Karin M. McCarthy

Glycogen synthase kinase 3 inhibitors induce the canonical WNT/β-catenin pathway to suppress growth and self-renewal in embryonal rhabdomyosarcoma

Significance Embryonal rhabdomyosarcoma (ERMS) is a cancer of skeletal muscle and is one of the most common pediatric cancers of soft tissue. There is no effective treatment for patients with relapsed ERMS, with less than 50% surviving the disease. The self-renewing and molecularly defined tumor propagating cells (TPCs) drive continued tumor growth and relapse. Yet to date, drugs targeting ERMS self-renewal and differentiation of TPCs have not been identified. Our study describes a large-scale chemical screen to identify targetable pathways essential for modulating self-renewal and differentiation of ERMS and demonstrates the feasibility of inducing differentiation of TPCs in ERMS by small molecules. Embryonal rhabdomyosarcoma (ERMS) is a common pediatric malignancy of muscle, with relapse being the major clinical challenge. Self-renewing tumor-propagating cells (TPCs) drive cancer relapse and are confined to a molecularly definable subset of ERMS cells. To identify drugs that suppress ERMS self-renewal and induce differentiation of TPCs, a large-scale chemical screen was completed. Glycogen synthase kinase 3 (GSK3) inhibitors were identified as potent suppressors of ERMS growth through inhibiting proliferation and inducing terminal differentiation of TPCs into myosin-expressing cells. In support of GSK3 inhibitors functioning through activation of the canonical WNT/β-catenin pathway, recombinant WNT3A and stabilized β-catenin also enhanced terminal differentiation of human ERMS cells. Treatment of ERMS-bearing zebrafish with GSK3 inhibitors activated the WNT/β-catenin pathway, resulting in suppressed ERMS growth, depleted TPCs, and diminished self-renewal capacity in vivo. Activation of the canonical WNT/β-catenin pathway also significantly reduced self-renewal of human ERMS, indicating a conserved function for this pathway in modulating ERMS self-renewal. In total, we have identified an unconventional tumor suppressive role for the canonical WNT/β-catenin pathway in regulating self-renewal of ERMS and revealed therapeutic strategies to target differentiation of TPCs in ERMS.

Flow-cytometric and ultrastructural analysis of alveolar macrophage maturation.

Alveolar macrophages (AM) from adult and newborn rats were studied by flow cytometry and ultrastructural morphometry. We observed that the laser scatter and autofluorescent properties of newborn macrophages were different from those of adult cells. Relative to the adult AM, the forward‐angle laser scatter obtained with the newborn AM was reduced; this optical measurement appeared to correlate with the smaller mean size, as determined by ultrastructural and electronic volume measurements. The diminished right‐angle laser scatter (90° angle) correlated with the presence of fewer small, irregularly shaped lysosomal structures in the newborn AM, compared with AM from adult animals. AM from 1–2‐day‐old rats displayed large vacuoles containing multilamellar structures, which proved to be less effective at scattering light. Cells from newborn rats were less autofluorescent, a finding that appeared to correlate best with the numbers of secondary lysosomes. Flow cytometry may be used to discern structural alterations that occur during the maturation of AM. These changes correlate well with quantitative ultrastructural analyses of these cells.

Lung dendritic cells are primed by inhaled particulate antigens, and retain MHC class II/antigenic peptide complexes in hilar lymph nodes for a prolonged period of time.

