Sophie Barillé-Nion

New Insights in Myeloma-induced Osteolysis

Abstract Multiple myeloma (MM) is a plasma cell malignancy localized in the bone marrow (BM) and characterized by a high capacity for bone destruction. Almost all patients with MM have early osteolytic lesions, which result mainly from increased bone resorption related to stimulation of osteoclast recruitment and activity in the immediate vicinity of myeloma cells. The recent discovery of Osteoprotegerin (OPG) and the subsequent identification of its ligand RANKL have provided new insights in the regulation of osteoclastogenesis. The ratio OPG/RANKL is critical for the regulation of bone remodeling maintaining the balance between osteoblastic and osteoclastic activity. This review summarizes the new concept that myeloma cells induce in bone environment an imbalance in the OPG/RANKL system responsible for osteolysis observed in patients. Indeed, myeloma cells increase in bone environment the expression of the potent osteoclastogenic factor RANKL and decrease the osteoprotective factor OPG production. Biological mechanisms involved in these processes are discussed. Furthermore, the chemokines MIP-1α and MIP-1β belonging to the RANTES family are potent osteoclastogenic factors produced by myeloma cells and participate in myeloma-associated bone disease. These data open new avenues for the treatment of bone disease in MM and highlight the promising therapeutical interest of RANKL inhibitors (OPG and RANK-Fc) and MIP-1 inhibitors in the management of myeloma-associated osteolysis, besides bisphosphonates.

Epithelial-to-mesenchymal transition Induced by TGF-β1 is mediated by Blimp-1-dependent repression of BMP-5

Induction of epithelial-to-mesenchymal transition (EMT) by TGF-β1 requires Ras signaling. We recently identified the transcriptional repressor Blimp-1 (PRDM1) as a downstream effector of the NF-κB, RelB/Bcl-2/Ras-driven pathway that promotes breast cancer cell migration. As the RelB/Blimp-1 pathway similarly required Ras signaling activation, we tested whether Blimp-1 plays a role in TGF-β1-mediated EMT. Here, TGF-β1 treatment of untransformed NMuMG mammary epithelial and MDA-MB-231 breast cancer cells was shown to induce Blimp-1 expression, which promoted an EMT signature and cell migration. TGFB1 and BLIMP1 RNA levels were correlated in patient breast tumors. BLIMP1 gene transcription was activated by TGF-β1 via a c-Raf (RAF1) to AP-1 pathway. Blimp-1 induced expression of the EMT master regulator Snail (SNAI1) via repressing BMP-5, which inhibited Snail expression upon TGF-β1 treatment. Interestingly, a similar cascade was observed during postnatal mouse mammary gland development. RelB expression was detected early in pregnancy followed progressively by Blimp-1 and then Snail; whereas, BMP-5 levels were high in nulliparous and regressing glands. Finally, lower BMP5 RNA levels were detected in patient breast tumors versus normal tissues, and correlated with cancer recurrence. Thus, the Ras effector Blimp-1 plays an essential role in TGF-β1-induced EMT via repression of BMP-5 in breast cancer.

ADAM8 expression in invasive breast cancer promotes tumor dissemination and metastasis

The transmembrane metalloprotease‐disintegrin ADAM8 mediates cell adhesion and shedding of ligands, receptors and extracellular matrix components. Here, we report that ADAM8 is abundantly expressed in breast tumors and derived metastases compared to normal tissue, especially in triple‐negative breast cancers (TNBCs). Furthermore, high ADAM8 levels predicted poor patient outcome. Consistently, ADAM8 promoted an aggressive phenotype of TNBC cells in culture. In a mouse orthotopic model, tumors derived from TNBC cells with ADAM8 knockdown failed to grow beyond a palpable size and displayed poor vascularization. Circulating tumor cells and brain metastases were also significantly reduced. Mechanistically, ADAM8 stimulated both angiogenesis through release of VEGF‐A and transendothelial cell migration via β1‐integrin activation. In vivo, treatment with an anti‐ADAM8 antibody from the time of cell inoculation reduced primary tumor burden and metastases. Furthermore, antibody treatment of established tumors profoundly decreased metastases in a resection model. As a non‐essential protein under physiological conditions, ADAM8 represents a promising novel target for treatment of TNBCs, which currently lack targeted therapies and frequently progress with fatal dissemination.

Canonical Nuclear Factor κB Pathway Inhibition Blocks Myeloma Cell Growth and Induces Apoptosis in Strong Synergy with TRAIL

