Arole of Rasgrf1 in Rhabdomyosarcoma Expansion and Metastasis

Maciej Tarnowski

“Arole of RasGRF1 in Rhabdomyosarcoma expansion and metastasis”

Bone marrow provides an environment for the survival and expansion of several tumor types. It is a place where several pediatric sarcomas, including rhabdomyosarcoma (RMS), metastasize from the primary tissue location to such a degree that, in rare situations it is difficult to distinguishbetween them and acute lymphoid leukemia and lymphoma. A bone marrow (BM) biopsy shows on such occasions so-called “small round blue cells,” which are sarcoma cells infiltrating the BM (1, 2, 3)

Accumulatingevidence indicates that some chemokines and growth factors play important roles in the metastasis of cancer cells to bone marrow. Among potential chemokine candidates, a crucial role in directing metastasis has been documented for the SDF-1-CXCR4 receptor axis (4, 5). CXCR4 is a Gαi-protein-coupled, seven transmembrane-spanning receptor that has important roles in the homing of hematopoietic stem cells to the BM and their retention in the BM microenvironment. The homing and engraftment of hematopoietic stem cells to BM is mediated by the interaction between CXCR4 expressed on these cells and SDF-1 secreted by BM stroma (4). It was demonstrated that CXCR4+ tumor cells (e.g., breast and prostate cancers, pediatric sarcomas) exploit this mechanism to metastasize to SDF-1-expressing tissues, such as BM, lungs, liver, or lymph nodes (5-8). A new receptor for SDF-1 named CXCR7 was recently described, and the role of SDF-1 in metastasis of CXCR4+ and CXCR7+ tumors become more complex (9-13). Furthermore, the presence of two SDF-1 binding receptors on cancer cells complicates the rationale to employ anti-CXCR4 blocking agents only in anti-metastatic therapy.

Rhabdomyosarcoma(RMS) is the most common sarcoma in children and there are two major histologicsubtypes: ARMS and ERMS. Clinical evidence indicates that ARMS is more aggressive and associated with a significantly worse outcome than ERMS (14-22) and is characterized by the t(2;13)(q35;q14) translocation in 55% of cases or the variant t(1;13)(p36;q14) translocation in 22% of cases. However, 23% of ARMS cases arefusion gene-negative (20). These translocations generate PAX3-FKHR or PAX7-FKHR fusion genes (17-22) that encode the fusion proteins PAX3-FKHR and PAX7-FKHR that demonstrate enhanced transcriptional activity as compared to wild-type PAX3 and PAX7 and play an important role in survival and proliferation of ARMS cells (14-22). In addition to PAX/FKHR fusion genes, the family of insulin factors, including insulin (Ins), insulin-like growth factor-1 (Igf-1), and insulin-like growth factor-2 (Igf-2), plays an important role both in proliferation and migration of RMS cells (23-26). Aberrant expression of several other genes such as p53, p16INK4A/p14ARF, and non-mutation-related activation of the H-Ras pathway have been postulated to function in RMS pathogenesis(27, 28). The potential role of Ras pathway activation in RMS was demonstrated in a zebrafish model where overexpression of H-RAS induced externally visible RMS tumors by 10 day of life (29). Furthermore, activation of H-Ras in MSCs that expressed dominant-negative p53 or SV40 early region and were transduced with PAX3-FKHR lead to formation of ARMS-like cells (30). However, because Ras family members are mutationally activated infrequently in pediatric RMS, it was postulated that H-Ras pathway activation in the majority of RMS results from “as-yet-unidentified” molecular mechanisms (29) and may involve other proteins like, for example, GTP exchange factors (GEFs).

The Ras superfamily of GTPases are regulated switches that control many intracellular pathways. The Ras family, which includes H-, K-, and N-Ras and other closely related isoforms, has been particularly associated with the control of cell proliferation and migration (31-33). Ras superfamily small GTPases function through their cycling between GTP-bound states that can couple to downstream events and guanosine diphosphate (GDP)-bound states that do not activate those pathways (31). The conversion between these states is governed by several groups of enzymes, including GEFs, which catalyze the release of GDP and subsequent binding of GTP activate these proteins, and GTPase-activating proteins (GAPs), which greatly stimulate the endogenous GTPase activity of Ras proteins and so stimulate their inactivation.

