Efficacy of Tamoxifen, Alone or in Combination with Vincristine and Temsirolimus, in in vitro and in vivo Models of Rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood, with an estimated five-year overall survival of less than 30% in patients with metastatic disease. Despite refinements in risk stratification and multimodality chemotherapy, improvements in RMS survival have been minimal and few molecularly targeted therapies are currently available. Previously, we demonstrated that RMS cells express estrogen receptor (ER) and are sensitive to 4-hydroxytamoxifen (4OHT), an active metabolite of tamoxifen. Here, we investigate the mechanisms by which 4OHT exert these effects. Previously, our group also found that 4OHT-induced apoptosis involves ER-dependent phosphorylation of c-Jun N-terminal kinase (JNK). Here, we found that inhibition of the pro-survival AKT/mTOR pathway is involved in 4OHT-induced apoptosis as well. These mechanisms can be exploited by combining 4OHT with other drugs acting on the JNK and AKT/mTOR pathways: vincristine (a core RMS chemotherapy agent) enhances JNK phosphorylation, while temsirolimus (an emerging RMS drug currently in phase III trials) inhibits mTOR complex 1. Combining tamoxifen with either of these drugs led to increased efficacy against cell proliferation in vitro. In an alveolar RMS (ARMS) xenograft mouse model, tamoxifen causes significantly reduced tumour growth and increased apoptosis. In contrast, long-term tamoxifen treatment led to enhanced tumour growth in an embryonal RMS (ERMS) xenograft model. A tolerable dose and schedule was optimized for tamoxifen in combination with vincristine and temsirolimus in vivo. However, preclinical phase II studies found that vincristine in combination with temsirolimus had similar efficacy with or without tamoxifen, at the given dose and schedule. Taken together, our work provides in vivo confirmation that tamoxifen may be an effective agent for treatment of ARMS, and provides a mechanistic framework that explains the anti-RMS effects of tamoxifen and its ability to potentiate vincristine and temsirolimus.Ph.D

Vangl2/RhoA signaling regulates stem cell self-renewal programs and growth in Rhabdomyosarcoma

International audienceTumor growth and relapse are driven by tumor propagating cells (TPCs). However, mechanisms regulating TPC fate choices, maintenance, and self-renewal are not fully understood. Here, we show that Van Gogh-like 2 (Vangl2), a core regulator of the non-canonical Wnt/planar cell polarity (Wnt/PCP) pathway, affects TPC self-renewal in rhabdomyosarcoma (RMS)-a pediatric cancer of muscle. VANGL2 is expressed in a majority of human RMS and within early mononuclear progenitor cells. VANGL2 depletion inhibited cell proliferation, reduced TPC numbers, and induced differentiation of human RMS in vitro and in mouse xenografts. Using a zebrafish model of embryonal rhabdomyosarcoma (ERMS), we determined that Vangl2 expression enriches for TPCs and promotes their self-renewal. Expression of constitutively active and dominant-negative isoforms of RHOA revealed that it acts downstream of VANGL2 to regulate proliferation and maintenance of TPCs in human RMS. Our studies offer insights into pathways that control TPCs and identify new potential therapeutic targets

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