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Sarcoma Knows No Borders

The Initiative's Research Grants Program encourages collaboration to fight a disease that knows no borders.

Leiomyosarcoma
Research Grants

Leiomyosarcoma Research Grants

The Initiative has funded five LMS research grants and one study open to several types of sarcoma patients, including those with LMS.

The Sarcoma MicroRNA Expression Database

MPNST Research Grants

This database allows researchers to identify and develop novel miRNA-based biomarkers for sarcoma diagnosis and classification.

miRNA Alterations in Leiomyosarcoma Pathogenesis

By Laura Danielson, PhD and Eva Hernando, PhD

Introduction

Leiomyosarcoma (LMS), one of the most commonly diagnosed subtypes of sarcoma, is an aggressive malignancy of smooth muscle (SM) origin for which the basis of transformation and mechanisms of progression are mostly unknown.  Smooth muscle cells (SMCs) make up the involuntary muscle tissues that provide structure and contractile forces to organs such as those found in the genitourinary and gastrointestinal systems, in addition to providing the essential contractile support for the vascular system.  Given the variety of anatomical locations of SM tissues, it is not surprising that LMS can arise in various regions of the body.  Within these sites of origin, the smooth muscle neoplasms are frequently associated with the vessels of the surrounding tissues.  The most common sites in which LMS can be found are the smooth muscle lining of the uterus and the retroperitoneal cavity.

Following diagnosis of LMS, the first line of defense is surgical resection of the primary tumor. Unfortunately, given the location and common involvement of adjacent tissues, wide margins of resection are not always possible, and recurrence rates are high. Patients may also undergo radiotherapy at this time, and chemotherapy is common in the case of systemic metastatic spread. Although tumors from the various regions are histologically similar, they often have different clinical courses and chemotherapeutic responses. While studies have shown that LMS of cutaneous origin have a favorable outcome,1 LMS of deep tissue origin, such as the frequent retroperitoneal tumors, are highly aggressive, commonly recur after resection, and despite therapeutic attempts, have a 5-year survival rate of less than 50%.2 It is clear that further study into the mechanisms contributing to the pathobiology of disease, as well as development of efficacious therapies, is needed to better manage the care of and treat those diagnosed with LMS.

LMS belongs to the group of sarcomas that are characterized by a high degree of genomic instability and complex chromosomal aberrations. Over the years studies have attempted to define the molecular characteristics of LMS through comparative genomic hybridization (CGH), mutational analyses, expression arrays.3 The earliest whole genome analyses of chromosomal rearrangements demonstrated mostly non-recurrent chromosomal gains and losses at multiple locations.4,5 A comprehensive review published in 2009 compiled the CGH studies to date and demonstrated that more than 20 chromosomes were affected, with regions encompassing over 2,200 genes.3 The most common regions of chromosomal loss identified were 10q and 13q, the genomic loci of PTEN and RB1 genes, respectively.  In some reported studies these aberrations were found as frequently as in 50% (10q) and 78% (13q) of the patient samples analyzed.5 These findings suggest that loss of PTEN and/or RB1 may contribute to initiation or progression of LMS.  Point mutations and small insertions or deletions are relatively rare in LMS, which can possibly be explained by the chromosomal losses and gains being the primary genetic alterations in these tumors (unpublished data from our lab).6 An example of some genetic mutations found are within the RB1, PTEN, p53 and MDM2 genes, but their prevalence is hard to discern given the rarity of these tumors and the small sample sizes analyzed.

With the advent of expression arrays, mRNA profiling provided further insight into the molecular classification of LMS.7,8 These initial studies demonstrated that on a molecular level independent LMS samples display a high degree of heterogeneity and have significant dysregulation of hundreds of genes.8 Subsequent to these publications, mRNA profiles have been used to categorize LMS samples based on the potential for metastasize,9 as well as to define 3 distinct subtypes based on a comprehensive genomic analysis.10 Again, these studies highlight the complexity of this disease, and further define the molecular alterations in LMS tissues.

