International audienceBackground: Circulating tumor cells (CTCs) are biomarkers for non-invasively measuring the evolution of tumor genotypes during treatment and disease progression. Recent technical progress has made it possible to detect and characterize CTCs at the single-cell level in blood. Content: Most current methods are based on epithelial cell adhesion molecule (EpCAM) detection, but numerous studies have demonstrated that EpCAM is not a universal marker for CTC detection since it fails to detect both carcinoma cells that undergo epithelial-mesenchymal transition (EMT), and CTCs of mesenchymal origin. Moreover, EpCAM expression has been found in patients with benign diseases. A large proportion of the current studies and reviews about CTCs describe EpCAM based methods, but there are evidences that not all tumor cells can be detected using this marker. Here we describe the most recent EpCAM-independent methods for enriching, isolating and characterizing CTCs, based on physical and biological characteristics, and point out their main advantages and disadvantages.Summary: CTCs offer an opportunity to obtain key biological information required for the development of personalized medicine. However there is no universal marker of these cells. To strengthen the clinical utility of CTCs, it is important to improve existing technologies and develop new, non-EpCAM based systems to enrich and isolate CTCs

The vicious cycle established between bone-associated tumours and bone resorption is the central problem with therapeutic strategies against primary bone tumours and bone metastasis. Here we report data to support inhibition of BET bromodomain proteins as a promising therapeutic strategy that target simultaneously the three partners of the vicious cycle. Treatment with JQ1, a BET bromodomain inhibitor, reduces cell viability of osteosarcoma cells and inhibits osteoblastic differentiation both in vitro and in vivo. These effects are associated with transcriptional silencing of MYC and RUNX2, resulting from the depletion of BRD4 from their respective loci. Moreover, JQ1 also inhibits osteoclast differentiation by interfering with BRD4-dependent RANKL activation of NFATC1 transcription. Collectively, our data indicate that JQ1 is a potent inhibitor of osteoblast and osteoclast differentiation as well as bone tumour development.

Ewing Sarcoma is a rare bone and soft tissue malignancy affecting children and young adults. Chromosomal translocations in this cancer produce fusion oncogenes as characteristic molecular signatures of the disease. The most common case is the translocation t (11; 22) (q24;q12) which yields the EWS-Fli1 chimeric transcription factor. Finding a way to directly target EWS-Fli1 remains a central therapeutic approach to eradicate this aggressive cancer. Here we demonstrate that treating Ewing Sarcoma cells with JQ1(+), a BET bromodomain inhibitor, represses directly EWS-Fli1 transcription as well as its transcriptional program. Moreover, the Chromatin Immuno Precipitation experiments demonstrate for the first time that these results are a consequence of the depletion of BRD4, one of the BET bromodomains protein from the EWS-Fli1 promoter. In vitro, JQ1(+) treatment reduces the cell viability, impairs the cell clonogenic and the migratory abilities, and induces a G1-phase blockage as well as a time- and a dose-dependent apoptosis. Furthermore, in our in vivo model, we observed a tumor burden delay, an inhibition of the global vascularization and an increase of the mice overall survival. Taken together, our data indicate that inhibiting the BET bromodomains interferes with EWS-FLi1 transcription and could be a promising strategy in the Ewing tumors context.

Primary cancer cell dissemination is a key event during the metastatic cascade, but context-specific determinants of this process remain largely undefined. Multiple reports have suggested that the p53 (TP53) family member p63 (TP63) plays an anti-metastatic role through its minor epithelial isoform containing the N-terminal transactivation domain (TAp63). However, the role and contribution of the major p63 isoform lacking this domain, ΔNp63α, remain largely undefined. Here, we report a distinct and TAp63-independent mechanism by which ΔNp63α-expressing cells within a TGFβ-rich microenvironment become positively selected for metastatic dissemination. Orthotopic transplantation of ΔNp63α-expressing human osteosarcoma cells into athymic mice resulted in larger and more frequent lung metastases than transplantation of control cells. Mechanistic investigations revealed that ΔNp63α repressed miR-527 and miR-665, leading to the upregulation of two TGFβ effectors, SMAD4 and TβRII (TGFBR2). Furthermore, we provide evidence that this mechanism reflects a fundamental role for ΔNp63α in the normal wound healing response. We show that ΔNp63α-mediated repression of miR-527/665 controls a TGFβ-dependent signaling node that switches off anti-migratory miR-198 by suppressing the expression of the regulatory factor, KSRP (KHSRP). Collectively, these findings reveal that a novel microRNA network involved in the regulation of physiological wound healing responses is hijacked and suppressed by tumor cells to promote metastatic dissemination.

