ES proliferation and survival is also determined by autocrine and paracrine activation of growth factor receptors and their ligands as insulin-like growth factor 1 [135]. EWS-FLI1 shRNA interference affects IGF-1/IGF-1R survival pathway and its downstream targets (Fig. 34.4) [136]. shRNAi clone showed a marked decrease in IGF-1 transcript and protein levels, both early and late stages (Fig. 34.4a, b), but there were no significant changes in IGF-1R protein level (Fig. 34.4b). shRNAi clone was more sensitive to the action of IGF-1R signaling pathway inhibitors (Fig. 34.4c). A higher apoptotic index was detected, as analyzed by FACS for apoptosis using annexin V (Fig. 34.4d) as well as the reduction of proteins involved in the activation of IGF-1R signaling pathway after treatment with AEW571 combined with inhibitors LY294002 and PD98059 (Fig. 34.4e).

Benini et al. analyzed the contribution of the 2 major pathways of the intracellular IGF-1R signaling cascade to the overall effects elicited by IGF-1 in Ewing sarcoma [137] Both the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3-K) signaling pathways appeared to be constitutively activated in Ewing sarcoma, likely due to the presence of the IGF-1R-mediated autocrine loop. Exogenous IGF-1 completely reverted LY294002-induced growth inhibition by abrogating anti-proliferative and pro-apoptotic effects of the PI3-K inhibitor. By contrast, IGF-1 could not rescue cells from growth inhibition induced by PD98059. MEK/MAPK blockade also significantly reduced the migratory ability of ES cells, both in basal and IGF-1-induced conditions, and increased chemosensitivity to doxorubicin, a leader drug in the treatment of ES patients.

Hernando et al. analyzed the PI3K-AKT signaling cascade in a cohort of sarcomas and found a new and critical role for the AKT-mTOR pathway in smooth muscle transformation and leiomyosarcoma genesis [35]. PTEN is a dual–function lipid and protein phosphatase originally identified as a protein encoded by a tumor suppressor gene, found to be frequently mutated in sporadic cancers and hereditary disorders. The main substrate of PTEN is phosphatidylinositol-3,4,5-triphosphate (PIP3), a second-messenger molecule generated by the action of PI3Ks. PIP3 activates the serine-threonine kinase AKT, involved in cell survival, proliferation, and growth. Other alterations, such as IGF-1 or IGF-1R overexpression, also frequently observed in leiomyosarcoma [138], could ultimately account for the observed upregulation of this pathway.

The putative aberrant signaling provided by c-kit overexpression may be dispensable for Ewing sarcoma development and is unlikely to constitute a critical therapeutic target [139, 140] Baird et al. found highly expressed tyrosine kinases or receptor tyrosine kinases associating with half of the tumor groups [59] Along with known highly expressed kinases, such as KIT in gastrointestinal stromal tumor and PDGFRB in dermatofibrosarcoma protuberans, they additionally found JAK1 in Ewing sarcoma, FLT1 in hemangiopericytoma, EGFR and PDGFRA in synovial sarcoma, and several FGFR in osteosarcoma.

The WNT signaling pathway (Fig. 34.3), involved in the development of brain and the peripheral nervous system, has been found to play a critical role in the formation of several cancers [141], including synovial sarcoma [142–144], Ewing sarcoma [145], and malignant fibrous histiocytoma [41]. Wnts activate a canonical pathway that is characterized by accumulation of β-catenin in the cytoplasm. In the absence of Wnt signaling, cytoplasmic b-catenin forms a complex with Axin, adenomatous polyposis coli (APC) gene product, glycogen synthase kinase-3b (GSK-3b), and casein kinase I-a (CKIa). Phosphorylation of β-catenin by CKIa and GSK-3b results in its ubiquitination and rapid degradation [146–148]. Wnt binding to Fz and LRP5 or LRP6 disrupts this degradation machinery by a process that involves Disheveled (Dvl)-dependent recruitment of Axin to the plasma membrane [147]. As a result, unphosphorylated β-catenin accumulates in the cytoplasm and nucleus, forming a complex with members of the TCF/LEF-1 family of transcription factors that activates transcription of target genes such as c-myc and cyclin-D1 [149–151]. Mutations of Axin, APC, and β-catenin have been characterized in numerous human malignancies including colon cancer, malignant melanoma, hepatocellular carcinoma, endometrial carcinoma, ovarian carcinoma, and prostate cancer [152, 153]. A common outcome of these mutations is the accumulation of free β-catenin, mimicking constitutive Wnt activation. Saito et al. showed that APC mutations also occur in sarcomas, especially in synovial sarcoma, and the possible inactivation of the APC gene by missense mutations was thought to contribute to the accumulation of b-catenin in synovial sarcoma [154]. Baird et al. also found e vidence implicating this pathway in synovial sarcoma [59]. WNT5A expression was pronounced, as was the expression of the WNT signaling targets FZD1, CDH4, EN2, TLE4, and TLE1. TLE1, WNT5A, FZD1, and TLE4 were ranked among the top 50 discriminating genes for the synovial sarcoma, and both WNT5A and FZD1 showed heavy staining on the tissue microarray as well. Uren et al. showed evidence that Wnt/Frizzled signaling is functional in Ewing sarcoma cell lines [145]. They observed a marked stimulation of the β-catenin/canonical Wnt pathway in ES cells treated with Wnt-3a. Wnt-3a induced morphologic changes characterized by the formation of long cytoplasmic extensions in ES cells. β-catenin/canonical Wnt signaling enhanced ES motility, contributing to metastasis, and it could occur through either autocrine or paracrine modes of Wnts since they are expressed in bone, muscle, and soft tissues. They also observed chemotaxis of ES cells in response to Wnt-3a. Both canonical and non-canonical Wnt pathways have been shown to modulate cell motility and tumor metastasis, but non-canonical Wnt pathway is pending of demonstration in ES.