MicroRNAs are small, non-coding RNAs that functionally repress target proteins via RNA::RNA binding at imperfect complementary sequences within the 3’untranslated region (3’UTR) of the target mRNA, causing either mRNA degradation or translational inhibition. More than 1000 miRNAs have been found within the human genome and they are predicted to regulate over one third of the human genes.11  

In the 11 years since the first human miRNA was cloned, it has become abundantly clear that they play fundamental roles in development and cancer.  A striking example of this is the early embryonic lethality of Dicer1 knockout mice.12 Furthermore, conditional deletion of Dicer1 in a multitude of systems has further defined the necessity for miRNA production in nearly all developmental systems, including but not limited to T-cell, skin, and heart development.13-15 These studies provide proof-of-principle that miRNAs as a whole are necessary for these processes, but, importantly, it has also been shown that singular miRNAs play key roles in various tissues and organ systems.15-18

It became clear that molecular characterization of miRNAs modulated during differentiation and disease would reveal miRNA candidates that may play a functional role in these processes. Profiles of large cohorts of tumor samples revealed that miRNAs held more power than mRNA expression arrays to subclassify tumor types and identify molecules altered between tumor and normal tissues.19,20 We postulated that alterations in miRNAs that regulate SM differentiation may contribute to LMS genesis and/or contribute to the characteristic aggressive and metastatic behavior of these tumors.

Results

Identification of MicroRNAs (MiRNAs) Involved in smooth muscle (SM) Differentiation and/or Neoplastic Transformation. The generation of miRNA profiles for SM differentiation and leiomyosarcoma (LMS), a previous project also sponsored by the Liddy Shriver Sarcoma Initiative grant program, allowed us to discriminate miRNAs that are exclusively associated with either SM maturation or neoplastic transformation, from miRNAs that appear to be modulated in both processes, suggesting they may play a dual role (Danielson et al., Am J Pathol 2010). We found that 20 out of 72 miRNAs altered in LMS also change during SM differentiation, suggesting that these miRNAs may play a role in both differentiation and transformation.  The remaining 52 miRNAs altered in LMS, but not modulated during differentiation, may be strictly associated to transformation. It should be noted that this information cannot be achieved by traditional profiling of ‘tumor’ versus ‘normal’ tissues, and provides insights into both processes and their intersection at LMS pathogenesis.

From this list of candidate miRNAs we chose to focus our subsequent investigations on miR-130b and miR-17-92.  These miRNAs were identified to be downregulated during smooth muscle cell (SMC) differentiation and upregulated in LMS when compared to myometrium (MM).   The expression levels of these miRNAs are higher in both LMS tissues and hMSCs when compared to normal MM and mature SMCs, supporting our findings that LMS is a mesenchymal stem cell related malignancy (Danielson et al., Am J Pathol 2010).  We hypothesized that these miRNAs, and subsequently their targets, would act as negative regulators of differentiation and, when altered, contribute to initiation or progression of LMS.

To test this hypothesis, experiments were then carried out to assess the role of miR-130b in SMC differentiation in vitro and the contribution to in vitro and in vivo tumorigenic properties of LMS cell lines. In addition to the miR-130b studies, an in vivo model of miR-17-92 overexpression in SMC tissues was setup to investigate the sufficiency of this miRNA cluster to impact on basic properties of SMC development and/or leiomyosarcoma-genesis.

A. Study of mIR-17-92 Dysregulation in LMS

miR-17-92 overexpression suppresses SM differentiation in vitro.

Figure 1. miR-17-92 overexpression inhibits SMC maturation.

Figure 1. miR-17-92 overexpression inhibits SMC maturation.

miR-17-92 cluster members were found downregulated during SM differentiation and overexpressed in LMS compared to MM. This lead us to hypothesize that aberrant miR-17-92 upregulation could impair SMC differentiation, thus contributing to LMS pathogenesis. Indeed, miR-17-92 ectopic expression blocked SMC differentiation in vitro (Fig. 1).

MiR-17-92 overexpression in the SM lineage was insufficient to drive LMS genesis in vivo.

Mice were engineered to overexpress mIR-17-92 in the smooth muscle lineage by crossing Tagln-cre mice with ROSA26-Lox-STOP-lox-miR-17-92 mice, which have the mir-17-92 cluster inserted in the ROSA26 locus after a STOP cassette which is excised by cre-mediated recombination. Upregulation of miR-17-92 cluster components in the smooth muscle was confirmed both in homozygous and heterozygous mice for the conditional allele. To our surprise, homozygous mice carrying two copies of the miR-17-92 transgene died premature of massive cardiomyopathy and arrhythmia (mean survival = 98.3 days ±42.5, p<0.0001) (Fig. 2A).

Figure 2. MiR-17-92 overexpression leads to decreased survival and cardiac hypertrophy.

