implicated in carcinogenesis. Disease-associated changes in miRNA expression in OS samples were determined in our laboratory and by other independent groups. Thus, miRNAs are anticipated to play significant roles in OS development. OS-related functional studies of miRNAs are growing in number but still fragmental. Uncovering of a complex network of miRNAs and functional interactions with protein-encoding genes, disturbance of which leads to OS development, would provide a basis for rational design of new therapies for OS.
Purpose of Investigation
Previously, we reported an OS-specific microRNA signature. Of particular interest, we found higher expression of miR-27a that is associated with clinical metastatic disease. Our working hypothesis was that miR-27a plays major roles in driving metastasis in OS. To test this, we set to 1) determine the effect of miR-27a manipulation on OS traits both in cell culture and in mice and 2) decipher the molecular mechanism by which miR-27a acts as a metastatic-miR.
Summary of Findings
Our study demonstrates that overexpression of miR-27a/miR-27a*, a microRNA pair derived from a single precursor, promotes pulmonary OS metastases formation. By contrast, sequestering miR-27a/miR-27a* by sponge technology suppressed OS cells invasion and metastases formation. miR-27a/miR-27a* directly repressed CBFA2T3 expression among other target genes. We demonstrated that CBFA2T3 is downregulated in majority of OS samples and its overexpression significantly attenuated OS metastatic process mediated by miR-27a/miR-27a* underscoring CBFA2T3 functions as a tumor suppressor in OS. These findings establish that miR-27a/miR-27a* pair plays a significant role in OS metastasis and proposes it as a potential diagnostic and therapeutic target in managing OS metastases.
Results
MiR-27a and miR-27a* co-overexpression promotes metastatic properties of OS cells.
We first determined the expression level of miR-27a in OS and OS cells. We found elevated levels of miR-27a in OS cases (Figure 1) and differential expression of miR-27a in all OS cells with a prominently high level of expression in LM7 cells, a known metastatic cell line. We also found that miR-27a*, the passenger strand derived from the same precursor as miR-27a, display high levels in metastatic OS cells. Since LM7 cells are of high metastatic potential and were originally derived from low metastatic potential SAOS2 cells, we mainly studied miR-27a and miR-27a* pro-metastatic effects in these two cell lines.
Manipulating the levels of MIR-27a gene, which encodes for both miR-27a and miR-27a*, revealed their prometastatic properties. This was evident by changes in cell morphology, colony formation ability in 2-D and 3-D cultures, and in migration and invasion potential (Figure 2). Interestingly, no change in proliferation rates were observed.
Furthermore, manipulated cells were examined for growth and seeding metastasis after implantation in immunocompromised mice.
The results showed that miR-27a and miR-27a* co-overexpression boosts SAOS2 cell metastatic properties suggesting pro-metastatic functions of these miRNAs while inactivation of miR-27a or miR-27a* significantly reduce the metastatic ability of LM7 cells without affecting primary tumor growth (Figure 3). Therefore, our findings suggest that both miRNAs – miR-27a and miR-27a* – possess pro-metastatic functions in OS cells.
CBFA2T3 is a target of miR-27a and miR-27a*.
In order to address the molecular mechanism underlying the pro-metastatic functions of miR-27a and miR-27a*, we studied the effect of overexpressing and inactivation of these miRNAs on expression of several putative miR-27a targets, which were identified as downregulated in OS versus healthy bones, from our previous studies. A candidate gene, which is downregulated in OS and is putatively targeted by miR-27a as predicted by TargetScan, is CBFA2T3. Indeed, gene expression of CBFA2T3 was altered by MIR-27a gene overexpression. Therefore, we decided to study this miR-27a target in greater details.
We first examined CBFA2T3 expression in several OS cell lines and found that its expression is very low in most OS cell lines but higher in SAOS2 cells. To further examine that miR-27a directly targets CBFA2T3, we tested the effect of miR-27a on CBFA2T3 protein expression. We found that the CBFA2T3 protein levels were significantly downregulated after expression of miR-27a mimetics. Moreover, Synthetic miR-27a precursor (containing miR-27a and miR-27a*) significantly reduces firefly luciferase activity when co-transfected with CBFA2T3-3’UTR while a mutation, which affects miR-27a binding site, nullifies this effect. Altogether, this suggest that miR-27a and miR-27a* can directly regulate CBFA2T3 expression through their binding sites in the 3’-UTR of CBFA2T3.