Purpose: Intrinsic activation of nuclear factor κB (NF-κB) characterizes various hematologic malignancies. In this study, we specifically address the role of NF-κB blockade in mediated antimyeloma activity using the IκB kinase-2 pharmacologic inhibitor, AS602868. Experimental Design: Human myeloma cell lines (n = 16) and primary myeloma cells (n = 10) were tested for their sensitivity to AS602868 in terms of proliferation and apoptosis. Both in vitro and in vivo experiments were conducted. Functional mechanisms regarding the apoptotic pathways triggered by AS602868 were studied. The potential proapoptotic synergy between AS602868 and tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) was also evaluated. Results: Our results show that AS602868 efficiently targeted the canonical NF-κB pathway in myeloma cells and potently inhibited their growth in inducing apoptosis through Bax and caspase-3 activation. AS602868 also induced apoptosis in primary myeloma cells even in the presence of bone marrow mononuclear cells. Moreover, the IκB kinase-2 inhibitor targeted the paracrine effect on the bone marrow environment. Indeed, it decreased the intrinsic and myeloma-induced secretion of interleukin-6 from bone marrow stromal cells. In addition, AS602868 inhibited myeloma cell growth in the MM.1S xenograft myeloma model. Of particular interest, AS602868 strongly increased myeloma sensitivity to TRAIL in blocking TRAIL-induced NF-κB activation and in decreasing the expression of antiapoptotic proteins such as cFLIP and cIAP-1/2. Conclusions: Taken together, our data point out the interest to inhibit the canonical NF-κB pathway in myeloma and clearly encourage clinical evaluation of novel therapies based on targeting NF-κB, especially in combination with TRAIL.

c-Myc dependent expression of pro-apoptotic Bim renders HER2-overexpressing breast cancer cells dependent on anti-apoptotic Mcl-1

BackgroundAnti-apoptotic signals induced downstream of HER2 are known to contribute to the resistance to current treatments of breast cancer cells that overexpress this member of the EGFR family. Whether or not some of these signals are also involved in tumor maintenance by counteracting constitutive death signals is much less understood. To address this, we investigated what role anti- and pro-apoptotic Bcl-2 family members, key regulators of cancer cell survival, might play in the viability of HER2 overexpressing breast cancer cells.MethodsWe used cell lines as an in vitro model of HER2-overexpressing cells in order to evaluate how anti-apoptotic Bcl-2, Bcl-xL and Mcl-1, and pro-apoptotic Puma and Bim impact on their survival, and to investigate how the constitutive expression of these proteins is regulated. Expression of the proteins of interest was confirmed using lysates from HER2-overexpressing tumors and through analysis of publicly available RNA expression data.ResultsWe show that the depletion of Mcl-1 is sufficient to induce apoptosis in HER2-overexpressing breast cancer cells. This Mcl-1 dependence is due to Bim expression and it directly results from oncogenic signaling, as depletion of the oncoprotein c-Myc, which occupies regions of the Bim promoter as evaluated in ChIP assays, decreases Bim levels and mitigates Mcl-1 dependence. Consistently, a reduction of c-Myc expression by inhibition of mTORC1 activity abrogates occupancy of the Bim promoter by c-Myc, decreases Bim expression and promotes tolerance to Mcl-1 depletion. Western blot analysis confirms that naïve HER2-overexpressing tumors constitutively express detectable levels of Mcl-1 and Bim, while expression data hint on enrichment for Mcl-1 transcripts in these tumors.ConclusionsThis work establishes that, in HER2-overexpressing tumors, it is necessary, and maybe sufficient, to therapeutically impact on the Mcl-1/Bim balance for efficient induction of cancer cell death.

Impact of XIAP protein levels on the survival of myeloma cells

XIAP is the best characterized and the most potent direct endogenous caspase inhibitor and is considered a key actor in the control of apoptotic threshold in cancer cells. The results of this study demonstrate that a decrease in XIAP level in myeloma cells shifts the balance toward apoptosis. Background XIAP is the best characterized and the most potent direct endogenous caspase inhibitor and is considered a key actor in the control of apoptotic threshold in cancer cells. In this report, we specifically addressed XIAP regulation and function in myeloma cells. Design and Methods XIAP and its endogenous inhibitor XAF-1 protein levels and their regulation were assessed by immunoblot analysis in myeloma cell lines or primary myeloma cells. XIAP knockdown by RNA interference was used to evaluate XIAP impact on in vitro drug sensitivity and in vivo tumor growth. Results Our results indicate that myeloma cells expressed high levels of XIAP protein that were tightly regulated during growth factor stimulation or stress condition. Of note, an increased XIAPlevel was evidenced during the blockade of the canonical cap-dependent translation by the mTOR inhibitor rapamycin, supporting the hypothesis of a functional IRES sequence in XIAP mRNA. In addition, caspase-mediated XIAP cleavage correlated to an apoptotic process occurring upon cell treatment with the proteasome inhibitor bortezomib. Importantly, XIAP knockdown using RNA interference enhanced drug sensitivity and decreased tumor formation in NOD/SCID mice. Finally, myeloma cells also expressed the XIAP inhibitor XAF-1 that interacted with XIAP in viable myeloma cells. Conclusions Altogether, our data argue for a delicate control of XIAP function in myeloma cells and stimulate interest in targeting XIAP in myeloma treatment.

