Exosomes secreted under hypoxia enhance stemness in Ewing's sarcoma through miR-210 delivery.

Intercellular communication between tumor cells within the hypoxic microenvironment promote aggressiveness and poor patient prognoses for reasons that remain unclear. Here we show that hypoxic Ewing's sarcoma (EWS) cells release exosomes that promote sphere formation, a stem-like phenotype, in EWS cells by enhancing survival. Analysis of the hypoxic exosomal miRNA cargo identified a HIF-1α regulated miRNA, miR-210, as a potential mediator of sphere formation in cells exposed to hypoxic exosomes. Knockdown of HIF-1α in hypoxic EWS cells led to decreased exosomal miR-210 levels and reduced the capacity of hypoxic exosomes to form spheres. Inhibition of miR-210 in hypoxic spheres attenuated sphere formation and overexpression of miR-210 in normoxic spheres significantly enhanced the number of EWS spheres. Our results indicate that hypoxic exosomal miR-210 targets the proapoptotic protein CASP8AP2 in recipient cells. Moreover, the suppression of CASP8AP2 led to a reduction in apoptotic cells and increased sphere formation. Together, the findings in this study suggest that hypoxic exosomes promote stemness in EWS cells by delivering enriched miR-210 that is capable of down-regulating apoptotic pathways, resulting in the survival of cells with increased sphere formation. Future studies will further investigate the effects of EWS derived exosomal miRNAs on target genes and the role these interactions play in driving aggressiveness in hypoxic EWS tumors.

Long non-coding RNA profiling of pediatric Medulloblastoma.

BACKGROUND: Medulloblastoma (MB) is one of the most common malignant cancers in children. MB is primarily classified into four subgroups based on molecular and clinical characteristics as (1) WNT (2) Sonic-hedgehog (SHH) (3) Group 3 (4) Group 4. Molecular characteristics used for MB classification are based on genomic and mRNAs profiles. MB subgroups share genomic and mRNA profiles and require multiple molecular markers for differentiation from each other. Long non-coding RNAs (lncRNAs) are more than 200 nucleotide long RNAs and primarily involve in gene regulation at epigenetic and post-transcriptional levels. LncRNAs have been recognized as diagnostic and prognostic markers in several cancers. However, the lncRNA expression profile of MB is unknown. METHODS: We used the publicly available gene expression datasets for the profiling of lncRNA expression across MB subgroups. Functional analysis of differentially expressed lncRNAs was accomplished by Ingenuity pathway analysis (IPA). RESULTS: In the current study, we have identified and validated the lncRNA expression profile across pediatric MB subgroups and associated molecular pathways. We have also identified the prognostic significance of lncRNAs and unique lncRNAs associated with each MB subgroup. CONCLUSIONS: Identified lncRNAs can be used as single biomarkers for molecular identification of MB subgroups that warrant further investigation and functional validation.

A Novel Combination Approach Targeting an Enhanced Protein Synthesis Pathway in MYC-driven (Group 3) Medulloblastoma.

The MYC oncogene is frequently amplified in patients with medulloblastoma, particularly in group 3 patients, who have the worst prognosis. mTOR signaling-driven deregulated protein synthesis is very common in various cancers, including medulloblastoma, that can promote MYC stabilization. As a transcription factor, MYC itself is further known to regulate transcription of several components of protein synthesis machinery, leading to an enhanced protein synthesis rate and proliferation. Thus, inhibiting enhanced protein synthesis by targeting the MYC and mTOR pathways together may represent a highly relevant strategy for the treatment of MYC-driven medulloblastoma. Here, using siRNA and small-molecule inhibitor approaches, we evaluated the effects of combined inhibition of MYC transcription and mTOR signaling on medulloblastoma cell growth/survival and associated molecular mechanism(s) in MYC-amplified (group 3) medulloblastoma cell lines and xenografts. Combined inhibition of MYC and mTOR synergistically suppressed medulloblastoma cell growth and induced G1 cell-cycle arrest and apoptosis. Mechanistically, the combined inhibition significantly downregulated the expression levels of key target proteins of MYC and mTOR signaling. Our results with RNA-sequencing revealed that combined inhibition synergistically modulated global gene expression including MYC/mTOR components. In addition, the combination treatment significantly delayed tumor growth and prolonged survival of MYC-amplified medulloblastoma xenografted mice by downregulating expression of MYC and the key downstream components of mTOR signaling, compared with single-agent therapy. Together, our findings demonstrated that dual inhibition of MYC (transcription) and mTOR (translation) of the protein synthesis pathway can be a novel therapeutic approach against MYC-driven medulloblastoma.

