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Abstract Background Anti-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. Methods We 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. Results We 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. Conclusions This 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.

Breast cancer stands as the most prevalent malignancy afflicting women. Despite significant advancements in its diagnosis and treatment, breast cancer metastasis continues to be a leading cause of mortality among women. To metastasize, cancer cells face numerous challenges: breaking away from the primary tumor, surviving in the circulation, establishing in a distant location, evading immune detection and, finally, thriving to initiate a new tumor. Each of these sequential steps requires cancer cells to adapt to a myriad of stressors and develop survival mechanisms. In addition, most patients with breast cancer undergo surgical removal of their primary tumor and have various therapeutic interventions designed to eradicate cancer cells. Despite this plethora of attacks and stresses, certain cancer cells not only manage to persist but also proliferate robustly, giving rise to substantial tumors that frequently culminate in the patient's demise. To enhance patient outcomes, there is an imperative need for a deeper understanding of the molecular and cellular mechanisms that empower cancer cells to not only survive but also expand. Herein, we delve into the intrinsic stresses that cancer cells encounter throughout the metastatic journey and the additional stresses induced by therapeutic interventions. We focus on elucidating the remarkable strategies adopted by cancer cells, such as cell-cell clustering and intricate cell-cell communication mechanisms, to ensure their survival., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Introduction: Tumor Associated Macrophages (TAM) are a major component of the tumor environment and their accumulation often correlates with poor prognosis by contributing to local inflammation, inhibition of anti-tumor immune response and resistance to anticancer treatments. In this study, we thus investigated the anti-cancer therapeutic interest to target ChemR23, a receptor of the resolution of inflammation expressed by macrophages, using an agonist monoclonal antibody, αChemR23., Methods: Human GM-CSF, M-CSF and Tumor Associated Macrophage (TAM)-like macrophages were obtained by incubation of monocytes from healthy donors with GM-CSF, M-CSF or tumor cell supernatants (Breast cancer (BC) or malignant pleural mesothelioma (MPM) cells). The effects of αChemR23 on macrophages were studied at the transcriptomic, protein and functional level. Datasets from The Cancer Genome Atlas (TCGA) were used to study CMKLR1 expression, coding for ChemR23, in BC and MPM tumors. In vivo , αChemR23 was evaluated on overall survival, metastasis development and transcriptomic modification of the metastatic niche using a model of resected triple negative breast cancer., Results: We show that ChemR23 is expressed at higher levels in M-CSF and tumor cell supernatant differentiated macrophages (TAM-like) than in GM-CSF-differentiated macrophages. ChemR23 activation triggered by αChemR23 deeply modulates M-CSF and TAM-like macrophages including profile of cell surface markers, cytokine secretion, gene mRNA expression and immune functions. The expression of ChemR23 coding gene ( CMKLR1 ) strongly correlates to TAM markers in human BC tumors and MPM and its histological detection in these tumors mainly corresponds to TAM expression. In vivo , treatment with αChemR23 agonist increased mouse survival and decreased metastasis occurrence in a model of triple-negative BC in correlation with modulation of TAM phenotype in the metastatic niche., Conclusion: These results open an attractive opportunity to target TAM and the resolution of inflammation pathways through ChemR23 to circumvent TAM pro-tumoral effects., Competing Interests: CT, VG, CM, and NP are inventors on a patent application (no. WO 2019/193029, filed 3 April 2019, published 8 October 2019) and product (WO2021/069709, filed 09 october 2019, published 15 April 2021) related to this work filed by OSE Immunotherapeutics and employees of OSE Immunotherapeutics with IG and SN. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This study was partially supported by OSE Immunotherapeutics funding., (Copyright © 2023 Lavy, Gauttier, Dumont, Chocteau, Deshayes, Fresquet, Dehame, Girault, Trilleaud, Neyton, Mary, Juin, Poirier, Barillé-Nion and Blanquart.)

Inflammation is a fundamental physiological response orchestrated by innate immune cells to restore tissue homeostasis. Specialized pro-resolving mediators (SPMs) are involved in active resolution of inflammation but when inflammation is incomplete, chronic inflammation creates a favorable environment that fuels carcinogenesis and cancer progression. Conventional cancer therapy also strengthens cancer-related inflammation by inducing massive tumor cell death that activate surrounding immune-infiltrating cells such as tumor-associated macrophages (TAMs). Macrophages are key actors of both inflammation and its active resolution due to their plastic phenotype. In line with this high plasticity, macrophages can be hijacked by cancer cells to support tumor progression and immune escape, or therapy resistance. Impaired resolution of cancer-associated inflammation supported by TAMs may thus reinforces tumor progression. From this perspective, recent evidence suggests that stimulating macrophage's pro-resolving functions using SPMs can promote inflammation resolution in cancer and improve anticancer treatments. Thus, TAMs' re-education toward an antitumor phenotype by using SPMs opens a new line of attack in cancer treatment. Here, we review SPMs' anticancer capacities with special attention regarding their effects on TAMs. We further discuss how this new therapeutic approach could be envisioned in cancer therapy., Competing Interests: ML, VG, and NP are employees and/or shareholders of OSE Immunotherapeutics, a company developing pro-resolutive agonist monoclonal antibodies. VG and NP are patent owners on ChemR23 agonist monoclonal antibodies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lavy, Gauttier, Poirier, Barillé-Nion and Blanquart.)