CBFA2T3 acts as a tumor suppressor in OS cells and its overexpression counteracts miR-27a overexpression in SAOS2 cells.
We next set to determine whether CBFA2T3 is a potential tumor suppressor in OS. We first examined its expression level, using immunohistochemistry, in OS and chondrosarcoma tissue microarrays using anti-CBFA2T3 antibody. We found that the CBFA2T3 protein is undetectable or reduced in majority of OS samples.
In order to directly address the question whether CBFA2T3 functions as a tumor suppressor in OS, we generated CBFA2T3-OS expressing cells and examined their behavior in vitro and in vivo. We found that overexpression of CBFA2T3 led to significant decrease in cell growth, colony formation ability and attenuated OS cells invasiveness and motility (Figure 4).
We next addressed whether overexpression of CBFA2T3 inhibits metastatic traits in vivo. Control or CBFA2T3-expressing OS cells were intratibialy-injected and lung metastatic foci were quantified. We found that indeed CBFA2T3 overexpression decreased metastatic potential of HOS cells in vivo without affecting growth of primary tumors (Figure 4). Therefore, CBFA2T3 exerts anti-metastatic properties in OS cells.
Conclusion
Overall, our findings of miR-27a and miR-27a* acting as pro-metastatic miRNAs contribute to a growing body of evidence that star miRNAs, which were originally mostly viewed as degraded byproducts, are functional and might have driver function in the tumorigenesis process. We also for the first time provide evidence that CBFA2T3 acts as a tumor suppressor in OS cells suggesting that deregulated CBFA2T3 expression by miR-27a and miR-27a*, at least in part, is accountable for their pro-metastatic action in OS. The presented data further suggest the potential of miR-27a and miR-27a* pair as diagnostic markers and therapeutic targets for OS.
Please see our manuscript recently published in Oncotarget, supported by the Liddy Shriver Sarcoma Initiative.
By Rami Aqeilan, PhD
Hebrew University of Jerusalem
References
1. Ward E, DeSantis C, Robbins A, Kohler B, Jemal A. Childhood and adolescent cancer statistics, 2014. CA Cancer J Clin. 2014 Mar-Apr;64(2):83-103.
2. Ottaviani G, Jaffe N. The epidemiology of osteosarcoma. Cancer Treat Res. 2010; 152: 3-13.
3. Ameres SL, Zamore PD. Diversifying microRNA sequence and function. Nat Rev Mol Cell Biol. 2013; 14: 475-488.
4. Ha M, Kim VN. Regulation of microRNA biogenesis. Nat Rev Mol Cell Biol. 2014; 15: 509-524.
5. Calin GA, Croce CM. MicroRNA signatures in human cancers. Nat Rev Cancer. 2006; 6: 857-866.
6. Jones KB, Salah Z, Del Mare S, Galasso M, Gaudio E, Nuovo GJ, Lovat F, LeBlanc K, Palatini J, Randall RL, Volinia S, Stein GS, Croce CM, et al. microRNA signatures associate with pathogenesis and progression of osteosarcoma. Cancer Research. 2012; 72: 1865-1877.
7. Pan W, Wang H, Jianwei R, Ye Z. MicroRNA-27a promotes proliferation, migration and invasion by targeting MAP2K4 in human osteosarcoma cells. Cell Physiol Biochem. 2014; 33: 402-412.
8. Gruber TA, Larson Gedman A, Zhang J, Koss CS, Marada S, Ta HQ, Chen SC, Su X, Ogden SK, Dang J, Wu G, Gupta V, Andersson AK, et al. An Inv(16)(p13.3q24.3)-encoded CBFA2T3-GLIS2 fusion protein defines an aggressive subtype of pediatric acute megakaryoblastic leukemia. Cancer Cell. 2012; 22: 683-697.
9. Masetti R, Pigazzi M, Togni M, Astolfi A, Indio V, Manara E, Casadio R, Pession A, Basso G, Locatelli F. CBFA2T3-GLIS2 fusion transcript is a novel common feature in pediatric, cytogenetically normal AML, not restricted to FAB M7 subtype. Blood. 2013; 121: 3469-3472.