Role of protein arginine methyltransferase 5 in group 3 (MYC-driven) Medulloblastoma.

BACKGROUND: MYC amplification or overexpression is common in Group 3 medulloblastoma and is associated with the worst prognosis. Recently, protein arginine methyl transferase (PRMT) 5 expression has been closely associated with aberrant MYC function in various cancers, including brain tumors such as glioblastoma. However, the role of PRMT5 and its association with MYC in medulloblastoma have not been explored. Here, we report the role of PRMT5 as a novel regulator of MYC and implicate PRMT5 as a potential therapeutic target in MYC-driven medulloblastoma. METHODS: Expression and association between PRMT5 and MYC in primary medulloblastoma tumors were investigated using publicly available databases. Expression levels of PRMT5 protein were also examined using medulloblastoma cell lines and primary tumors by western blotting and immunohistochemistry, respectively. Using MYC-driven medulloblastoma cells, we examined the physical interaction between PRMT5 and MYC by co-immunoprecipitation and co-localization experiments. To determine the functional role of PRMT5 in MYC-driven medulloblastoma, PRMT5 was knocked-down in MYC-amplified cells using siRNA and the consequences of knockdown on cell growth and MYC expression/stability were investigated. In vitro therapeutic potential of PRMT5 in medulloblastoma was also evaluated using a small molecule inhibitor, EPZ015666. RESULTS: We observed overexpression of PRMT5 in MYC-driven primary medulloblastoma tumors and cell lines compared to non-MYC medulloblastoma tumors and adjacent normal tissues. We also found that high expression of PRMT5 is inversely correlated with patient survival. Knockdown of PRMT5 using siRNA in MYC-driven medulloblastoma cells significantly decreased cell growth and MYC expression. Mechanistically, we found that PRMT5 physically associated with MYC by direct protein-protein interaction. In addition, a cycloheximide chase experiment showed that PRMT5 post-translationally regulated MYC stability. In the context of therapeutics, we observed dose-dependent efficacy of PRMT5 inhibitor EPZ015666 in suppressing cell growth and inducing apoptosis in MYC-driven medulloblastoma cells. Further, the expression levels of PRMT5 and MYC protein were downregulated upon EPZ015666 treatment. We also observed a superior efficacy of this inhibitor against MYC-amplified medulloblastoma cells compared to non-MYC-amplified medulloblastoma cells, indicating specificity. CONCLUSION: Our results reveal the regulation of MYC oncoprotein by PRMT5 and suggest that targeting PRMT5 could be a potential therapeutic strategy for MYC-driven medulloblastoma.

Improved therapy for medulloblastoma: targeting hedgehog and PI3K-mTOR signaling pathways in combination with chemotherapy.

Aberrant activation and interactions of hedgehog (HH) and PI3K/AKT/mTOR signaling pathways are frequently associated with high-risk medulloblastoma (MB). Thus, combined targeting of the HH and PI3K/AKT/mTOR pathways could be a viable therapeutic strategy to treat high-risk patients. Therefore, we investigated the anti-MB efficacies of combined HH inhibitor Vismodegib and PI3K-mTOR dual-inhibitor BEZ235 together or combined individually with cisplatin against high-risk MB. Using non-MYC- and MYC-amplified cell lines, and a xenograft mouse model, the in vitro and in vivo efficacies of these therapies on cell growth/survival and associated molecular mechanism(s) were investigated. Results showed that combined treatment of Vismodegib and BEZ235 together, or with cisplatin, significantly decreased MB cell growth/survival in a dose-dependent-fashion. Corresponding changes in the expression of targeted molecules following therapy were observed. Results demonstrated that inhibitors not only suppressed MB cell growth/survival when combined, but also significantly enhanced cisplatin-mediated cytotoxicity. Of these combinations, BEZ235 exhibited a significantly greater efficacy in enhancing cisplatin-mediated MB cytotoxicity. Results also demonstrated that the MYC-amplified MB lines showed a higher sensitivity to combined therapies compared to non-MYC-amplified cell lines. Therefore, we tested the efficacy of combined approaches against MYC-amplified MB growing in NSG mice. In vivo results showed that combination of Vismodegib and BEZ235 or their combination with cisplatin, significantly delayed MB tumor growth and increased survival of xenografted mice by targeting HH and mTOR pathways. Thus, our studies lay a foundation for translating these combined therapeutic strategies to the clinical setting to determine their efficacies in high-risk MB patients.

Heterogeneity of functional properties of Clone 66 murine breast cancer cells expressing various stem cell phenotypes.