Figure 2. MiR-17-92 overexpression leads to decreased survival and...

Ectopic expression of miR-17-92 in the smooth muscle lineage during development did not have a major phenotypic impact on smooth muscle containing tissues.  Expression analysis of dissected aortic tissues revealed a significant increase in expression of miR-17-92 in both heterozygous and homozygous animals.  This, however, did not result in altered smooth muscle marker expression, tissue architecture, or thickness. No significant increase in aortic diameter was observed (data not shown).

Figure 3. Overexpression of miR-17-92 does not result in frank SM defect.

Figure 3. Overexpression of...

Additionally, histological analysis of other SM-containing organs such as the bladder, intestine, and myometrium did not reveal alterations in smooth muscle marker expression (ASMA), proliferation (PCNA) or thickness, nor overt functional defects (Fig. 3). 

B. STUDY OF MIR-130B DYSREGULATION IN LMS

Figure 4. Expression analyses of miR-130b in LMS.

Figure 4. Expression analyses of miR-130b in LMS.

Validation of miR-130b overexpression in LMS tumor samples. 

Validation of the original array data was achieved by performing expression analyses on an independent cohort of samples.  Array data found miR-130b to be statistically significantly upregulated in LMS tissues when compared to normal MM (5.47 fold, FDR 0.0000113) (Fig. 4, left).  In an independent cohort of paired samples (i.e. normal MM and LMS tissues collected from the same patient) miR-130b was found statistically significantly upregulated in a majority of the cases (10 out of 15, average 2.56 fold change ± 0.36) (Fig. 4, right).  In an effort to address a possible mechanism for this upregulation, we sought to determine if the genomic locus of miR-130b was amplified in another independent data set.  Primers were designed to amplify the region directly flanking the miR-130b genomic sequence located at 22q11.21 in the human genome.  This analysis revealed at least 1 out of the 9 patient tumor samples had 4 fold higher levels of miR-130b genomic DNA, while 3 other patient tumor specimens had nearly a 2 fold increase (data not shown).  This additional human data strongly supports that miR-130b is significantly upregulated in patient samples of LMS, possibly in part via genomic amplification, and warranted further investigation into how its dysregulation may be contributing to this disease.

Validation and functional testing of miR-130b in SMC differentiation. 

Figure 5. miR-130b overexpression inhibits SMC maturation.

Figure 5. miR-130b overexpression inhibits SMC maturation.

Array data demonstrated that miR-130b is downregulated during SMC differentiation (3.09 fold change, FDR 0.0000023).  To test the hypothesis that miR-130b plays in active role in SMC differentiation, TxA2 differentiation in the presence of ectopic miR-130b overexpression was performed.  To accomplish this, hMSCs were transiently transfected with miR-130b mimic oligonucleotides (oligos), and then induced to differentiate 24 hours later by addition of 1uM TxA2.  Brightfield images of the cells at the end timepoint of differentiation demonstrated a decrease in aligned parallel and perpendicular cell patterns, commonly seen in SMC cultures, and instead a more random fibroblast appearance (Fig. 5, upper panels). Accordingly, miR-130b overexpression significantly impaired the upregulation of SM-MHC expression when compared to the control (Fig. 5, lower panels). In sum, miR-130b acts as a negative regulator of SMC maturation.  These data suggests that if miR-130b is aberrantly overexpressed in SMC progenitors during development, this may inhibit/block SMC maturation, potentially providing an avenue for initiation or progression of LMS.

miR-130b overexpression promotes the in vitro invasive potential of LMS cells

To study the molecular mechanisms miR-130b may contribute towards promotion of LMS, we first generated a LMS cell line stably overexpressing miR-130b.  This was achieved by transducing the cell line SK-LMS1 (LMS1) with a lentiviral construct containing the hairpin sequence of pre-miR-130b under the ubiquitous CMV promoter.  The lentiviral construct also contained the GFP sequence downstream of an independent promoter allowing for assessment of transduction efficiency by monitoring the number of GFP positive cells.  Successful transduction rendered a cell line with levels of miR-130b ~20 fold higher than control vector-transduced cells, and resulted in little alteration of levels of stem cell or SMC differentiation markers (data not shown).

Figure 6. MiR-130b overexpression enhances invasion.

Figure 6. MiR-130b overexpression enhances invasion.