10. Cleton-Jansen A-M, Callen DF, Seshadri R, Goldup S, McCullum B, Crawford J, Powell J A, Settasatian C, van Beerendonk H, Moerland EW, Smit VT, Harris WH, Millis R, et al. Loss of heterozygosity mapping at chromosome arm 16q in 712 breast tumors reveals factors that influence delineation of candidate regions. Cancer Res. 2001; 61: 1171-1177.
Copyright © 2015 Liddy Shriver Sarcoma Initiative.
MicroRNAs as Driver Genes in the Pathogenesis of Osteosarcoma
Osteosarcoma (OS) is the most common type of bone sarcoma and remains a too-frequent cause of death during childhood and adolescence.1 Significant improvement in overall survival of OS patients was achieved after implementation of cytotoxic chemotherapy in the 1970s, but a third of patients still die within 5 years of diagnosis.1 Hence, there is urgent need for further improvement in OS treatment. MicroRNAs (miRNAs) are small non-coding RNAs that have been widely implicated in carcinogenesis.2 Disease-associated changes in miRNA expression in OS samples were determined in our laboratory3 and by other independent groups.4-6 Thus, miRNAs are anticipated to play significant roles in OS development. OS-related functional studies of miRNAs are growing in number but still fragmental. Uncovering of a complex network of miRNAs and functional interactions with protein-encoding genes, disturbance of which leads to OS development, would provide a basis for rational design of new therapies for OS.
Of particular interest, some of these miRNAs were found to be altered in metastatic OS including miR-27a and miR-181c*. However, little is known about how these miRNAs are regulated or their molecular targets in OS. It can be anticipated that some of these miRNA genes with altered expression in OS are drivers and play causative roles in the genesis and progression of this cancer type. Hence, precise roles of these miRNAs, in particular miR-27a, in OS cell proliferation, survival, invasion, metastatic potential and resistance to chemotherapy will be examined in this study.
The present research is designed to decipher one complex network of miRNAs and their functional interactions with protein-encoding genes that underlie OS development and progression. This goal will be accomplished through:
- Identifying miR-27a family members (including miR-23a and miR-24-2) with altered expression in OS that exhibit oncogenic properties
- Determining whether miR-27afamily members (hereafter referred to as miR-27s) are upstream drivers in the pathogenesis and progression of OS
- Identifying functionally relevant targets of miR-27a and
- Proposing anti-miRNAs that could potentially be used as therapeutic targets and biomarkers.
Taken together, our study will reveal driver miRNAs that are associated with pathogenesis of OS as well as critical pre-treatment biomarkers of metastasis and responsiveness to therapy.
Aims of This Study
Earlier work in our laboratory led to identification of miRNA genes with altered expression in OS in comparison with normal bones.3 To further examine the specific roles of miRNAs in osteosarcomagenesis, we propose to:
1. Determine whether miR-27s play a driver role in OS.
We hypothesize that altered miR-27s expression impacts OS development and metastasis. Expression of miR-27s will be determined in OS cells and in patient samples. MiR-27s overexpression and/or inactivation with miR sponges in OS cell lines with subsequent assessment of cell proliferation, survival, invasion, metastatic potential and chemotherapy resistance will be applied to accomplish this aim.
2. Identify OS-relevant targets of miR-27s.
We hypothesize that miRNAs with demonstrable biological impact on the character of OS will also have discernible impact on the expression of predicted target genes. Taking into account the fact that each miRNA can regulate, on average, the expression of 100–200 target genes,2 it will be necessary to gain insight into the biological role of miR-27s and identify the full repertoire of its mRNA and miRNA targets. To achieve this aim, we shall combine affymetrix cDNA profiling3 and computational analysis to identify and validate potential targets. We will overexpress and/or silence putative miRNAs’ (including thoseidentified from Aim 1) and targets in OS cell lines with consequent assessment of relevant cell properties both in vitro and in vivo. We will also examine whether upstream driver miR-27s target expression of downstream miRNAs that we identified previously.3
Research Design
Determine whether miR-27s is a driver metastatic gene in OS.