γ-Secretase inhibition promotes cell death, Noxa upregulation, and sensitization to BH3 mimetic ABT-737 in human breast cancer cells

IntroductionInappropriate Notch signaling, downstream of γ-secretase activity, is understood to have tumor-promoting function and to be associated with poor outcome in cancer, of the breast in particular. The molecular basis of antitumoral effects of its inhibitors, however, remains poorly characterized. Moreover, the effects of their combination with the pro-apoptotic pharmacologic inhibitor of Bcl-2/Bcl-xL, ABT-737, have never been evaluated. In this study, we thus specifically addressed the biologic consequences of targeting γ-secretase and Bcl-2/Bcl-xL, alone or simultaneously, in breast cancer cell lines as well as in a novel human breast cancer ex vivo assay.MethodsBy using in vitro 2D or 3D cultures of breast cancer cells plus a novel preclinical short-term ex vivo assay that correctly maintains human mammary tissue integrity and preserves tumor microenvironment, we tested the effects of the pharmacologic γ-secretase inhibitor GSIXII used as a single agent or in combination with ABT-737.ResultsWe show herein that the γ-secretase inhibitor, GSIXII, efficiently induces apoptosis in breast cancer cell lines by a process that relies on the induction of Noxa, a pro-apoptotic Bcl2-homology 3 domain (BH3)-only protein of the Bcl-2 family that functions as an inhibitor of antiapoptotic Mcl1. GSIXII also targets mammary cancer stem-like cells because it dramatically prevents in vitro mammosphere formation. Moreover, combining GSIXII treatment with ABT-737, a BH3-mimetic inhibitor of additional antiapoptotic proteins, such as Bcl-2 and Bcl-xL, leads to both a synergistic apoptotic response in breast cancer cells and to an inhibitory effect on mammosphere formation. These effects are also found when a Notch transcriptional inhibitor, SAHM1, is used. Finally, we evaluated individual human tumor responses to γ-secretase inhibition alone or in combination with ABT-737 in ex vivo assays. Analysis of a series of 30 consecutive tumors indicated that a majority of tumors are sensitive to apoptosis induction by GSIXII and that association of GSIXII with ABT-737 leads to an enhanced induction of apoptosis in tumor cells.ConclusionsWe thus provide evidence that γ-secretase, and downstream Notch signaling, are relevant targets in breast cancer. GSIXII, used as single agent or in combination with clinically relevant BH3-mimetics, is a promising innovative proapoptotic strategy to treat mammary tumors.

Bcl-xL controls a switch between cell death modes during mitotic arrest

Antimitotic agents such as microtubule inhibitors (paclitaxel) are widely used in cancer therapy while new agents blocking mitosis onset are currently in development. All these agents impose a prolonged mitotic arrest in cancer cells that relies on sustained activation of the spindle assembly checkpoint and may lead to subsequent cell death by incompletely understood molecular events. We have investigated the role played by anti-apoptotic Bcl-2 family members in the fate of mitotically arrested mammary tumor cells treated with paclitaxel, or depleted in Cdc20, the activator of the anaphase promoting complex. Under these conditions, a weak and delayed mitotic cell death occurs that is caspase- and Bax/Bak-independent. Moreover, BH3 profiling assays indicate that viable cells during mitotic arrest are primed to die by apoptosis and that Bcl-xL is required to maintain mitochondrial integrity. Consistently, Bcl-xL depletion, or treatment with its inhibitor ABT-737 (but not with the specific Bcl-2 inhibitor ABT-199), during mitotic arrest converts cell response to antimitotics to efficient caspase and Bax-dependent apoptosis. Apoptotic priming under conditions of mitotic arrest relies, at least in part, on the phosphorylation on serine 62 of Bcl-xL, which modulates its interaction with Bax and its sensitivity to ABT-737. The phospho-mimetic S62D-Bcl-xL mutant is indeed less efficient than the corresponding phospho-deficient S62A-Bcl-xL mutant in sequestrating Bax and in protecting cancer cells from mitotic cell death or yeast cells from Bax-induced growth inhibition. Our results provide a rationale for combining Bcl-xL targeting to antimitotic agents to improve clinical efficacy of antimitotic strategy in cancer therapy.

The imbalance between Survivin and Bim mediates tumour growth and correlates with poor survival in patients with multiple myeloma

Survivin is selectively expressed in most of common human cancers and is now viewed as a potent modulator of the cell death/proliferation balance in tumour cells. We previously found that myeloma cells expressed high levels of Survivin protein in correlation with disease progression and that Survivin knock‐down by RNA interference decreased myeloma cell growth. We now demonstrate that Survivin overexpression promotes the proliferation and survival of human myeloma cells both in vitro and in vivo in the absence of their major growth factor, interleukin 6. Of particular interest, this effect correlates with the down regulation of Bim, a critical BH3‐only cell death activator during cytokine deprivation, mainly at transcriptional level. The tight link between Survivin and Bim expression, reported for the first time here in myeloma cells and in other cell lines, is further confirmed in a panel of newly diagnosed patients with myeloma, and BIRC5 is validated as a gene significantly associated with short survival in these patients. Altogether, our findings provide evidence that Survivin directly contributes to malignant progression of myeloma and strongly suggest that targeting Survivin may disrupt the delicate balance controlling cell survival and proliferation, opening new avenues for the therapy of this still difficult‐to‐treat cancer.

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