In order to elucidate a potential role for miR-130b in the tumorigenic properties of LMS, we carried out a number of in vitro assays.  We found that increasing the expression of miR-130b in the LMS1 cell line had no impact on proliferation (both in normal and low serum conditions), capacity for colony formation, wound healing/migration, ability to grow in suspension or to form sarcospheres (data not shown).  These results suggest that miR-130b does not provide stem-cell like features nor a proliferative or survival advantage to LMS cells.  Importantly, however, ectopic expression of miR-130b did result in a statistically significant increase in invasion through fibronectin or matrigel coated trans-wells (Fig. 6).  Taken together these data suggest that the increased expression of miR-130b identified in patient samples may be directly contributing to the invasive nature of this malignancy.

Ectopic expression of miR-130b potentiates metastasis in vivo.

Stably transduced LMS1 cells overexpressing miR-130b injected into the flanks of immunocompromised mice showed accelerated tumor growth when compared to control cells.  Furthermore, tumor weights at the end of experiment confirmed miR-130b overexpressing tumors to be larger in size.

Figure 7. Overexpression of miR-130b enhances metastasis in vivo.

Figure 7. Overexpression of miR-130b enhances metastasis in vivo.

Important to confirming the role of miR-130b on the metastatic potential of this cell line, whole lungs were removed and examined for presence of GFP positive cells indicating lung infiltration and colonization by LMS cells.  A striking increase in macrometastases was observed both by whole organ imaging (Fig. 7), as well as GFP staining of histological lung sections.  Of note, no other sites of metastasis, such as liver, were found colonized by control or miR-130b expressing cells.  We have now confirmed that miR-130b contributes to both invasive properties in vitro as well as enhancing metastasis in vivo. We are currently study the direct molecular targets and pathways regulated by this miRNA that mediate its pro-metastatic behavior in LMS.

Conclusions

In this study we first developed a method of in vitro SMC differentiation, and with this tool defined the miRNA signature of this process. In addition, we defined a miRNA signature of LMS using human patient samples. With these profiles we were able to then molecularly classify LMS as a mesenchymal stem cell related malignancy, and define a list of candidate miRNAs that may play an active role in SMC differentiation and simultaneously impact upon differentiation and LMS initiation and/or progression when aberrantly expressed. We further characterized the role of two of these candidates, miR-17-92 and 130b. Furthermore, we found that miR-130b expression enhances invasive and metastatic properties of a LMS cell line in vitro and in vivo, respectively. This may be due to targeting TSC1 because independent repression of this protein partially phenocopies this effect. We have further elucidated that miR-130b directly targets essential molecules that govern cytoskeletal structure, motility, and invasion. These findings place miR-130b as an important factor that not only may contribute to the undifferentiated nature of LMS, but also regulate its ability to migrate and invade, essential attributes for metastatic spread of this aggressive malignancy.

By Laura S. Danielson, PhD
Eva Hernando, PhD
Department of Pathology, NYU School of Medicine
NYU Langone Medical Center

References

1. Svarvar C, Böhling T, Berlin O, Gustafson P, Follerås G, Bjerkehagen B et al (2007). Clinical course of nonvisceral soft tissue leiomyosarcoma in 225 patients from the Scandinavian Sarcoma Group. Cancer 109: 282-373.

2. Bonvalot S, Rivoire M, Castaing M, Stoeckle E, Le Cesne A, Blay J et al (2009). Primary retroperitoneal sarcomas: a multivariate analysis of surgical factors associated with local control. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 27: 31-38.

3. Yang J, Du X, Chen K, Ylipää A, Lazar A, Trent J et al (2009). Genetic aberrations in soft tissue leiomyosarcoma. Cancer letters 275: 1-9.

4. Packenham J, du Manoir S, Schrock E, Risinger J, Dixon D, Denz D et al (1997). Analysis of genetic alterations in uterine leiomyomas and leiomyosarcomas by comparative genomic hybridization. Molecular carcinogenesis 19: 273-282.

5. Otaño-Joos M, Mechtersheimer G, Ohl S, Wilgenbus K, Scheurlen W, Lehnert T et al (2000). Detection of chromosomal imbalances in leiomyosarcoma by comparative genomic hybridization and interphase cytogenetics. Cytogenetics and cell genetics 90: 86-178.

6. Barretina J, Taylor B, Banerji S, Ramos A, Lagos-Quintana M, Decarolis P et al (2010). Subtype-specific genomic alterations define new targets for soft-tissue sarcoma therapy. Nature genetics 42: 715-736.

