Camille Jacques

Selective inhibition of BET bromodomain epigenetic signalling interferes with the bone-associated tumour vicious cycle

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.

Phytochemicals isolated from leaves of Chromolaena odorata: impact on viability and clonogenicity of cancer cell lines.

The leaves of Chromolaena odorata (Asteraceae) are exploited extensively in West and Central African ethnopharmacy for the treatment of a wide range of conditions, despite this being a non‐native species established in the last 50 years. With the objective of seeking bioactive principles, the nonvolatile compounds, an ethanolic (80% v/v) extract was made and fractionated. From the hexane‐soluble fraction, three compounds were isolated. Two of these, 5‐hydroxy‐7,4′‐dimethoxyflavanone and 2′‐hydroxy‐4,4′,5′,6′‐tetramethoxychalcone, have previously been identified in C. odorata leaves. The third was fully characterised spectroscopically and found to be 1,6‐dimethyl‐4‐(1‐methylethyl)naphthalene (cadalene), not previously isolated from the Asteraceae. All three compounds were tested for their cytotoxicity and anticancer properties. 2′‐Hydroxy‐4,4′,5′,6′‐tetramethoxychalcone was found to be both cytotoxic and anticlonogenic at 20 µm in cell lines Cal51, MCF7 and MDAMB‐468, and to act synergistically with the Bcl2 inhibitor ABT737 to enhance apoptosis in Cal51 breast cancer cells. Copyright

ΔNp63α silences a microRNA program to aberrantly initiate a wound healing program that promotes TGFβ-induced metastasis.

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 antimetastatic 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 antimigratory miR-198 by suppressing the expression of the regulatory factor, KSRP (KHSRP). Collectively, these findings reveal that a novel miRNA network involved in the regulation of physiologic wound-healing responses is hijacked and suppressed by tumor cells to promote metastatic dissemination. Cancer Res; 76(11); 3236-51. ©2016 AACR.

Targeting the epigenetic readers in Ewing Sarcoma inhibits the oncogenic transcription factor EWS/Fli1

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.

Small animal models for the study of bone sarcoma pathogenesis: characteristics, therapeutic interests and limitations

Osteosarcoma, Ewing sarcoma and chondrosarcoma are the three main entities of bone sarcoma which collectively encompass more than 50 heterogeneous entities of rare malignancies. In contrast to osteosarcoma and Ewing sarcoma which mainly affect adolescents and young adults and exhibit a high propensity to metastasise to the lungs, chondrosarcoma is more frequently observed after 40 years of age and is characterised by a high frequency of local recurrence. The combination of chemotherapy, surgical resection and radiotherapy has contributed to an improved outcome for these patients. However, a large number of patients still suffer significant therapy related toxicities or die of refractory and metastatic disease. To better delineate the pathogenesis of bone sarcomas and to identify and test new therapeutic options, major efforts have been invested over the past decades in the development of relevant pre-clinical animal models. Nowadays, in vivo models aspire to mimic all the steps and the clinical features of the human disease as accurately as possible and should ideally be manipulable. Considering these features and given their small size, their conduciveness to experiments, their affordability as well as their human-like bone-microenvironment and immunity, murine pre-clinical models are interesting in the context of these pathologies. This chapter will provide an overview of the murine models of bone sarcomas, paying specific attention for the models induced by inoculation of tumour cells. The genetically-engineered mouse models of bone sarcoma will also be summarized.

Abstract B46: miRNA-193a-5p repression of p73 induces Cisplatine chemoresistance in bone-related sarcomas