INTRODUCTION: Breast cancer grows, metastasizes and relapses from rare, therapy resistant cells with a stem cell phenotype (cancer stem cells/CSCs). However, there is a lack of studies comparing the functions of CSCs isolated using different phenotypes in order to determine if CSCs are homogeneous or heterogeneous. METHODS: Cells with various stem cell phenotypes were isolated by sorting from Clone 66 murine breast cancer cells that grow orthotopically in immune intact syngeneic mice. These populations were compared by in vitro functional assays for proliferation, growth, sphere and colony formation; and in vivo limiting dilution analysis of tumorigenesis. RESULTS: The proportion of cells expressing CD44(high)CD24(low/neg), side population (SP) cells, ALDH1(+), CD49f(high), CD133(high), and CD34(high) differed, suggesting heterogeneity. Differences in frequency and size of tumor spheres from these populations were observed. Higher rates of proliferation of non-SP, ALDH1(+), CD34(low), and CD49f(high) suggested properties of transit amplifying cells. Colony formation was higher from ALDH1(-) and non-SP cells than ALDH1(+) and SP cells suggesting a progenitor phenotype. The frequency of clonal colonies that grew in agar varied and was differentially altered by the presence of Matrigel™. In vivo, fewer cells with a stem cell phenotype were needed for tumor formation than "non-stem" cells. Fewer SP cells were needed to form tumors than ALDH1(+) cells suggesting further heterogeneities of cells with stem phenotypes. Different levels of cytokines/chemokines were produced by Clone 66 with RANTES being the highest. Whether the heterogeneity reflects soluble factor production remains to be determined. CONCLUSIONS: These data demonstrate that Clone 66 murine breast cancer cells that express stem cell phenotypes are heterogeneous and exhibit different functional properties, and this may also be the case for human breast cancer stem cells.

Novel therapy for therapy-resistant mantle cell lymphoma: multipronged approach with targeting of hedgehog signaling.

Mantle cell lymphoma (MCL) is one of the most aggressive B-cell lymphomas with a median patient survival of only 5-7 years. The failure of existing therapies is mainly due to disease relapse when therapy-resistant tumor cells remain after chemotherapy. Therefore, development and testing of novel therapeutic strategies to target these therapy-resistant MCL are needed. Here, we developed an in vivo model of therapy-resistant MCL by transplanting a patient-derived MCL cell line (Granta 519) into NOD/SCID mice followed by treatment with combination chemotherapy. Cytomorphologic, immunophenotypic, in vitro and in vivo growth analyses of these therapy-resistant MCL cells confirm their MCL origin and resistance to chemotherapy. Moreover, quantitative real-time PCR revealed the upregulation of GLI transcription factors, which are mediators of the hedgehog signaling pathway, in these therapy-resistant MCL cells. Therefore, we developed an effective therapeutic strategy for resistant MCL by treating the NOD/SCID mice bearing Granta 519 MCL with CHOP chemotherapy to reduce tumor burden combined with GLI-antisense oligonucleotides or bortezomib, a proteosome inhibitor, to target therapy-resistant MCL cells that remained after chemotherapy. This regimen was followed by treatment with MCL-specific cytotoxic T lymphocytes to eliminate all detectable leftover minimal residual disease. Mice treated with this strategy showed a significantly increased survival and decreased tumor burden compared to the mice in all other groups. Such therapeutic strategies that combine chemotherapy with targeted therapy followed by tumor-specific immunotherapy are effective and have excellent potential for clinical application to provide long-term, disease-free survival in MCL patients.

A comparative evaluation of teaching methods in an introductory neuroscience course for physical therapy students.

BACKGROUND: The use of computer-based instruction (CBI) in physical therapy education is growing, but its effectiveness compared to lecture is undefined. This study compared CBI to lecture in an introductory neuroscience course for students in their first year of a 3 year professional program leading to the Doctor of Physical Therapy Degree. SUBJECTS: Twenty-eight students participated in 2003 and 34 in 2004. METHODS: A randomized, cross-over design was employed. The course was divided into two sections with an exam after each. Students in one group participated in CBI during the first half of the course and lecture during the second half with the order of participation reversed for the other group. A 6 months post-course review exam was also administered. Exam scores, study time, and student opinions regarding teaching methods were collected after each half of the course. Course development costs for both teaching approaches were also documented. RESULTS: There were no statistically significant differences in exam scores between participant groups. CBI students spent less time studying. Student did not distinguish a major preference for either instruction method. Many students preferred that CBI be used as a complementary rather than mutually exclusive instructional method. Lecture-based instruction was much less expensive than CBI. CONCLUSION: Lecture-based instruction was more cost effective than CBI, but CBI was more time efficient in terms of student learning.