Preliminary studies identified an array of miRNAs that associate with OS.3 These miRNAs were identified using RNA isolated from incisional OS biopsies and normal bones from trauma patients making our study unique in its nature.3 Of particular interest, miR-27a was found to be significantly upregulated in metastatic OS. Our preliminary data suggest that miR-27s behaves as a pro-metastatic gene in OS. Both in vitro and in vivo traits of OS cells were affected upon overexpression of miR-27a (Figure 1).
To determine if other members are also affected, we will perform qRT-PCR analysis of miR-27s (including miR-23a and miR-24-2) using RNA isolated from OS cells and patient samples (used in our paper3 and newly isolated from FFPE). Furthermore, we shall use an independent public available miRNA datasets ([ArrayExpress,6 for example) to investigate the levels of these miRs. Next, we will examine which of these validated miRNAs function as drivers in the pathogenesis and/or progression of OS. Lentiviral vectors to overexpress each miRNA and/or miRNA sponges to suppress their function will be generated. OS cell lines (HOS, KHOS, 143B, LM-7) will be transduced with these lentiviruses and tested with regard to proliferation, migration, Boyden chamber invasion, soft agar colony formation and apoptotic assays, which are routinely performed in our laboratory.3,7 Assay of lung metastases after tail vein (IV) and orthotopic intratibial (IT) injection of transduced OS cells in immunocompromised mice will also be conducted. These models will reveal whether miR-27s affect metastasis (IT) or colonization (IV) in vivo.
Since aggressive metastatic OS is usually associated with chemoresistance, we will examine whether high levels of miR-27s family member associate with chemoresistance in OS. Methotrexate, doxorubicin and platinum-based chemotherapies are the main drugs utilized in OS treatment.1 To examine the effects of these miRNAs in OS chemosensitivity, miR-27s-depleted OS cells, using sponge constructs, will be generated. Sensitivity to methotrexate and doxorubicin will be assessed using XTT proliferation assays, clonogenic assays and in vivo tumorigenesis assays using NOD-SCID mice. Validation of each miRNA's role in this context will further our understanding of OS-related chemoresistance mechanisms and will suggest whether expression of certain miRNAs can be used as biomarkers for prediction of chemoresistance, a major pitfall in OS management. If successful, the ability of miR-27s anti-mimics will be employed in future in vivo studies to determine its clinical and therapeutic potential in OS treatment and metastasis eradication.
Identify OS-relevant targets of driver miRNAs.
Gaining insight into the biological role of miR-27s and identify the full repertoire of its mRNA and miRNA targets is our second goal in this proposal. Through studying the molecular mechanism of miR-27a, we shall decipher the relevant mRNA targets that are regulated by miR-27a either directly or indirectly. Uncovering these targets will certainly help identify the pathways regulated by these miRNAs. To address these points, we shall modulate miR-27a by overexpression in SaOS2 cells, which express low levels of endogenous miR-27a, or deplete it in LM-7 cells, which express abundant levels of miR-27a. Total RNA and protein lysates will be collected and analyzed by RNA sequencing and mass spectrometry (MS), respectively. Significant hits will be validated by real-time PCR and immunoblotting. Enriched pathways will be determined using bioinformatics tools such as David and KEGG.
Expression of predicted targets of miR-27a that are downregulated in OS cases, as assessed by cDNA profiling, were found to be downregulated in OS cells following miR-27a overexpression (Figure 2).
We will focus our work on those targets that were consistently affected in more than one OS cell type and those that changed at the RNA or protein levels as described above. For example, hCBFA2T3 levels are downregulated upon miR-27a expression. Furthermore, miR-27a inhibition using miR-27a sponges is associated with upregulation of endogenous CBFA2T3 expression in LM-7 OS cell line, as assessed by qRT-PCR (Figure 3).
CBFA2T3 was suggested to be a putative tumor suppressor in breast cancer since the gene is located at 16q24.3, a region that is associated with loss of heterozygosity in breast cancer. In fact, reduced expression in breast cancer primary tumors and tumor suppressor properties in in vitro assays were observed.8 In addition, CBFA2T3 is involved in translocations in hematopoietic malignancies.9,10 Of note, no tumorogenesis was reported in CBFA2T3 knockout mice.11 Nonetheless, CBFA2T3 is a promising target of miR-27a and its role in miR-27a pro-metastatic and pro-survival action in OS cells will be tested.