The purpose of this study is to better delineate the implication of the TAp73β/miRNA-193a-5p9s axis in the Cisplatine chemoresistance, in the context of Osteosarcoma and Ewing Sarcoma, the two most common Bone Sarcomas. The last decade9s improvement in the biomedical research field has widely contributed to improve the Bone Sarcomas outcome. Among the arsenal of chemotherapeutic-drugs currently used in those pathologies, the Cisplatine is a very common therapeutic option. Unfortunately, chemoresistance against this drug remains, often leading to the development of pulmonary metastases and the consequent relapse of the young patients suffering from these tumors. In the hope of eradicating these aggressive kinds of cancer, a better understanding of the molecular origins of such chemoresistance is needed. Through their gene9s expression inhibitory role, the implication of the miRNAs in the Bone Sarcomas9 chemoresistance is already clearly established. Furthermore, it has previously been demonstrated that through targeting the p53-family member TAp73β, the miR-193a-5p is implicated in the Cisplatine resistance in a carcinoma model. Thus, the aim of this work is to assess if TAp73β is also a mediator of the Cisplatine-induced apoptosis in Bone Sarcomas and consequently, if its modulation by the miR-193a-5p could be implicated in the chemoresistance mechanisms against this drug. In addition, this study also strives to bring the proof of concept of miR-193a-5p9s as a new therapeutic-target to potentiate the Cisplatine anti-tumor9s effects in Bone Sarcomas and override chemoresistance. Our qRT-PCR and viability assays results reveal that human Osteosarcoma and Ewing Sarcoma cell lines express the miR-193a-5p and TAp73β at various levels and display different Cisplatine sensitivities. In addition, an interesting correlation was found between the Cisplatine IC 50 values and the miR-193a-5p expression levels. In line with these results, we have also demonstrated for the first time in this model that Cisplatine represses the miR-193a-5p expression and consequently induces TAp73β and its target-genes. Through TAp73β expression level modulation, we validated its implication in both the apoptosis and the clonogenic capabilities of the Bone Sarcoma cells. Our cellular viability assays also confirm the predicted role of TAp73β-mediated Cisplatine-sensitivity. In the same way, we show that modulating the miR-193a-5p expression level through transient transfections with synthesis miRNAs (pre-miR) or their inhibitory counterparts (anti-miRs) has a functional impact on the Cisplatine sensitivity and the apoptosis-induction (caspase 3/7 activity assay, PARPs cleavage). In conclusion, our body of data confirms that through targeting TAp73β, the miRNA-193a-5p modulates the viability, the clonogenic capabilities and the Cisplatine-induced apoptosis in Bone Sarcoma cells. Such results shed light on the implication of the miR-193a-5p in the Bone Sarcomas9 Cisplatine-chemoresistance and open the road to new therapeutic opportunities provided by targeting the miR-193a-5p/TAp73β axis in the context of these pediatric cancers. In a fundamental approach, studying the Cisplatine-resistance might lead to better understand the role of miRNAs in therapeutic resistance. However, this miRNAs could also be used as “chemo-sensitizer” molecule in this context, improving the Bone Sarcoma outcome. Citation Format: Camille Jacques, Francois Lamoureux, Marc Baud9huin, Lidia Rodriguez Calleja, Dominique Heymann, Benjamin Ory. miRNA-193a-5p repression of p73 induces Cisplatine chemoresistance in bone-related sarcomas. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr B46.

Abstract 1911: ΔNp63α promotes TGFβ-induced metastasis by silencing a microRNA network restraining wound healing

Primary cancer cell dissemination is the key event in metastasis, yet context-specific determinants remain largely undefined. Multiple reports have suggested an anti-metastatic role for the p53 family member p63 linked to its minor epithelial isoform, TAp63. However, the role and contribution of the major p63 isoform, ΔNp63α, remain largely undefined. Here we report a distinct and TAp63-independent mechanism in which ΔNp63α-expressing cells, in a TGFβ-rich microenvironment, are positively selected for metastatic dissemination. We define the molecular mechanism of this effect, through ΔNp63α-mediated repression of miR-527 and miR-665 and the subsequent upregulation of two TGFβ pathway major effectors, SMAD4 and TβRII. Furthermore, we provide evidence that this mechanism reflects a fundamental role for ΔNp63α in the normal wound healing response. We show that ΔNp63α and miR-527/665 control the TGFβ-dependent signaling that switches off anti-migratory miR-198 by downregulating the regulatory factor KSRP. Collectively, these findings reveal a novel microRNA network orchestrated selectively by ΔNp63α that is involved in the physiological regulation of wound healing but is hijacked by tumors to promote metastatic dissemination. Citation Format: Lidia Rodriguez Calleja, camille Jacques, Francois Lamoureux, Marc Baud’huin, Thibault Quillard, Franck Verrecchia, Dominique Heymann, Leif Ellisen, Benjamin Ory. ΔNp63α promotes TGFβ-induced metastasis by silencing a microRNA network restraining wound healing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1911.