7. Nielsen T, West R, Linn S, Alter O, Knowling M, O'Connell J et al (2002). Molecular characterisation of soft tissue tumours: a gene expression study. Lancet 359: 1301-1308.

8. Skubitz K, Skubitz A (2003). Differential gene expression in leiomyosarcoma. Cancer 98: 1029-1067.

9. Lee EK, Lee MJ, Abdelmohsen K, Kim W, Kim MM, Srikantan S et al (2010). miR-130 Suppresses Adipogenesis by Inhibiting Peroxisome Proliferator-Activated Receptor   Expression. Molecular and cellular biology 31.

10. Beck A, Lee CH, Witten D, Gleason B, Edris B, Espinosa I et al (2010). Discovery of molecular subtypes in leiomyosarcoma through integrative molecular profiling. Oncogene 29: 845-899.

11. Bartel D (2009). MicroRNAs: target recognition and regulatory functions. Cell 136: 215-248.

12. Bernstein E, Kim S, Carmell M, Murchison E, Alcorn H, Li M et al (2003). Dicer is essential for mouse development. Nature genetics 35: 215-222.

13. Muljo S, Ansel K, Kanellopoulou C, Livingston D, Rao A, Rajewsky K (2005). Aberrant T cell differentiation in the absence of Dicer. The Journal of experimental medicine 202: 261-270.

14. Yi R, O'Carroll D, Pasolli H, Zhang Z, Dietrich F, Tarakhovsky A et al (2006). Morphogenesis in skin is governed by discrete sets of differentially expressed microRNAs. Nature genetics 38: 356-418.

15. Zhao Y, Ransom J, Li A, Vedantham V, von Drehle M, Muth A et al (2007). Dysregulation of cardiogenesis, cardiac conduction, and cell cycle in mice lacking miRNA-1-2. Cell 129: 303-320.

16. Stefani G, Slack F (2008). Small non-coding RNAs in animal development. Nature reviews Molecular cell biology 9: 219-249.

17. Rodriguez A, Vigorito E, Clare S, Warren M, Couttet P, Soond D et al (2007). Requirement of bic/microRNA-155 for normal immune function. Science (New York, NY) 316: 608-619.

18. Thai TH, Calado DP, Casola S, Ansel KM, Xiao C, Xue Y et al (2007). Regulation of the Germinal Center Response by MicroRNA-155. Science 316.

19. Lu J, Getz G, Miska E, Alvarez-Saavedra E, Lamb J, Peck D et al (2005). MicroRNA expression profiles classify human cancers. Nature 435: 834-842.

20. Volinia S, Calin G, Liu C-G, Ambs S, Cimmino A, Petrocca F et al (2006). A microRNA expression signature of human solid tumors defines cancer gene targets. Proceedings of the National Academy of Sciences of the United States of America 103: 2257-2318.

V9N4 ESUN Copyright © 2012 Liddy Shriver Sarcoma Initiative.

Effect of Mir-17-92 Dysregulation in Leiomyosarcoma-Genesis

By Eva Hernando, PhD

Abstract

Leiomyosarcomas (LMS) derive from the smooth-muscle lineage, appear in the uterus, retroperitoneum or extremities, and are prone to both local recurrence and metastasis with often fatal outcomes. LMS display extensive heterogeneity at the cellular level and often contain cells that express markers of immature cells, suggesting that LMS may be direct descendants of smooth muscle progenitors, not of mature smooth-muscle cells (SMC). We propose to test this theory of LMS origin, differentiation and transformation by probing a subset of microRNA candidates. MicroRNAs (miRNAs) are critical regulators of differentiation, development and tissue homeostasis, as indicated by the loss of miRNAs and subsequent embryonic lethality of mice with engineered null mutations of the Dicer gene. MiRNA alterations have been found to contribute to tumorigenesis by deregulating oncogenes and tumor suppressors. Furthermore, composite miRNA expression profiles, or ‘signatures’, have been used to successfully classify many tumor types.

MicroRNA basic concepts

  • miRNAs do not encode for proteins, but are key regulators of gene expression.
  • miRNAs can modulate multiple genes simultaneously.
  • miRNAs are key regulators of differentation and developmental processes.
  • miRNA profiles are cell-type specific.