Additional experiments to verify that CBFA2T3, or any other identified target, is indeed a direct target of miR-27a will be conducted. An intact or mutated 3’-UTR region of CBFA2T3 cDNA with putative miR-27a binding sites (Figure 4) will be cloned 3’ of the firefly luciferase reporter gene (pGL-3). Effects of miR-27s on expression of the reporter with and without putative miR-27 binding sites of CBFA2T3 cDNA will be studied to determine whether CBFA2T3 is a direct target of miR-27s in these cells.
Lentiviruses for overexpression of CBFA2T3 and CBFA2T3 shRNA will be generated. OS cell lines expressing CBFA2T3 cDNA or shRNA will be generated, according to endogenous levels. Both proliferation and metastatic properties of CBFA2T3-overexpressing OS cells will be compared to control OS cells in XTT, soft agar colony formation, cell migration and Boyden chamber invasion assays. If any difference is noticed, then intratibial and intravenous cell injections in immunosuppressed mice will be also conducted to determine the effects of CBFA2T3 overexpression on primary tumor growth and pulmonary metastases. Results of these experiments will clarify whether CBFA2T3 expression down-regulation contributes to the miR-27a oncogenic potential. The effect of other members of the miR-27s family on CBFA2T3 expression similar to the approach above. In the same venue other novel targets of miR-27s will be assessed in the same manner.
If functional significance of CBFA2T3 down-regulation by miR-27s will be verified in the first set of experiments then we will test whether down-regulation of CBFA2T3 expression gives a similar readout to miR-27s overexpression functional outcomes. To this end we will transduce the OS cells expressing miR-27s with lentivirus of CBFA2T3 or control cDNA. Transduced cells will be compared in cell migration, Boyden chamber invasion, and soft agar colony formation assays. In the case of encouraging results, experiments with cell injections in immunosuppressed mice will be performed. These experiments will clarify whether upregulation of CBFA2T3 can functionally compensate for increased levels of miR-27s expression. These data will be further validated in OS patient samples using RNA isolated from OS cells and patient samples (used in our paper3 and newly isolated from FFPE) and using an independent public available miRNA datasets.6 In our recently published paper, we have used 29 samples collected from fresh tumor and control samples. We shall also aim to isolate additional RNA from ~50 FFPE tissues from primary OS with known clinical and response information. Future studies shall aim to further increase the number of cases analyzed with known clinical and response behavior.
Since miRNAs are known to target on average 200 genes, we speculate that there will be other functionally relevant genes targeted by miR-27s. To explore other possible protein-coding targets for miR-27s, we will use our Affymetrix array-derived mRNA expression data in OS,3 outcome of RNA seq and MS analysis experiments from above and computational approaches such as Sanger, miRanda, PicTar, Targetscan. While no single method is perfect, we will consider those target that are predicted by more than one tool. We will choose those putative miR-27s targets, whose mRNA expression negatively correlates with miR-27s expression in OS samples, and study them analogous to CBFA2T3.
Of major significance, we will be interested in identifying potential common targets of miR-27s, the expression of which is inversely correlated with chemotherapeutic treatment response.(3 and new OS cases) Identifying protein-coding genes that are targeted by miR-27s will facilitate a better understanding of the molecular basis of chemoresistance in OS. Novel target genes identified in this context will be further investigated in assays to address chemosensitivity as outlined in Aim 1. Targets will be manipulated in OS cells in the presence or absence of differential expression of miR-27s. Effect on survival and apoptosis will be explored. If proven in vitro, in vivo experiments will be designed to treat mice with manipulated cells and chemotherapeutic drugs.
Another possibility that we will test is whether miR-27s acts as upstream miRNAs affecting expression of (target) downstream ones. Changes in expression of endogenous miRNAs upon overexpression of miR-27a will be further validated by quantitative RT-PCR and/or northern blot. If changes in expression or functional levels of an miR-27a will cause appropriate changes in expression of other miRNAs then it will suggest that the miR-27a works upstream of the other miRNAs. Upstream miRNAs are more likely to play causative (driver) roles than downstream ones. Success of these experiments will provide proof of principle for the fact that some miRNAs indeed function upstream of others and can drive tumorigenesis.
