Embryonic Signaling Pathways and Rhabdomyosarcoma: Contributions to Cancer Development and Opportunities for Therapeutic Targeting

Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood and adolescence, accounting for approximately 7% of childhood cancers. Current therapies include nonspecific cytotoxic chemotherapy regimens, radiation therapy, and surgery; however, these multimodality strategies are unsuccessful in the majority of patients with high-risk disease. It is generally believed that these tumors represent arrested or aberrant skeletal muscle development, and, accordingly, developmental signaling pathways critical to myogenesis such as Notch, WNT, and Hedgehog may represent new therapeutic targets. In this paper, we summarize the current preclinical studies linking these embryonic pathways to rhabdomyosarcoma tumorigenesis and provide support for the investigation of targeted therapies in this embryonic cancer

Imaging Primary Mouse Sarcomas After Radiation Therapy Using Cathepsin-Activatable Fluorescent Imaging Agents

Purpose: Cathepsin-activated fluorescent probes can detect tumors in mice and in canine patients. We previously showed that these probes can detect microscopic residual sarcoma in the tumor bed of mice during gross total resection. Many patients with soft tissue sarcoma (STS) and other tumors undergo radiation therapy (RT) before surgery. This study assesses the effect of RT on the ability of cathepsin-activated probes to differentiate between normal and cancerous tissue. Methods and Materials: A genetically engineered mouse model of STS was used to generate primary hind limb sarcomas that were treated with hypofractionated RT. Mice were injected intravenously with cathepsin-activated fluorescent probes, and various tissues, including the tumor, were imaged using a hand-held imaging device. Resected tumor and normal muscle samples were harvested to assess cathepsin expression by Western blot. Uptake of activated probe was analyzed by flow cytometry and confocal microscopy. Parallel in vitro studies using mouse sarcoma cells were performed. Results: RT of primary STS in mice and mouse sarcoma cell lines caused no change in probe activation or cathepsin protease expression. Increasing radiation dose resulted in an upward trend in probe activation. Flow cytometry and immunofluorescence showed that a substantial proportion of probe-labeled cells were CD11b-positive tumor-associated immune cells. Conclusions: In this primary murine model of STS, RT did not affect the ability of cathepsin-activated probes to differentiate between tumor and normal muscle. Cathepsin-activated probes labeled tumor cells and tumor-associated macrophages. Our results suggest that it would be feasible to include patients who have received preoperative RT in clinical studies evaluating cathepsin-activated imaging probes.Damon Runyon Cancer Research Foundation (Damon Runyon-Rachleff Innovation Award

Efficacy of Phosphatidylinositol-3 Kinase Inhibitors in a Primary Mouse Model of Undifferentiated Pleomorphic Sarcoma

Recent advances in sarcoma genomics have identified novel mutations in the PI3K pathway in human sarcomas. Here, we use a mouse model of primary soft-tissue sarcoma for preclinical testing of doxorubicin and inhibitors of the PI3K pathway: BKM120 (PI3K inhibitor) and BEZ235 (a dual PI3K/mTOR inhibitor). Doxorubicin-treated tumors (n = 15) showed a partial response rate of 6.6%, just as the majority of human sarcomas do not respond to doxorubicin. Treatment with BKM120 elicited a partial response in 50% of tumors (n = 10), which was also seen in combination with doxorubicin (n = 10). Additionally, BKM120 treatment produced a robust delay in tumor growth kinetics. BEZ235-treated tumors (n = 9) showed a complete response rate of 11.1%. Combining BEZ235 with doxorubicin (n = 10) increased the complete response rate to 50% (P = 0.035). These studies demonstrate that PI3K pathway inhibition is a viable and attractive target for soft-tissue sarcomas

A Novel Imaging System Permits Real-time in Vivo Tumor Bed Assessment After Resection of Naturally Occurring Sarcomas in Dogs

Background Treatment of soft tissue sarcoma (STS) includes complete tumor excision. However, in some patients, residual sarcoma cells remain in the tumor bed. We previously described a novel hand-held imaging device prototype that uses molecular imaging to detect microscopic residual cancer in mice during surgery. Questions/purposes To test this device in a clinical trial of dogs with naturally occurring sarcomas, we asked: (1) Are any adverse clinical or laboratory effects observed after intravenous administration of the fluorescent probes? (2) Do canine sarcomas exhibit fluorescence after administration of the cathepsin-activated probe? (3) Is the tumor-to-background ratio sufficient to distinguish tumor from tumor bed? And (4) can residual fluorescence be detected in the tumor bed during surgery and does this correlate with a positive margin? Methods We studied nine dogs undergoing treatment for 10 STS or mast cell tumors. Dogs received an intravenous injection of VM249, a fluorescent probe that becomes optically active in the presence of cathepsin proteases. After injection, tumors were removed by wide resection. The tumor bed was imaged using the novel imaging device to search for residual fluorescence. We determined correlations between tissue fluorescence and histopathology, cathepsin protease expression, and development of recurrent disease. Minimum followup was 9 months (mean, 12 months; range, 9–15 months). Results Fluorescence was apparent from all 10 tumors and ranged from 3 × 107 to 1 × 109 counts/millisecond/cm2. During intraoperative imaging, normal skeletal muscle showed no residual fluorescence. Histopathologic assessment of surgical margins correlated with intraoperative imaging in nine of 10 cases; in the other case, there was no residual fluorescence, but tumor was found at the margin on histologic examination. No animals had recurrent disease at 9 to 15 months. Conclusions These initial findings suggest this imaging system might be useful to intraoperatively detect residual tumor after wide resections. Clinical Relevance The ability to assess the tumor bed intraoperatively for residual disease has the potential to improve local control