Does the number or quality of pluripotent bone marrow stem cells decrease with age?

In the next 25 years, as the 37 million baby boomers age, we can expect a 400% increase in total joint arthroplasties that will challenge surgeons, the healthcare system, and the scientific community. The burden may be eased if we are able to manipulate side population stem cells and enhance peri-prosthetic bone remodeling thereby reducing the incidence of revisions. Therefore, as a preliminary question, we asked if the number and quality of side population stem cells, with the ability to proliferate into multiple cell lineages for long periods, correlates with age and can be evaluated in peripheral blood. Using flow cytometry we analyzed the quantity and quality of side population stem cells from bone marrow and peripheral blood in 54 patients (20 under 60 years of age, 34 over) undergoing THA. The total side population of stem cells decreased with age, but their long-term repopulating ability (quality) remained constant. The total count of side population stem cells in marrow correlated with the number found in peripheral blood. If these populations can be manipulated, periprosthetic remodeling may be beneficially enhanced.

Promises and pitfalls of stem cell therapy for promotion of bone healing.

UNLABELLED: There is promise in combining stem cells with allogeneic bone matrix to promote bone healing. Murine bone marrow, peripheral blood, and compact bone cells were transplanted ectopically under the kidney capsule in mice, alone or in combination with allogeneic matrix products: powder and putty to determine their bone forming potential in comparison to transplanted femoral bone fragments and long-term cultured bone marrow cells. The end point was the amount of bone formed as determined by quantitative histology. Mononuclear cells from marrow, peripheral blood, or bone alone transplanted under the kidney capsule did not form bone. Mononuclear cell populations did not combine readily with matrix products and there was in vivo migration of the transplanted combinations. Kidney subcapsular transplanted cultured bone marrow cells formed bone in proportion to the culture period, but after 9 weeks, the extent was only 20% by area of that of similarly transplanted femoral bone fragments. An inductive stimulus for bone formation seemed necessary. Osteoprogenitor cells were not detected in significant numbers in blood unless high doses of cytokines were administered. A better definition of the optimal cell populations and manipulations required for promotion of bone healing is needed along with new (transplant) models that allow for cell tracking. Much work remains to overcome current pitfalls in the use of stem cells to promote allograft integration and bone healing. LEVEL OF EVIDENCE: Therapeutic study, Level V (expert opinion). See the Guidelines for Authors for a complete description of levels of evidence.

Cells derived from the circulation contribute to the repair of lung injury.

Bone marrow (stem/progenitor) cells have been shown to "differentiate" into cells in multiple tissues, including lung. A low number of hematopoietic stem/progenitor cells also circulate in peripheral blood. The physiologic roles of these cells are still uncertain. This study was designed to test, using parabiotic mice that were joined surgically, whether stem/progenitor cells in blood contributed to the regeneration of lung after injury. Parabiotic mice were generated surgically by joining green fluorescent protein transgenic mice and wild-type littermates. These mice developed a common circulation (approximately 50% green cells in blood) by 2 weeks after surgery. The wild-type mouse was either uninjured or lethally irradiated or received intratracheal elastase or the combination of radiation with intratracheal elastase injection. Radiation or the combination of radiation with elastase significantly increased the proportion of bright green cells in the lungs of the wild-type mice. Morphologically, interstitial monocytes/macrophages, subepithelial fibroblast-like interstitial cells, and additionally type I alveolar epithelial cells immunostained for green fluorescent protein in wild-type mice. Approximately 5 to 20% of lung fibroblasts primary cultured from injured wild-type mice were green fluorescent protein expressing cells, indicating their blood derivation. This study demonstrates that stem/progenitor cells in blood contribute to the repair of lung injury in irradiated mice.

Lung cells transplanted to irradiated recipients generate lymphohematopoietic progeny.

Bone marrow (stem) cells can differentiate into cells in multiple tissues, including lung. Conversely, there are reports that cells of nonhematopoietic tissues (brain, muscle) can give rise to lymphohematopoietic cells. Here we show that the lung contains cells capable of giving rise to lymphohematopoietic cells when transplanted to irradiated recipients. Whole lung cell suspensions, lung side population (SP) cells, and CD45(+/-) lung cells obtained from male transgenic enhanced green fluorescent protein-expressing mice were transplanted intravenously to total body irradiated female mice. Green fluorescent cells were recovered from the circulation and phenotyped for their expression of lymphohematopoietic markers (CD3, CD4, CD8, B220, Gr-1, and Mac-1). Lung SP cells were composed of heterogeneous populations and had less ability to give rise to lymphohematopoietic cells than did bone marrow SP cells. Furthermore, the ability of cells from the lung of aged mice to generate lymphohematopoietic progeny was equivalent to that of cells from young mice. Cells from lung with radioprotective and lymphohematopoietic reconstituting abilities were CD45(+). CD45(+) cells in the lung cells have lymphohematopoietic stem/progenitor cell characteristics, and this has implications for cell or gene therapy applications.