We propose a new study to assess the role of miRNAs in the development of LMS, in order to advance our objectives to uncover molecular events that contribute to LMS and ultimately, open new doors to better strategies for prognosis and treatment. Thus far, my laboratory has made several contributions on the way to our goal:

  1. We have established two efficient models of SM differentiation from bone-marrow progenitor-derived human mesenchymal stem cells (hMSCs).
  2. We demonstrated that miRNAs are required for smooth muscle (SM) differentiation.
  3. We have identified a miRNA signature associated with this process (ref. 1).
  4. We have defined miRNA signatures of uterine LMS (ULMS), compared to normal myometrium (MM).

Juxtaposing the two miRNA signatures (SM differentiation and ULMS) has allowed us to identify a set of miRNAs modulated both in the differentiation and transformation processes. Those selected miRNAs include miR29c, which is upregulated during SM differentiation and downregulated in ULMS compared to MM, and several miRNAs of the miR-17-92 subset that are repressed during SM differentiation while overexpressed in ULMS compared to MM. Moreover, ectopic expression of miR-17-92 members impaired SM differentiation of hMSCs. We therefore hypothesize upon this evidence that alterations in miRNAs which control MSC regulation or SM differentiation such as miR-17-92 may contribute to LMS genesis or progression. Results from our following aims will provide new insights into the LMS cell-of-origin.

miRNAs in cancer

  • miRNA genes are frequently located at fragile sites and chromosomal regions altered in human cancer (e.g. amplifications, deletions, translocations).
  • miRNA patterns are able to sub-classify tumor types.
  • Some altered miRNAs can act as tumor suppressors (e.g. let7, miR-34) and some can be oncogenic (e.g. miR-21); these functions are cell-type specific.
  • Some miRNAs are able to predict patient prognosis (e.g. let-7 and lung cancer).
  • Some miRNAs are able to contribute to metastasis (e.g. miR-10b, miR-335, miR-182).

Introduction and Preliminary Data

Identification of MiRNAs Involved in SM Differentiation and Altered in LMS

Plan Figure 1: Histology of human (A) and mouse (B) uterine LMS.

Figure 1: Histology of human (A) and mouse (B) uterine LMS...

We have generated miRNA profiles for smooth muscle (SM) differentiation and uterine leiomyosarcoma (ULMS).1 This is allowing us to investigate miRNAs that are exclusively associated with either SM maturation or neoplastic transformation, as well as miRNAs that appear to be modulated in both processes. We found that 20 out of 73 miRNAs altered in ULMS are also modulated during SM differentiation, suggesting that these miRNAs may play a dual role in both differentiation and transformation. Of the 20 miRNAs, 6 miRNAs increase during SM differentiation and are downregulated in ULMS, which display many characteristics of undifferentiated progenitor cell types, further confirming that low expression level of those miRNAs is strongly associated with an immature phenotype. One of those miRNAs is miR29c, which has been found to contribute to the oncogenic properties of rhabdomyosarcoma.2-4 Conversely, 8 miRNAs downregulated during SM differentiation, including several components of the miR-17-92 cluster, are overexpressed in ULMS compared to normal myometrium (MM) (Fig. 1). These correlations may indicate candidate miRNAs that play a causative role in transformation by impacting the differentiation stage of SMCs. miRNAs that we hypothesize may play a dual role in SM differentiation and LMS pathogenesis (i.e. miR-17-92), will be the focus of our proposed investigation.

miRNAs With a Dual Role in Differentiation and Oncogenesis

miR-29c, miR-206 and rhabdomyosarcoma miRNAs-29 and -206 have been recently shown to contribute to normal skeletal muscle maturation and to the oncogenic properties of tumors of skeletal muscle origin (rhabdomyosarcomas) by altering the tumor cell’s state of differentiation and proliferation.2-4 These studies highlight the importance of studying oncogenesis in relation to differentiation, and a need to investigate the duality of miRNA function in both processes.

Role of miR-17-92 in Cancer

In the human genome, the miR-17-92 cluster encodes six miRNAs that are tightly grouped within an 800 base-pair region of human chromosome 13. Both the sequences of these mature miRNAs and their organization are highly conserved in all vertebrates. The human genomic locus encoding these miRNAs, 13q31.3, undergoes amplification in several types of lymphoma and solid tumors, including LMS.5 Expression and functional studies have shown that the miR-17-92 cluster can act as a bona fide oncogene in solid tumors,6-8 and is directly transactivated by the oncogene c-Myc.7-9 Verified targets of the miR-17-92 cluster include the E2F family of transcription factors,9-11 the cyclin-dependent kinase inhibitor CDKN1A (p21),7 the pro-apoptotic gene Bim7,12 and the Pten tumor suppressor.13 Enforced expression of the miR-17-92 cluster resulted in premature death of transgenic animal models of lymphoproliferative disease and autoimmunity.14

Overexpression of the miR-17-92 Cluster Impairs SM Differentiation of hMSCs in vitro

Plan Figure 2: Schematic presentation of uterine LMS development in mice...