Osteosarcoma and Ewing Sarcoma are the two most common types of Bone Sarcomas, principally localized at the long bones of the extremities and mainly affecting adolescents and young adults. Cisplatin is one of the current options in the therapeutic arsenal of drugs available to cure these aggressive cancers. Unfortunately, chemoresistance against this agent is still a major cause of patient relapse. Thus, a better understanding of the molecular pathways by which these drugs induce cancer cell death, together with a better delineation of the origins of chemoresistance are required to improve the success rate of current treatments. Furthermore, as p53 is frequently mutated in Bone Sarcomas, other pathways in these cancers must mediate drug-induced cell death. Here, we demonstrate for the first time that TAp73β, a p53-family protein, is implicated in Cisplatin-induced apoptosis of Bone Sarcomas'. Furthermore, while acquired resistance developed by cancer cells against such drugs can have multiple origins, it is now well accepted that epigenetic mechanisms involving microRNAs (miRNAs) are one of them. We show that miRNA-193a-5p modulates the viability, the clonogenic capacity and the Cisplatin-induced apoptosis of the Bone Sarcoma cells through inhibition of TAp73β. Collectively, these results shed light on the involvement of miR-193a-5p in Cisplatin chemoresistance of Bone Sarcomas', and they open the road to new therapeutic opportunities provided by targeting the miR-193a-5p/TAp73β axis in the context of these malignancies.

Histone modifications are important for maintaining the transcription program. BET proteins, an important class of "histone reading proteins", have recently been described as essential in bone biology. This study presents the therapeutic opportunity of BET protein inhibition in osteoporosis. We find that the pharmacological BET protein inhibitor JQ1 rescues pathologic bone loss in a post-ovariectomy osteoporosis model by increasing the trabecular bone volume and restoring mechanical properties. The BET protein inhibition suppresses osteoclast differentiation and activity as well as the osteoblastogenesis in vitro. Moreover, we show that treated non-resorbing osteoclasts could still activate osteoblast differentiation. In addition, specific inhibition of BRD4 using RNA interference inhibits osteoclast differentiation but strongly activates osteoblast mineralization activity. Mechanistically, JQ1 inhibits expression of the master osteoclast transcription factor NFATc1 and the transcription factor of osteoblast Runx2. These findings strongly support that targeting epigenetic chromatin regulators such as BET proteins may offer a promising alternative for the treatment of bone-related disorders such as osteoporosis.

The metastatic dissemination is a complex multistep process by which tumor cells from a primary site enter into the systemic circulation to finally spread at distant sites. Even if this mechanism is rare at the tumor level, it remains the major cause of Osteosarcoma-patients’ relapse and mortality. MicroRNAs (miRNAs) have recently been described as novel epigenetics’ genes’ expression regulators actively implicated in cancer progression and dissemination. The understanding of their implication in the metastatic spreading could help clinicians to improve the outcome of osteosarcoma. We established the miRNA’s expression-profile between primary bone-tumors (PTs), circulating tumor cells (CTCs) and lung metastatic (META) samples from in vivo mice xenograft models. Our results show that the expression level of the miR-198 and -206 was decreased in META samples, in which the expression of the metastasis-related receptor C-Met was up-regulated. Those expression variations were validated in osteosarcoma patient biopsies from matching primary tumors and lung metastasis. We validated in vitro the endogenous miRNAs inhibitory effects on both migration and invasion, as well as we confirmed by luciferase assays that the C-Met receptor is one of their bona-fide targets. The anti-metastatic effect of these miRNAs was also validated in vivo, as their direct injections into the tumors reduce the number of lung-metastases and prolongs the overall survival of the treated animals. All together, our results suggest the absence of the miR-198 and -206 as powerful predictive biomarkers of the tumor cell dissemination and the rationale of their potential therapeutic use in the treatment of Osteosarcoma.

TP53 (TP53), p73 (TP73), and p63 (TP63) are members of the p53 transcription factor family, which has many activities spanning from embryonic development through to tumor suppression. The utilization of two promoters and alternative mRNA splicing has been shown to yield numerous isoforms in p53, p63, and p73. TAp73 is thought to mediate apoptosis as a result of nuclear accumulation following chemotherapy-induced DNA damage, according to a number of studies. Overexpression of the nuclear ΔNp63 and ΔNp73 isoforms, on the other hand, suppresses TAp73’s pro-apoptotic activity in human malignancies, potentially leading to metastatic spread or inhibition. Another well-known pathway that has been associated to metastatic spread is the TGF pathway. TGFs are a family of structurally related polypeptide growth factors that regulate a variety of cellular functions including cell proliferation, lineage determination, differentiation, motility, adhesion, and cell death, making them significant players in development, homeostasis, and wound repair. Various studies have already identified several interactions between the p53 protein family and the TGFb pathway in the context of tumor growth and metastatic spread, beginning to shed light on this enigmatic intricacy.