Intratracheal (IT) administration of heat‐killed Listeria monocytogenes (HKL) results in an influx of macrophage and dendritic cell (DC) precursors into the lung interstitium. Low‐density, FcR+, interstitial lung cells isolated from rats instilled 24 hr before with HKL or vehicle alone, were > 90% Mar1+. After culturing with granulocyte–macrophage colony‐stimulating factor (GM‐CSF) for 3 days, up to 24% of the loosely adherent cells were DC that stimulated allogeneic T‐cell proliferation in an mixed lymphocyte reaction (MLR) assay. After only an overnight incubation with GM‐CSF, however, the capacity of interstitial Mar1+ cells to stimulate HKL immune T‐cell proliferation without exogenous antigen was low. By contrast, when DC were isolated as major histocompatibility complex (MHC) class II+ cells from rat lungs at 1, 3, 7 and 14 days after HKL instillation and cultured overnight with GM‐CSF, their antigen presentation capacity without added exogenous antigen was robust, but declined over the 2‐week period. Interestingly, hilar lymph node DC maintained their HKL antigen‐presenting capacity for up to 2 weeks after instillation of HKL. Following IT administration of PKH‐26 labelled HKL, fluorescent or immunolabelled organisms were detected in OX62+ DC in airway epithelium, lung interstitium and hilar lymph nodes in situ and in MHC class II+ DC isolated from these sites. We conclude that newly immigrated Mar1+ lung DC precursors, while efficient in endocytosing particulate antigens, are incapable of eliciting a significant proliferative response from HKL‐sensitized T cells. By contrast, MHC class II+ DC isolated from lungs and incubated overnight with GM‐CSF induce vigorous antigen‐specific T‐cell proliferation. Antigen‐loaded lung DC in hilar lymph nodes maintain their antigen presentation capacity for up to 2 weeks.

Cholesterol efflux stimulates metalloproteinase-mediated cleavage of occludin and release of extracellular membrane particles containing its C-terminal fragments.

That changes in membrane lipid composition alter the barrier function of tight junctions illustrates the importance of the interactions between tetraspan integral tight junction proteins and lipids of the plasma membrane. Application of methyl-beta-cyclodextrin to both apical and basolateral surfaces of MDCK cell monolayers for 2 h, results in an approximately 80% decrease in cell cholesterol, a fall in transepithelial electrical resistance, and a 30% reduction in cell content of occludin, with a smaller reduction in levels of claudins-2, -3, and -7. There were negligible changes in levels of actin and the two non-tight junction membrane proteins GP-135 and caveolin-1. While in untreated control cells breakdown of occludin, and probably other tight junction proteins, is mediated by intracellular proteolysis, our current data suggest an alternative pathway whereby in a cholesterol-depleted membrane, levels of tight junction proteins are decreased via direct release into the intercellular space as components of membrane-bound particles. Occludin, along with two of its degradation products and several claudins, increases in the basolateral medium after incubation with methyl-beta-cyclodextrin for 30 min. In contrast caveolin-1 is detected only in the apical medium after adding methyl-beta-cyclodextrin. Release of occludin and its proteolytic fragments continues even after removal of methyl-beta-cyclodextrin. Sedimentation and ultrastructural studies indicate that the extracellular tight junction proteins are associated with the membrane-bound particles that accumulate between adjacent cells. Disruption of the actin filament network by cytochalasin D did not diminish methyl-beta-cyclodextrin-induced release of tight junction proteins into the medium, suggesting that the mechanism underlying their formation is not actin-dependent. The 41- and 48-kDa C-terminal occludin fragments formed during cholesterol depletion result from the action of a GM6001-sensitive metalloproteinase(s) at some point in the path leading to release of the membrane particles.

Myogenic regulatory transcription factors regulate growth in rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is a pediatric malignacy of muscle with myogenic regulatory transcription factors MYOD and MYF5 being expressed in this disease. Consensus in the field has been that expression of these factors likely reflects the target cell of transformation rather than being required for continued tumor growth. Here, we used a transgenic zebrafish model to show that Myf5 is sufficient to confer tumor-propagating potential to RMS cells and caused tumors to initiate earlier and have higher penetrance. Analysis of human RMS revealed that MYF5 and MYOD are mutually-exclusively expressed and each is required for sustained tumor growth. ChIP-seq and mechanistic studies in human RMS uncovered that MYF5 and MYOD bind common DNA regulatory elements to alter transcription of genes that regulate muscle development and cell cycle progression. Our data support unappreciated and dominant oncogenic roles for MYF5 and MYOD convergence on common transcriptional targets to regulate human RMS growth. DOI: http://dx.doi.org/10.7554/eLife.19214.001

The NOTCH1/SNAIL1/MEF2C Pathway Regulates Growth and Self-Renewal in Embryonal Rhabdomyosarcoma

