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:
- We have established two efficient models of SM differentiation from bone-marrow progenitor-derived human mesenchymal stem cells (hMSCs).
- We demonstrated that miRNAs are required for smooth muscle (SM) differentiation.
- We have identified a miRNA signature associated with this process (ref. 1).
- 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
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
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).
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).