Soft-tissue sarcomas are rare malignant tumors arising from connective tissues and have an overall incidence of about five per 100,000 per year. While this diverse family of malignancies comprises over 100 histological subtypes and many molecular aberrations are prevalent within specific sarcomas, very few are therapeutically targeted. Instead of utilizing molecular signatures, first-line sarcoma treatment options are still limited to traditional surgery and chemotherapy, and many of the latter remain largely ineffective and are plagued by disease resistance. Currently, the mechanism of sarcoma oncogenesis remains largely unknown, thus necessitating a better understanding of pathogenesis. Although substantial progress has not occurred with molecularly targeted therapies over the past 30 years, increased knowledge about sarcoma biology could lead to new and more effective treatment strategies to move the field forward. Here, we discuss biological advances in the core molecular determinants in some of the most common soft-tissue sarcomas – liposarcoma, angiosarcoma, leiomyosarcoma, rhabdomyosarcoma, Ewing’s sarcoma, and synovial sarcoma – with an emphasis on emerging genomic and molecular pathway targets and immunotherapeutic treatment strategies to combat this confounding disease.

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Primary effusion lymphoma (PEL) is an aggressive type of non-Hodgkin lymphoma localized predominantly in body cavities. Kaposi’s sarcoma-associated herpes virus is the causative agent of PEL. PEL is an incurable malignancy and has extremely poor prognosis when treated with conventional chemotherapy. Immunomodulatory drugs (IMiDs) lenalidomide and pomalidomide are FDA approved drugs for the treatment of various ailments. IMiDs display pronounced anti-proliferative effect against majority of PEL cell lines within their clinically achievable concentrations, by arresting cells at G0/G1 phase of cell-cycle, and without any induction of KSHV lytic-cycle reactivation. Although microarray examination of PEL cells treated with lenalidomide revealed activation of interferon (IFN) signaling, blocking the IFN pathway did not block the anti-PEL activity of IMiDs. The anti-PEL effects of IMiDs involved cereblon-dependent suppression of IRF4 and rapid degradation of IKZF1, but not IKZF3. Small hairpin-RNA (shRNA) mediated knockdown of MYC enhanced the cytotoxicity of IMiDs. Bromodomain and extraterminal domain (BET) proteins are epigenetic readers which perform a vital role in chromatin remodeling and transcriptional regulation. BRD4, a widely expressed transcriptional coactivator, belongs to BET family of proteins, which has been shown to co-occupy the super-enhancers associated with MYC. Specific BRD4 inhibitors were developed which suppress MYC transcriptionally. Lenalidomide displayed synergistic cytotoxicity with several structurally distinct BRD4 inhibitors (JQ-1, IBET151, and PFI-1). Furthermore, combined administration of lenalidomide and BRD4 inhibitor JQ-1 significantly increased the survival of PEL bearing NOD.SCID mice in an orthotopic xenograft model as compared to either agent alone. These results provide compelling evidence for clinical testing of IMiDs alone and in combination with BRD4 inhibitors for PEL.

Primary effusion lymphoma (PEL) is an aggressive form of non-Hodgkin's B cell lymphoma associated with infection by Kaposi's sarcoma associated herpesvirus (KSHV). (+)-JQ1 and I-BET151 are two recently described novel small molecule inhibitors of BET bromodomain chromatin-associated proteins that have shown impressive preclinical activity in cancers in which MYC is over-expressed at the transcriptional level due to chromosomal translocations that bring the MYC gene under the control of a super-enhancer. PEL cells, in contrast, lack structural alterations in the MYC gene, but have deregulated Myc protein due to the activity of KSHV-encoded latent proteins. We report that PEL cell lines are highly sensitive to BET bromodomain inhibitors-induced growth inhibition and undergo G0/G1 cell-cycle arrest, apoptosis and cellular senescence, but without the induction of lytic reactivation, upon treatment with these drugs. Treatment of PEL cell lines with BET inhibitors suppressed the expression of MYC and resulted in a genome-wide perturbation of MYC-dependent genes. Silencing of BRD4 and MYC expression blocked cell proliferation and cell-cycle progression, while ectopic expression of MYC from a retroviral promoter rescued cells from (+)-JQ1-induced growth arrest. In a xenograft model of PEL, (+)-JQ1 significantly reduced tumor growth and improved survival. Taken collectively, our results demonstrate that the utility of BET inhibitors may not be limited to cancers in which genomic alterations result in extremely high expression of MYC and they may have equal or perhaps greater activity against cancers in which the MYC genomic locus is structurally intact and c-Myc protein is deregulated at the post-translational level and is only modestly over-expressed.