Figure 2: Schematic presentation of uterine LMS development in mice...

We infected hMSCs with a lentiviral vector carrying the miR-17-92 cluster. Cells were set-up to differentiate into SM by 24h of serum starvation, followed by Thromboxane A2 treatment.1 MiR-17-92-overexpressing hMSCs failed to acquire the morphological features of SMCs (appearance of cytoplasmic actin filaments) (Fig. 2A), and did not upregulate smooth muscle myosin heavy chain (SM-MHC), relative to vector-transduced hMSCs (Fig. 2B). These results indicate that miR-17-92 downregulation is required for SM differentiation, and could be necessary to exit the pluripotent stage, to arrest cell proliferation or to directly mediate differentiation.

Specific Aims and Experimental Strategy

1. Evaluate the ability of miR-17-92 altered gene expression to induce neoplastic transformation of SMCs or hMSCs.

First, we will test the ability of miR-17-92 overexpression to transform immortal SMCs and hMSCs stably transduced with hTERT15 using in vitro assays for altered cell proliferation or transformation. DKK115 and E1a/ras will serve as positive controls. Then, we will apply a more stringent test of tumorigenicity by subcutaneously injecting transduced cells into the flanks of immunodeficient mice, and monitoring tumor formation and growth. We will analyze morphology and pathology features (i.e. differentiation, pleomorphism) of the xenografts generated, and their resemblance to human LMS.

2. Investigate the capacity of miR-17-92 modulation to alter the oncogenic properties of established LMS cells.

Modulating candidate miRNAs in established LMS cell lines may alter their differentiation state and/or oncogenic properties. To address the role of miR-17-92 in the maintenance of the oncogenic phenotype, we will either induce overexpression (using mimic oligos for transient transfection; lentiviral vectors for stable transduction) or downregulation (using anti-miRs or miRZips) in LMS cell lines (SK-LMS1, SK-UT1, CNIO-AA and CNIO-AY16). To test the properties of transformation, we will use assays for cell proliferation (MTT or WST-1), clonogenicity, and colony formation in soft-agar. To assess metastatic behavior, we will measure invasion (matrigel or fibronectin transwell assays), apoptosis by low serum or hypoxia (measured by Caspase3, PARP cleavage, Annexin V staining), and anoikis assays (ability to survive and grow in absence of attachment).

3. Determine how deregulating the mir-17-92 cluster in vivo impacts smooth muscle differentiation and leiomyosarcoma pathogenesis.

We will manipulate the expression of miR-17-92 at distinct developmental stages to ascertain whether impairing SM differentiation or imposing a ‘de-differentiated’ state in adult SMCs can have a distinct impact on the initiation or biological behavior of LMS. Mice carrying the miR-17~92 sequence after a loxP-STOP-loxP cassette will be crossed with Tagln::cre,17 or SM-MHC::creERT2 mice18 (generously shared by Dr. Offermanns, Max Planck Institute) in order to express miR-17~92 in undifferentiated (Tagln-transgelin-positive) or mature (SM-MHC positive) cells of the SM lineage, respectively. SM-MHC::creERT2 mice will ectopically express the miR-17-92 cluster in a temporally controlled manner upon addition of 4-hydroxy-tamoxifen (4-OHT).

Significance

There is very little understanding of the biology of leiomyosarcomas (LMS), although they are among the most common sarcomas of soft tissue (STS). It is believed that differentiating smooth muscle cells are the precursors of LMS,19 but we do not know the significance of the differentiation stage at which transformation occurs, nor do we know the underlying molecular events leading to LMS. Our results will: 1) lead to a better understanding of the molecular basis of LMS, providing the first in vivo functional evidence of altered miRNAs that contribute to this tumor type; and 2) reveal potential new therapeutic targets for LMS treatment (specific miRNAs and their targets).