Summary Tumor-propagating cells (TPCs) share self-renewal properties with normal stem cells and drive continued tumor growth. However, mechanisms regulating TPC self-renewal are largely unknown, especially in embryonal rhabdomyosarcoma (ERMS)—a common pediatric cancer of muscle. Here, we used a zebrafish transgenic model of ERMS to identify a role for intracellular NOTCH1 (ICN1) in increasing TPCs by 23-fold. ICN1 expanded TPCs by enabling the de-differentiation of zebrafish ERMS cells into self-renewing myf5+ TPCs, breaking the rigid differentiation hierarchies reported in normal muscle. ICN1 also had conserved roles in regulating human ERMS self-renewal and growth. Mechanistically, ICN1 up-regulated expression of SNAIL1, a transcriptional repressor, to increase TPC number in human ERMS and to block muscle differentiation through suppressing MEF2C, a myogenic differentiation transcription factor. Our data implicate the NOTCH1/SNAI1/MEF2C signaling axis as a major determinant of TPC self-renewal and differentiation in ERMS, raising hope of therapeutically targeting this pathway in the future.

Abstract 4733: Notch signaling increases the number of relapse-driving tumor propagating cells in embryonal rhabdomyosarcoma

Embryonal rhabdomyosarcoma (ERMS) is a devastating pediatric muscle cancer with extremely poor prognosis at relapse. Work from our group has identified the tumor-propagating cell (TPC) in a transgenic zebrafish model of kRASG12D-induced ERMS that is responsible for driving continued tumor growth and relapse. The TPC is molecularly similar to an activated muscle satellite-cell and expresses myf5, c-met, and m-cadherin. Building on these observations, we have identified the Notch pathway as a potent enhancer of ERMS self-renewal and TPC number. Specifically, bulk tumor limiting dilution cell transplantation experiments revealed that TPCs are increased 10-fold in kRASG12D expressing ERMS that co-express activated intracellular Notch1 (ICN1). This increase in TPC number is partly the result of ICN1 expressing ERMS exhibiting a 3-fold expansion of relapse-driving myf5-GFP+/mylz2-mCherry-negative ERMS-cell population. Unexpectedly, cell transplantation experiments revealed that Notch pathway activation also conferred tumor-propagating ability to the myf5-GFP+/mylz2-mCherry+ mid-differentiated ERMS cells - a population of cells previously shown to lack self-renewal capacity. Single cell engraftment studies uncovered that NOTCH activation caused cells to oscillate between the TPC and mid-differentiated ERMS molecular states that was not observed in kRASG12D-expressing ERMS, suggesting that Notch has important roles in both self-renewal and cell state transitions. Next, we validated our findings in human ERMS where NOTCH1 is highly expressed both in tumors and ERMS-cell lines. We show important roles for NOTCH in regulating self-renewal and differentiation in human ERMS. Specifically, human ERMS cells that expressed activated NOTCH1 had elevated sphere-colony formation, a surrogate for self-renewal in vitro. By contrast, shRNA knockdown of NOTCH1 resulted in decreased sphere-colony formation and robust terminal differentiation of ERMS cells into late-stage, myosin-expressing myoblasts. Moreover, we identified that NOTCH1 directly activated SNAI1 expression and was required for both efficient sphere formation and differentiation in ERMS cell lines. SNAI1 is commonly over-expressed in human ERMS and its expression is positively correlated with NOTCH1. Taken together, our data indicate that Notch signaling is an important modifier of human ERMS acting to regulate both TPC self-renewal and differentiation. Notch and/or SNAl1 pathway inhibition may have potential benefit for a subset of patients with relapsed ERMS. Citation Format: Myron Ignatius, Riadh Lobbardi, Madeline Hayes, Eleanor Chen, Karin McCarthy, G. Petur Nielsen, Brian Beleyea, Corinne Linardic, Javed Khan, Charles Keller, David M. Langenau. Notch signaling increases the number of relapse-driving tumor propagating cells in embryonal rhabdomyosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4733. doi:10.1158/1538-7445.AM2015-4733

Abstract A14: Canonical WNT/β-catenin pathway activation suppresses embryonal rhabdomyosarcoma growth and self-renewal