RasGRF1 (or CDC25Mm) is a protein endowed with GEF activity that contains a pleckstrin homology domain,which binds to phosphatidylinositol lipids within biological membranes and assembles signaling proteins in membrane lipid rafts, coiled coil and ilimaquinon regions, as well as catalytic Ras and Rac exchange factor domains. It has been reported that RasGRF1 knockout mice are smaller than normal littermates and display defects in memory consolidation associated with different areas of the brain, as well as defects in -cell development and glucose homeostasis (34). It has been demonstrated that RasGRF1 is a c-Jun-regulated gene necessary for promoting non-adherent growth of c-Myc- or c-Jun-transduced fibroblasts. However, not much attention has been paid to a potential role of RasGRF1 in tumorogenesis, despite this protein having been observed to be expressed in several tumor types (35, 36).

In this proposal, we will test the hypothesis that a GEF protein,RasGRF1, plays a crucial role in Ras hyperactivation in RMS. This hypothesis results from observation that very small embryonic-like stem cells (VSELs) identified in adult tissues are kept quiescent in adult murine tissues due to erasure of DNA methylation on differentially methylated regions (DMRs) for two paternally imprinted genes, Igf2-H19 and RasGRF1, which leads to downregulation of Igf2 and RasGRF1 expression respectively (37). In contrast, the reverse situation is observed in human RMS (spontaneous or part of Beckwith-Wiedemann syndrome), where imprinting of DMRs for Igf2 on chromosome 11p15.5 at the H19-Igf2 loci is re-established leading to overexpression of Igf2 (38-44).

Based on this and our preliminary data showing high expression of RasGRF1 in RMS cells, we would like to askifRasGRF1 could be overexpressed in RMS similarly to the paternally imprinted Igf2 and if RasGRF1 is involved in SDF-1/I-TAC signaling and metastasis.

To address these questions we would like to propose the following aims:

1. By employing Real-Time RQ-PCR we will evaluate RasGRF1 expression on all 10 available to us RMS cell lines and compare its expression in RMS cells to normal human skeletal muscles.
2. We will downregulate RasGRF1 in selected ARMS and ERMS cells lines via a shRNA strategy. Clones with more than 90% of RMS downregulation will be eomployed for further studies.
3. We will use in vitro assays (13, 45-48) to study the effect of RasGRF1 downregulation on RMS cell motility, directional chemotaxis, adhesion and secretion of metalloproteinases.
4. We will also assess whether downregulation of RasGRF1 affects serum-dependent proliferation and whether addition of strong growth stimulators like IGF2 or Insulin will provide rescue these cells from serum starvation and will affect cell proliferation.
5. In parallel, we will study SDF-1/I-TAC-mediated activation of MAPKp42/44, AKT, and Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathways in RMS cells with downregulated RasGRF1 as compared to unmanipulated wild type cells. The rationale for selecting these pathways is their involvement on cell migration/adhesion and our data showing that they are activated in RMS cells after stimulation by SDF-1 or I-TAC (6, 13).
6. We will also perform pull-down experiments using H-Ras-specific Abs to show the effect of RasGRF1 on its phospohorylation/activation (Ras activation kit from Upstate-Milipore). Based on data that RasGRF1 is also a GEF for Rac (49), similarly as for H-Ras, we will employ a Rac1 activation assay kit (Upstate-Milipore) to study effect of RasGRF1 on Rac1 activation.
7. In parallel experiments, we will employ farnesyl transferase inhibitor (FTI) to block H-Ras and NSC23766 inhibitor (Calbiochem) to inhibit Rac alone or in combination. These investigations will allow us to dissect which SDF-1–CXCR4/CXCR7-directed steps of metastasis involve RasGRF1.
8. Finally, we will employ RMS cells with downregulated RasGRF1 and evaluate their tumorogenic potential by employing a long-term metastasis/tumor formation assay, xenotranplants in SCID-Beige mice. At set time intervals, we will measure tumor size and, at time of sacrifice, we will isolate BM, liver, lungs, peripheral blood to evaluate dissemination of RMS cells. Human DNA in these tissues will be detected and quantified by RQ-PCR of human α-satellite DNA sequences as described by us (46-48).

Based on our data, we expect to better explain a role of Ras pathway activation in pathogenesis of RMS and to confirm our hypothesis that downregulation of RasGRF1 will impair in vitro and in vivo metastatic properties of RMS cells. The successful perturbation of RasGRF1/Ras/Rac signaling in RMS could find immediate translational application. We anticipate that high expression of RasGRF1 in patient samples will correlate with a more malignant and metastatic phenotype of RMS, which may have some prognostic value.

The impact of the proposed studies is identification of another potentially paternally imprinted gene, RasGRF1, in RMS pathogenesis, the molecular explanation of H-Ras activation pathway observed in RMS, and evaluation of RasGRF1 as a new potential RMS therapeutic target and prognostic factor.

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