Abstract 4472: Targeting the oncogenic transcription factor EWS-Fli1 by BET bromodomain inhibition in Ewing sarcoma

Objective Ewing Sarcoma is the second most common primitive malignant bone tumor after osteosarcoma. This childhood cancer is defined by a chromosomal translocation leading to the production of a chimerical transcription factor, EWS-Fli1, which is implicated in the progression of this malignancy. As current treatments improve its outcome, therapeutic resistances remain and reduce the survival rates. Despite protocols’ optimization efforts, patients still relapse and it is important to develop new therapeutic approaches. The recently synthesized molecule JQ1, an inhibitor of the BET bromodomain proteins could be a new original weapon against this cancer. BET proteins interact with acetylated histones to regulate the chromatin accessibility to transcription factors and RNA polymerases. As JQ1 was recently shown to reduce the transcription of “super-enhancers”-dependent-genes such as oncogenes, which are highly sensitive to the BET proteins presence, it sounds relevant to hypothesize that EWS-Fli19s expression depends on the activity of these proteins. Method The cell viability, the cell proliferation and the cell9s clonogenic potential consequent to a JQ1 treatment were studied in vitro. Using a Ewing Sarcoma nude mice model, the JQ1 effects on both the tumor growth and the animals overall survival were assessed. The EWS-Fli1 expression and ones of its transcriptional targets were evaluated by RT-qPCR and Western blotting and the direct regulation of EWS-Fli1 expression by BRD4 was checked by ChIP-qPCR. Results We demonstrated that JQ1 reduces the viability and the clonogenic capabilities of the cells as well as it leads to a G1-phase blockade, delays the burden of the tumor growth and improves the mice overall survival. Histological analysis shows that JQ1 reduces the vascularization and the cell proliferation within the tumors. Those effects are correlated to the associated silencing of EWS-Fli1 and the consequent modulation of some of its target-genes resulting from the depletion of BRD4 from the EWS-Fli1 promoter. Conclusion Our results shed light on the BET bromodomains’ role as essential regulators of Ewing Sarcoma carcinogenesis. These proteins are indeed required to control EWS-Fli19s expression, consequently impacting its downstream signaling, which is functionally decisive for the maintenance of the tumorigenic features of these cancer-cells. Thus, the BET bromodomain proteins could be promising therapeutic targets in the Ewing tumors context. Citation Format: Camille JACQUES, Francois LAMOUREUX, Marc BAUD’HUIN, Lidia RODRIGUEZ-CALLEJA, Thibaut QUILLARD, Pierre PERROT, Franck TIRODE, Francoise REDINI, James E. BRADNER, Dominique HEYMANN, Benjamin ORY. Targeting the oncogenic transcription factor EWS-Fli1 by BET bromodomain inhibition in Ewing sarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4472.

MicroRNA implication in therapeutic resistance and metastatic dissemination of bone-associated tumors

Abstract Despite the last decade’s improvements in the development of new treatments, mortality of cancer patients is still highly linked to chemoresistance and further occurrence of distant metastasis. The metastatic dissemination is a complex process composed of different steps in which only tumor cells with the highest capacity of adaptation will succeed in establishing and growing at distant sites. Through genetic mutations those cells acquire the chemoresistance features needed to survive in new environments. Nowadays, breast and prostate cancer patients have a risk of being bad responders to chemotherapeutic drugs and to consequently relapse through the development of bone metastasis. Indeed, bone is the most attractive niche for the homing of those cancer cell types. MicroRNAs (miRNAs), a class of small non-coding single strand RNAs, have been recognized as crucial regulatory factors implicated in various cellular processes including cancer initiation and spreading. As miRNAs intensively regulate mediators of migratory and survival pathways, it’s now well established that this class of molecules consequently influences both metastatic dissemination and chemoresistance. Here, we aim to overview the recent research progress on the role of dissemination and chemoresistance-related miRNAs, in the bone-associated tumor context. These pieces of evidence support the clinical use of miRNAs as diagnosis and prognosis markers, as well as potential therapeutic tools against cancer progression, resistance, recurrence and metastatic dissemination.