Impact Statement and Future Directions
Success of the proposal will uncover a complex network of miRNAs and functional interactions with protein-encoding genes, disturbance of which leads to OS development. The outcome of the current proposal shall pave the road for genetic studies addressing the role of miR-27s in osteosarcomagenesis using mouse models. Our study will also provide a basis for rational design of new therapies for OS; this shall include use of anti-mimics in pre-clinical animal models for OS. Future research will further implement strategies of use of miRNAs (including miR-27a) for diagnosis and prognosis in serum/plasma of OS patients.
By Rami Aqeilan, PhD
Hebrew University of Jerusalem
References
1. Ottaviani G, Jaffe N. The epidemiology of osteosarcoma. Cancer Treat Res. 2010; 152: pp. 3-13.
2. Croce C.M. Causes and consequences of microRNA dysregulation in cancer. Nat Rev Genet. 2009; 10(10): 704-714.
3. Jones K.B., et al…and Aqeilan R.I. miRNA signatures associate with pathogenesis and progression of osteosarcoma. Cancer Res. 2012; V. 72(7): pp. 1865-1877.
4. Kobayashi E, Hornicek FJ, Duan Z. MicroRNA Involvement in Osteosarcoma. Sarcoma. 2012; 2012:359739.
5. Sarver AL, Thayanithy V, Scott MC, Cleton-Jansen AM, Hogendoorn PC, Modiano JF, Subramanian S. MicroRNAs at the human 14q32 locus have prognostic significance in osteosarcoma. Orphanet J Rare Dis. 2013 Jan 11;8:7.
6. Kelly AD, Haibe-Kains B, Janeway KA, Hill KE, Howe E, Goldsmith J, Kurek K, Perez-Atayde AR, Francoeur N, Fan JB, April C, Schneider H, Gebhardt MC, Culhane A, Quackenbush J, Spentzos D. MicroRNA paraffin-based studies in osteosarcoma reveal reproducible independent prognostic profiles at 14q32. Genome Med. 2013 Jan 22;5(1):2.
7. Kurek K.C., et al and Aqeilan R.I. Frequent attenuation of the WWOX tumor suppressor in osteosarcoma is associated with increased tumorigenicity and aberrant RUNX2 expression. Cancer Res. 2010; 70(13): pp. 5577-5586.
8. Kochetkova M., et al. and Callen D.F. CBFA2T3 (MTG16) is a putative breast tumor suppressor gene from the breast cancer loss of heterozygosity region at 16q24.3. Cancer Res. 2002; 62(16):4599-4604.
9. Gamou T., Kitamura E., Hosoda F., Shimizu K., Shinohara K., Hayashi Y., Nagase T., Yokoyama Y., Ohki M. The partner gene of AML1 in t(16;21) myeloid malignancies is a novel member of the MTG8(ETO) family. Blood 1998; V. 91: pp. 4028-4037.
10. Athanasiadou A., Stalika E., Sidi V., Papaioannou M., Gaitatzi M., Anagnostopoulos A. RUNX1-MTG16 fusion gene in de novo acute myeloblastic leukemia with t(16;21)(q24;q22). Leuk Lymphoma. 2011; V. 52(1): pp. 145-147.
11. Chyla B.J., …et al and Hiebert S.W. Deletion of Mtg16, a target of t(16;21), alters hematopoietic progenitor cell proliferation and lineage allocation. Mol Cell Biol. 2008; V. 28(20): pp. 6234-6247. .
V11N2 ESUN Copyright © 2014 Liddy Shriver Sarcoma Initiative.
New Grant Funds Research on the Role of MicroRNAs in Osteosarcoma Development and Progression
The Liddy Shriver Sarcoma Initiative is pleased to partner with the Alan B. Slifka Foundation in the funding of a $62,500 grant for promising osteosarcoma research at the Hebrew University of Jerusalem. In the study, Rami Aqeilan, PhD and his research team will explore pre-treatment biomarkers of osteosarcoma metastasis and their responsiveness to therapy. They hope that the study's results will suggest a new anti-miRNA treatment, therapeutic target and/or biomarker that can be used in the management of osteosarcoma.