p53 Regulates Progenitor Cell Quiescence and Differentiation in the Airway

SummaryMechanisms that regulate progenitor cell quiescence and differentiation in slowly replacing tissues are not fully understood. Here, we demonstrate that the tumor suppressor p53 regulates both proliferation and differentiation of progenitors in the airway epithelium. p53 loss decreased ciliated cell differentiation and increased the self-renewal and proliferative capacity of club progenitors, increasing epithelial cell density. p53-deficient progenitors generated a pseudostratified epithelium containing basal-like cells in vitro and putative bronchioalveolar stem cells in vivo. Conversely, an additional copy of p53 increased quiescence and ciliated cell differentiation, highlighting the importance of tight regulation of p53 levels. Using single-cell RNA sequencing, we found that loss of p53 altered the molecular phenotype of progenitors and differentially modulated cell-cycle regulatory genes. Together, these findings reveal that p53 is an essential regulator of progenitor cell behavior, which shapes our understanding of stem cell quiescence during homeostasis and in cancer development

Bone Protection by Inhibition of MicroRNA-182

Targeting microRNAs recently shows significant therapeutic promise; however, such progress is underdeveloped in treatment of skeletal diseases with osteolysis, such as osteoporosis and rheumatoid arthritis (RA). Here, we identified miR-182 as a key osteoclastogenic regulator in bone homeostasis and diseases. Myeloid-specific deletion of miR-182 protects mice against excessive osteoclastogenesis and bone resorption in disease models of ovariectomy-induced osteoporosis and inflammatory arthritis. Pharmacological treatment of these diseases with miR-182 inhibitors completely suppresses pathologic bone erosion. Mechanistically, we identify protein kinase double-stranded RNA-dependent (PKR) as a new and essential miR-182 target that is a novel inhibitor of osteoclastogenesis via regulation of the endogenous interferon (IFN)-β-mediated autocrine feedback loop. The expression levels of miR-182, PKR, and IFN-β are altered in RA and are significantly correlated with the osteoclastogenic capacity of RA monocytes. Our findings reveal a previously unrecognized regulatory network mediated by miR-182-PKR-IFN-β axis in osteoclastogenesis, and highlight the therapeutic implications of miR-182 inhibition in osteoprotection

Deception in Research on the Placebo Effect

A common feature of research investigating the placebo effect is deception of research participants about the nature of the research. Miller and colleagues examine the ethical issues surrounding such deception

Expression of Distal-less, dachshund, and optomotor blind in Neanthes arenaceodentata (Annelida, Nereididae) does not support homology of appendage-forming mechanisms across the Bilateria

The similarity in the genetic regulation of arthropod and vertebrate appendage formation has been interpreted as the product of a plesiomorphic gene network that was primitively involved in bilaterian appendage development and co-opted to build appendages (in modern phyla) that are not historically related as structures. Data from lophotrochozoans are needed to clarify the pervasiveness of plesiomorphic appendage forming mechanisms. We assayed the expression of three arthropod and vertebrate limb gene orthologs, Distal-less (Dll), dachshund (dac), and optomotor blind (omb), in direct-developing juveniles of the polychaete Neanthes arenaceodentata. Parapodial Dll expression marks premorphogenetic notopodia and neuropodia, becoming restricted to the bases of notopodial cirri and to ventral portions of neuropodia. In outgrowing cephalic appendages, Dll activity is primarily restricted to proximal domains. Dll expression is also prominent in the brain. dac expression occurs in the brain, nerve cord ganglia, a pair of pharyngeal ganglia, presumed interneurons linking a pair of segmental nerves, and in newly differentiating mesoderm. Domains of omb expression include the brain, nerve cord ganglia, one pair of anterior cirri, presumed precursors of dorsal musculature, and the same pharyngeal ganglia and presumed interneurons that express dac. Contrary to their roles in outgrowing arthropod and vertebrate appendages, Dll, dac, and omb lack comparable expression in Neanthes appendages, implying independent evolution of annelid appendage development. We infer that parapodia and arthropodia are not structurally or mechanistically homologous (but their primordia might be), that Dll’s ancestral bilaterian function was in sensory and central nervous system differentiation, and that locomotory appendages possibly evolved from sensory outgrowths

The Use of Radiation Therapy in Well-Differentiated Soft Tissue Sarcoma of the Extremities: An NCDB Review

Objective. This study investigated patterns of utilization of radiation therapy (RT) and correlated this with overall survival by assessing patients with well-differentiated soft tissue sarcoma of the extremity (STS-E) in the National Cancer Database (NCDB). Methods. All patients diagnosed with well-differentiated STS-E between 1998 and 2006 were identified in the NCDB. Patients were stratified by use of surgery alone versus use of adjuvant RT after surgery and analyzed using multivariate analysis, Kaplan-Meier analysis, and propensity matching. Results. 2113 patients with well-differentiated STS-E were identified in the NCDB for inclusion with a mean follow-up time of 74 months. 69% of patients were treated with surgery alone, while 26% were treated with surgery followed by adjuvant RT. Patients undergoing amputation were less likely to receive adjuvant RT. There was no difference in overall survival between patients with well-differentiated STS treated with surgery alone and those patients who received adjuvant RT. Conclusions. In the United States, adjuvant RT is being utilized in a quarter of patients being treated for well-differentiated STS-E. While the use of adjuvant RT may be viewed as a means to facilitate limb salvage, this large national database review confirms no survival benefit, regardless of tumor size or margin status