Embryonal rhabdomyosarcoma (ERMS) is a common pediatric malignancy of muscle with relapse being the major clinical challenge. Self-renewing tumor-propagating cells (TPCs) drive cancer relapse and are confined to a molecularly definable subset of ERMS cells. To identify drugs that suppress TPC function, a large-scale chemical screen comprising of ~40,000 compounds including 47% FDA-approved drugs was completed, identifying GSK3-inhibitors as potent suppressors of ERMS growth through inducing terminal differentiation of TPCs into myosin-expressing cells. In support of GSK3 inhibitors functioning through activation of the canonical WNT pathway, recombinant WNT3A and a stabilized β-catenin enhanced differentiation and reduced self-renewal in human ERMS cell lines. Moreover, treatment of tumor-bearing zebrafish with a GSK3 inhibitor activated the WNT/β-catenin pathway, resulting in suppressed ERMS growth, depleted TPCs, and diminished self-renewal capacity in vivo. As canonical WNT signaling is essential for transition from muscle stem cell proliferation to myogenic differentiation during regeneration, our findings suggest that the same developmental pathways that regulate muscle stem cell self-renewal also contribute to tumorigenesis in ERMS. GSK3 inhibitors are being assessed for their efficacy in inhibiting growth as well as inducing differentiation of human ERMS in xenograft mouse models. Our work has identified the vital role of canonical WNT pathway in regulating differentiation and self-renewal of ERMS and demonstrated the effective application of small molecule inhibitors to target differentiation of TPCs in ERMS. Citation Format: Eleanor Chen, Michael DeRan, Katherine Brooke Grandinetti, Myron Ignatius, Ryan Clagg, Karin McCarthy, Riadh Lobbardi, Xu Wu, David Langenau. Canonical WNT/β-catenin pathway activation suppresses embryonal rhabdomyosarcoma growth and self-renewal. [abstract]. In: Proceedings of the AACR Special Conference: The Translational Impact of Model Organisms in Cancer; Nov 5-8, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(11 Suppl):Abstract nr A14.

Abstract A67: Canonical WNT/β;-catenin pathway activation suppresses embryonal rhabdomyosarcoma growth and self-renewal

Embryonal rhabdomyosarcoma (ERMS) is a common pediatric malignancy of muscle with relapse being the major clinical challenge. Self-renewing tumor-propagating cells (TPCs) drive cancer relapse and are confined to a molecularly definable subset of ERMS cells. To identify drugs that suppress TPC function, a large-scale chemical screen comprising of ∼40,000 compounds including 47% FDA-approved drugs was completed, identifying GSK3-inhibitors as potent suppressors of ERMS growth through inducing terminal differentiation of TPCs into myosin-expressing cells. In support of GSK3 inhibitors functioning through activation of the canonical WNT pathway, recombinant WNT3A and a stabilized β-catenin enhanced differentiation and reduced self-renewal in human ERMS cell lines. Moreover, treatment of tumor-bearing zebrafish with a GSK3 inhibitor activated the WNT/β-catenin pathway, resulting in suppressed ERMS growth, depleted TPCs, and diminished self-renewal capacity in vivo. As canonical WNT signaling is essential for transition from muscle stem cell proliferation to myogenic differentiation during regeneration, our findings suggest that the same developmental pathways that regulate muscle stem cell self-renewal also contribute to tumorigenesis in ERMS. GSK3 inhibitors are being assessed for their efficacy in inhibiting growth as well as inducing differentiation of human ERMS in xenograft mouse models. Our work has identified the vital role of canonical WNT pathway in regulating differentiation and self-renewal of ERMS and demonstrated the effective application of small molecule inhibitors to target differentiation of TPCs in ERMS. Citation Format: Eleanor Chen, Michael DeRan, Katherine Brooke Grandinetti, Myron Ignatius, Ryan Clagg, Karin McCarthy, Riadh Lobbardi, Xu Wu, David Langenau. Canonical WNT/β;-catenin pathway activation suppresses embryonal rhabdomyosarcoma growth and self-renewal. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A67.