Dr. Aqeilan's team has investigated the role of small, noncoding genes known as microRNAs in the pathogenesis of osteosarcoma. After discovering disease-associated changes in the miRNA of osteosarcoma samples, the researchers came to suspect that miRNA plays a significant role in osteosarcoma development.
The team recently reported a unique signature of miRNA that differentiates between normal bones and osteosarcoma. They also found that miR-27a was highly upregulated in the primary tumors of metastatic osteosarcoma patients.
In this study, the molecular function and regulation of miR-27a in osteosarcoma metastasis will be explored. The study should reveal driver miRNAs that are associated with pathogenesis of osteosarcoma, as well as critical pre-treatment biomarkers of metastasis and disease responsiveness to therapy.
MiRNAs and Osteosarcoma Metastasis
Metastasis is a major pitfall of osteosarcoma management. Until recently the central dogma of genetics was that RNA played the role of messenger between the gene and the final proteins codified by the gene, and non-coding RNAs were ignored in the field of genome sequencing. The dogma was challenged by the cumulative evidence that non-coding RNAs, including miRNAs, have critical roles in human diseases. The overarching hypothesis of our proposal is that miRNAs acts as driver genes in the pathogenesis of osteosarcoma. Of particular intrest, we shall focus on characterizing those miRNAs that are involved in osteosarcoma metastasis, or in other words "metastamiRs in osteosarcoma."
The Funding
This $62,500 grant is co-funded by the Alan B. Slifka Foundation and the Liddy Shriver Sarcoma Initiative. The Alan B. Slifka Foundation is a private grant-making foundation that is dedicated to making a world safe for difference and healing.
This study is also made possible by generous gifts in memory of Hallie Brown and Nathan Burgess, gifts in honor of Grace Buckel and Natalie Flechsig, and a contribution from Sarah’s Garden of Hope. Together, we are making a difference.
From the Investigator
Rami Aqeilan, PhD: In my laboratory we investigate the genetic and molecular basis of cancer development. My research interest is to uncover the genes (coding and non-coding) and to elucidate the pathways responsible for cancer initiation and progression. Hence, I became interested in revealing the key genes responsible for pediatric osteosarcomagenesis and developing rational, effective therapeutic approaches to this aggressive malignancy.
Osteosarcoma arises from a mesenchymal cell of origin, as do other types of sarcoma. This feature makes sarcomas different from most types of researched cancers that originate from epithelial cells. Further, osteosarcoma has a complex karyotype and displays chromosomal instability. These unique features make it necessary to study the significance of miRNAs in osteosarcomagenesis. The outcomes of this study might also be relevant to other sarcomas.
V11N2 ESUN Copyright © 2014 Liddy Shriver Sarcoma Initiative.
After lentivirus transduction, stable LM-7 clones overexpressing either miR-27a sponge with the SinGFP reporter (Sponge5) or the SinGFP reporter alone (SinGFP5) were created. Invassion ability of the Sponge5 clone in Boyden chamber invasion assay was remarkably reduced as compared to the control SinGFP5 clone.
Functional validation of miR-27a versus scrambled control miR in osteoblasts and OS cell lines show downregulation of TargetScan predicted target gene mRNAs, shown here by qRT-PCR.
After lentivirus transduction, stable LM-7 clones overexpressing either miR-27a sponge with the SinGFP reporter (Sponge1-4) or the SinGFP reporter alone (SIN1-4) were created. Quantitative RT-PCR was performed on RNA extracted from these clones to assess expression of endogenous CBFA2T3 gene. CBFA2T3 expression normalized to GAPDH expression and compared to UBC expression is shown on the figure. There can be seen prominent and specific upregulation of CBFA2T3 expression in SPOGE1-4 clones versus SIN1-4 clones.
Quantitative RT-PCR was conducted on RNA extracted from OS cell lines and bone marrow (BM) to assess expression of CBFA2T3. CBFA2T3 expression normalized to GAPDH expression and compared to UBC expression is shown. There can be seen downregulation of CBFA2T3 expression in metastatic OS cell lines LM7, KHOS and HOS in comparison with non-metastatic OS cell line SAOS2 and bone marrow.