The homeobox genes are involved in early embryonic development and in the determination of cell fate. There is interest in defining the relationship between deviant expression of these early developmental genes and their role in cancer. Specifically, aberrant homeobox gene expression has been linked to the development of leukemia, testicular cancer, breast carcinoma, as well as several other tumors [155]. Several homeobox genes have been identified in the gene expression profiles of various sarcomas: MEOX2 in synovial sarcoma [144], HOXA5 in liposarcoma [60], and MEOX1 in dermatofibrosarcoma protuberans [156].

Alternative proteomics approaches can also combine the disciplines of protein chemistry, molecular biology, and histopathology to paint a portrait of the protein circuitry in diseased cells. It allows the identification of the molecular circuitry of various proteins in a tumor by noting their state of activation (translocation and phosphorylation) and correlative expressions. So far, this approach has been only utilized to depict the mTOR pathway in mesenchymal chondrosarcoma [157].

The combination of 2-DE, MALDI-ToF MS, and bioinformatics offers to the biomedical researcher an opportunity to study the profile of changes in protein levels. Taking advantage of such combination of proteomic tools, and to better understand the mechanisms by which ascochlorin (ASC) regulates physiological or pathological events and induces responses in the pharmacological treatment of cancer, Chang and collaborators performed differential analysis of the proteome of the human osteosarcoma cells U2-OS in response to ASC [158]. In addition, the authors established the first two-dimensional map of the U2-OS proteome. They identified 117 proteins whose expression showed consistent differences in their expression patterns with ASC treatment. Most of the proteins downregulated in U2-OS cells treated with ASC are associated with tumor growth, suggesting that ASC may be useful as a potent clinical suppressor of tumor invasion, a topic of considerable interest in the biological chemistry of chemotherapeutic agents.

In many cases, as seen in the previous examples, sarcoma cell lines are employed as model systems wherein mechanistic molecular schemes are studied. Control of cell-cycle progression, mediation of invasion and migration, evasion of apoptosis, etc. constitute checkpoints that are usually investigated in cell line models of different types of tumors in response to different agents. E2F1, for example, is an essential transcription factor that regulates cell-cycle progression and apoptosis. Overexpression of E2F1 sensitizes neoplastic cells to apoptosis and leads to tumor growth suppression, making it an interesting target for anticancer therapy. Li et al. performed a differential proteome analysis to identify proteins associated with E2F1 activity in inducible p53-deficient Saos-2ERE2F1 osteosarcoma cells [159]. Thirty-three proteins were reproducibly identified, and most of them were the products of genes known to be cancer related. Proteome analysis provided, in this case, new information that may be considered when using E2F1 as a drug.

Using different models of sarcoma xenografts (SK-N-MC and IMR32 (neuroblastoma), RH1 and RH30 (RMS), and KHOS/NP (osteosarcoma), Izbicka et al. evaluated and compared the effects of docetaxel and paclitaxel [160]. The approach used immunoblotting and surface-enhanced laser desorption/ionization (SELDI) MS to assess the drug effects on the expression of the beta-tubulin isotypes and apoptotic markers (Bcl-2, Bax, Bcl-XL). However, the results of this anticancer activity showed no apparent correlation with drug effects on those proteins. The drugs had significantly different, yet highly heterogeneous effects on the tumor levels of the proteins. In contrast, six protein species identified by proteomic profiling were consistently and differentially regulated by docetaxel and paclitaxel in all xenografts.

The mechanisms underlying the clinical outcome of interferon alpha (IFNalpha) treatment of pleomorphic sarcoma (PS) were also investigated using proteomic approaches. A proteomic analysis of 120 signaling components in growth arrested, apoptotic PS cells showed that the relative endogenous expression levels of the IFNAR2 (the IFNalpha/beta receptor) isoforms influence the cytostatic and pro-apoptotic effect of IFNalpha on PS cells [161]. The levels of the receptors may dictate the signaling pathways triggered by the ligand, such as to cause exclusively cell cycle arrest or induce programmed cell death.

The study of ES has provided, as well, a few examples of how proteomics may deepen our understanding about the mechanisms regulating the response to different treatments. The disease expresses several deregulated autocrine loops mediating cell survival and proliferation that contribute to its pathogenesis. Insulin-like growth factor I receptor (IGF1R) and KIT are transmembrane receptors that mediate two of these loops [139, 162], and are therefore directly involved in the growth and survival properties of ES [163–165]. Their blockade is a promising therapeutic approach for this neoplasm. Martins et al. reported the in vitro impact of IGF1R/KIT pathway blockade on ES cell lines [166] and afterwards they extended their observations to the level of proteomic changes induced by this treatment, to find and validate new possible therapeutic targets. Two-dimensional PAGE analysis and MALDI-ToF studies of ES cell lines treated with ADW742 and/or Imatinib (specific IGF1R/KIT inhibitors) revealed a large panel of differentially expressed proteins, some of them related to stress-induced response. Among them, the changes in protein expression between cell lines sensitive and resistant to IGF1R/KIT inhibitors were particularly significant in the case of HSP90 [167].