Circulating cell-free DNA (cfDNA) released by tumor cells, termed ctDNA, closely reflects the heterogeneity of primary cancers and their metastases. As a noninvasive, real-time monitoring biomarker, ctDNA is a promising tool for detecting driver gene mutations, assessing tumor burden and acquired resistance, and early diagnosis. However, isolation and enrichment of cfDNA is a big challenge due to the high degree of DNA fragmentation and its relatively low abundance in the bloodstream. This review aims to provide insights into the recent technological advances in acquisition of optimal quality cfDNA, the use of preservatives, isolation methods, processing timelines, and detection techniques. It also describes clinical applications of ctDNA in cancer patient management.

The mechanism of Sonic Hedgehog (Shh) pathway activation in non-small cell lung cancer (NSCLC) is poorly described. Using an antibody against the Shh C-terminal domain, we found a small population of Shh-positive (Shh+) cells in NSCLC cells. The objective of this study was to characterize these Shh+ cells. Shh+ and Shh- cells were sorted by using Fluorescence Activated Cell Sorting (FACS) on 12 commercial NSCLC cell lines. Functional analyses on sorted cells were performed with gene expression assays (qRT-PCR and microarray) and cells were treated with cytotoxic chemotherapy and a targeted inhibitor of Shh signaling (GDC0449). We used in vivo models of nude mice inoculated with Shh+ and Shh- sorted cells and drug-treated cells. Finally, we confirmed our results in fresh human NSCLC samples (n=48) paired with normal lung tissue. We found that Shh+ cells produced an uncleaved, full-length Shh protein detected on the membranes of these cells. Shh+ cells exerted a paracrine effect on Shh- cells, inducing their proliferation and migration. Shh+ cells were chemo-resistant and showed features of cancer stem cells (CSCs) in vitro and in vivo. Pharmacological inhibition of the Shh pathway suppressed their CSC features. A high percentage of Shh+ cells was associated with poor prognosis in early-stage NSCLC patients. In conclusion, we describe for the first time the presence of an abnormal membrane-bound full-length Shh protein in human cancer cells that allows the identification of CSCs in vitro and in vivo.

BackgroundSquamous cell carcinomas (SCC) account for approximately 30% of non-small cell lung cancer (NSCLC). Current staging methods do not adequately predict outcome for this disease. EMX2 is a homeo-domain containing transcription factor known to regulate a key developmental pathway. This study assessed the significance of EMX2 as a prognostic and predictive marker for resectable lung SCC.MethodsTwo independent cohorts of patients with lung SCC undergoing surgical resection were studied. EMX2 protein expression was examined by immunohistochemistry, Western blot, or immunofluorescence. EMX2 expression levels in tissue specimens were scored and correlated with patient outcomes. Chemo-sensitivity of lung SCC cell lines stably transfected with EMX2 shRNAs to cisplatin, carboplatin, and docetaxel was examined in vitro.ResultsEMX2 expression was down-regulated in lung SCC tissue samples compared to their matched adjacent normal tissues. Positive EMX2 expression was significantly associated with improved overall survival in stage I lung SCC patients, and in stage II/IIIA lung SCC patients receiving adjuvant chemotherapy. EMX2 expression was also associated with expression of EMT markers in both lung SCC cell lines and tissue samples. Knock-down of EMX2 expression in lung SCC cells promoted chemo-resistance and cell migration.ConclusionsEMX2 expression is down-regulated in lung SCC and its down-regulation is associated with chemo-resistance in lung SCC cells, possibly through regulation of Epithelial-to-Mesenchymal Transition (EMT). EMX2 may serve as a novel prognostic marker for stage I lung SCC patients and a prediction marker for stage II/IIIA lung SCC patients receiving adjuvant chemotherapy.

IMPORTANCEKaposi's sarcoma-associated herpesvirus-encoded vFLIP K13 is believed to protect virally infected cells against death receptorinduced apoptosis and to activate the NF-B pathway by binding to adaptor protein NEMO/IKK␥. However, whether K13 can also induce gene expression independently of NEMO and the minimum region of NEMO that is sufficient for supporting K13-induced NF-B remain to be delineated. Furthermore, the contribution of NEMO and NF-B to the protective effect of K13 against death receptor-induced apoptosis is not clear. We demonstrate that NEMO is required for modulation of K13-induced genes and its N-terminal 251 amino acids are sufficient for supporting K13-induced NF-B. The ability of K13 to protect against TNF-␣-induced cell death is critically dependent on its ability to interact with NEMO and activate NF-B. Our results suggest that K13-based gene therapy approaches may have utility for the treatment of patients with NEMO mutations and immunodeficiency.