Occult tumor cells detected in autologous blood stem cell harvests have no impact on 5 year outcomes for breast cancer patients with 4-9 positive nodes treated with adjuvant high-dose therapy and stem cell transplantation.

Breast cancer cells have been detected in autologous blood stem cell collections of early stage breast cancer patients, but their clinical significance is undefined. From October 1993 to August 1998, 32 consecutive Stage II breast cancer patients with 4-9-positive nodes underwent stem cell apheresis. The patients were treated with cyclophosphamide 1.75 gm/m(2), etoposide 400 mg/m(2) and cisplatin 50 mg/m(2) daily for 3 days, followed by infusion of the autologous cells. Cytospins of cells from each apheresis collections and from an aliquot of three pooled collections were examined for cytokeratin expression using an immunocytochemical assay. The cells were considered positive for tumor if at least one cell with tumor morphology stained positively for cytokeratin. Negative aliquots were confirmed with RT-PCR. Six patients (19%) had positive collections. In total, 24 patients (75%) were disease free a median of 61 (30-86) months after transplant. Eight patients relapsed at a median of 17 (8-27) months after transplant. Four of the disease-free patients and two of the relapsed patients had positive apheresis collections. There was no significant correlation between the presence of detectable tumor cells in the graft product and outcome.

A murine model of bone marrow micrometastasis in breast cancer.

Bone marrow (BM) is one of the most common sites and often the first clinical indication of metastatic progression of breast cancer. Multivariate analyses have shown that the presence of cytokeratin positive tumor cells in the marrow of women with newly diagnosed stage I, II or III breast cancer is an independent predictor of survival. The objective of this study was to develop an orthotopic model of spontaneous BM metastasis to facilitate studies of this process. A murine mammary adenocarcinoma cell line, Clone 66, was transduced with the neomycin resistance gene (Cl66neo) and injected orthotopically into female Balb/c mice. Polymerase chain reaction (PCR) for the neo gene performed on BM cells harvested from tumor bearing mice demonstrated as few as 10(2) injected tumor cells produced BM micrometastases at 4 weeks postinjection. Small foci of tumor cells were identified in the mammary fatpad (mfp) without gross evidence of primary tumors. Higher doses of tumor cells produced BM micrometastases, detectable by PCR, at one week post-injection. Constructs containing green fluorescent protein (GFP) and the neomycin resistance gene (neo) were also transduced into Clone 66 cells (Cl66-GFPneo) and injected into the mfp. GFP transduced tumor cells were identified in multiple tissues in addition to BM by flow cytometric analysis (FACS) but less 13% of the animals developed gross metastases. This model is a clinically relevant tool for the analysis of organ specificity of metastasis.

Effect of prior therapy and bone marrow metastases on progenitor cell content of blood stem cell harvests in breast cancer patients.

This study was designed to examine the relationship of prior therapy, bone marrow metastases, mobilization, and blood progenitor/stem cell (BSC) collection in breast cancer patients. Cells were collected from 19 breast cancer patients during steady state (nonmobilized group) and from 69 breast cancer patients after cytokine administration (mobilized group). Characteristics of the patients were compared with the cells obtained. A significant inverse association was found between the number of chemotherapy regimens the patients had received prior to BSC collection and the mononuclear cell (MNC) count of the product per liter of blood processed (LBP) with apheresis (P = .0006) and the granulocyte monocyte/macrophage colony-forming cell (GM-CFC) numbers per LBP (P = .0002). This association was evident in both mobilized and nonmobilized patients. Similar results were seen in those 25 patients who had received prior radiation therapy (MNC/LBP, P = .0003; GM-CFC/LBP, P = .0004). Patients in both the mobilized and nonmobilized groups with marrow metastases at the time of collection also had significantly lower levels of MNC/LBP (P = .0039) and GM-CFC/LBP (P = .0001) than did those without marrow metastases. The findings suggest that prior administration of radiation therapy and/or chemotherapy and the presence of marrow metastases all negatively impacted the collection of mobilized and nonmobilized progenitor cells from breast cancer patients. The mechanisms of this impact are not understood.