By Eva Hernando, PhD
 Assistant Professor
NYU School of Medicine

References

1. Danielson LS, et al. (2010) A differentiation-based miRNA signature identifies leiomyosarcoma as a mesenchymal stem cell-related malignancy. American Journal of Pathology.

2. Taulli R, et al. (2009) The muscle-specific microRNA miR-206 blocks human rhabdomyosarcoma growth in xenotransplanted mice by promoting myogenic differentiation. (Translated from eng) J Clin Invest 119(8):2366-2378 (in eng).

3. Wang H, et al. (2008) NF-kappaB-YY1-miR-29 regulatory circuitry in skeletal myogenesis and rhabdomyosarcoma. (Translated from eng) Cancer Cell 14(5):369-381 (in eng).

4. Yan D, et al. (2009) MicroRNA-1/206 targets c-Met and inhibits rhabdomyosarcoma development. (Translated from Eng) J Biol Chem  (in Eng).

5. Svarvar C, et al. (2006) Do DNA copy number changes differentiate uterine from non-uterine leiomyosarcomas and predict metastasis? Mod Pathol 19(8):1068-1082.

6. He L, et al. (2005) A microRNA polycistron as a potential human oncogene. Nature 435(7043):828-833.

  • Experimental Plan Figure 1
  • Experimental Plan Figure 2
    A. Brightfield and immunofluorescence micrographies of hMSCs transduced with vector (control) and miR-17-92-expressing lentivirus after 4 days in TxA2 differentiation media. B. Quantitative PCR of SM-MHC at specific time points of SM differentiation of vector (control) and miR-17-92 transduced hMSCs.
  • Study Report Figure 1. miR-17-92 overexpression inhibits SMC maturation.
    Upper panels: Representative images of day 4 timepoint of SCR and miR-17-92 transduced hMSCs induced with TxA2. Lower panels: SM-MHC expression analysis. FC (fold change) is relative to respective t=1 and normalized to GAPDH.
  • Study Report Figure 2. MiR-17-92 overexpression leads to decreased survival and cardiac hypertrophy.
    A Kaplan meier curve of percent survival versus time (days) (median = 82 days, mean= 98.3 days +/- 42.5, p<0.0001). B) Gross images (upper panels) and histology (lower panel) of WT versus TG/TG hearts at ~2 months of age.
  • Study Report Figure 3. Overexpression of miR-17-92 does not result in frank SM defect.
    Immunohistochemical analysis of representative 3 month old WT (left) and TG/TG (right) mice for markers of SM differentiation (ASMA, top panel) and proliferation (PCNA, bottom panel).
  • Study Report Figure 4. Expression analyses of miR-130b in LMS.
    Left, expression of original array data of normal myometrium (MM, n=10) and LMS (n=10). Right, qPCR expression analysis of matched MM and LMS samples (n=15 each). FC (fold change) relative to mean of all MM and normalized to RNU44.
  • Study Report Figure 5. miR-130b overexpression inhibits SMC maturation.
    Upper panels: Representative images of day 4 timepoint of SCR and miR-130b transfected hMSCs induced with TxA2. Lower, left: miR-130b expression analysis confirms overexpression. Lower, right. SM-MHC expression analysis. FC (fold change) is relative to respective t=1 and normalized to RNU44 (for miR-130b expression) and GAPDH (for SM-MHC expression).
  • Study Report Figure 6. MiR-130b overexpression enhances invasion.
    Number of cells able to invade through fibronectin (left) or matrigel (right) coated transwell membrane inserts (top panels). Representative images of fixed and crystal violet stained cells on the bottom of the insert used for counting (bottom panels). Number of miR-130b invading cells is relative to control. Error bars equal standard deviation of experimental replicates. * p<0.05
  • Study Report Figure 7. Overexpression of miR-130b enhances metastasis in vivo.
    Ex vivo images of whole lungs under fluorescent light to detect GFP positive tumor cells colonizing the lungs of mice injected with Control (left) versus miR-130b (right) cell lines.
 

V8N2 ESUN Copyright © 2011 Liddy Shriver Sarcoma Initiative.

Grant Funding

The Liddy Shriver Sarcoma Initiative and the Leiomyosarcoma Direct Research Foundation co-funded this $50,000 grant in April 2011. The study was made possible, in part, by donations made to the Liddy Shriver Sarcoma Initiative from the Jim Hauser Sarcoma Foundation in memory of Jim Hauser.

 

 

 

 

 

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