The natural abundance of heavy stable isotopes ( 13 C, 15 N, 18 O, etc.) is now of considerable importance in many research fields, including human physiology. In fact, it varies between tissues and metabolites due to isotope effects in biological processes, that is, isotope discriminations between heavy and light isotopic forms during enzyme or transporter activity. The metabolic deregulation associated with many diseases leads to alterations in metabolic fluxes, resulting in changes in isotope abundance that can be identified easily with current isotope ratio technologies. In this review, we summarize the current knowledge on changes in natural isotope composition in samples (including various tissues, hair, plasma, saliva) found in patients compared to controls, caused by human diseases. We discuss the metabolic origin of such isotope fractionations and highlight the potential of using isotopes at natural abundance for medical diagnosis and/or prognostic.

The imbalance between BCL-2 homologues and pro-death counterparts frequently noted in cancer cells endows them with a cell autonomous survival advantage. To eradicate ectopic cells, inhibitors of these homologues (BH3 mimetics) were developed to trigger, during anticancer treatment, full activation of the canonical mitochondrial apoptotic pathway and related caspases. Despite efficiency in some clinical settings, these compounds do not completely fulfill their initial promise. We herein put forth that a growing body of evidence indicates that mitochondrial integrity, controlled by BCL-2 family proteins, and downstream caspases regulate other cell death modes and influence extracellular signaling by committed cells. Moreover, intercellular communications play a key role in spreading therapeutic response across cancer cell populations and in engaging an immune response. We thus advocate that BH3 mimetics administration would be more efficient in the long term if it did not induce apoptosis in all sensitive cells at the same time, but if it could instead allow (or trigger) death signal production by non-terminally committed dying cell populations. The development of such a trade-off strategy requires to unravel the effects of BH3 mimetics not only on each individual cancer cell but also on homotypic and heterotypic cell interactions in dynamic tumor ecosystems.

We recently identified a previously unappreciated ability of antimitotics to propagate apoptotic priming across cancer cell populations. The underlying paracrine cytotoxic signal, fueled by undead cells activating the cGAS/STING pathway, is required for in vivo antitumor response and it can be further exploited by delayed, but not synchronous, BCL-xL inhibition., (© 2020 Taylor & Francis Group, LLC.)

A fascinating but uncharacterized action of antimitotic chemotherapy is to collectively prime cancer cells to apoptotic mitochondrial outer membrane permeabilization (MOMP), while impacting only on cycling cell subsets. Here, we show that a proapoptotic secretory phenotype is induced by activation of cGAS/STING in cancer cells that are hit by antimitotic treatment, accumulate micronuclei and maintain mitochondrial integrity despite intrinsic apoptotic pressure. Organotypic cultures of primary human breast tumors and patient-derived xenografts sensitive to paclitaxel exhibit gene expression signatures typical of type I IFN and TNFα exposure. These cytokines induced by cGAS/STING activation trigger NOXA expression in neighboring cells and render them acutely sensitive to BCL-xL inhibition. cGAS/STING-dependent apoptotic effects are required for paclitaxel response in vivo, and they are amplified by sequential, but not synchronous, administration of BH3 mimetics. Thus anti-mitotic agents propagate apoptotic priming across heterogeneously sensitive cancer cells through cytosolic DNA sensing pathway-dependent extracellular signals, exploitable by delayed MOMP targeting.

We describe the first examples of small molecules able to disrupt the nanomolar interaction between the pro-apoptotic protein PUMA and its anti-apoptotic counterpart BcL-xL in malignant cells. Based on molecular modelling studies, we propose a rationale to this result, through a new "bottle-opener"-type strategy which could be of general use in the area of protein-protein interaction studies., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

In this study, we assessed the sensitivity of myeloma cells to the oncolytic measles virus (MV) in relation to p53 using 37 cell lines and 23 primary samples. We showed that infection and cell death were correlated with CD46 expression, which was associated with TP53 status; TP53 abn cell lines highly expressed CD46 and were preferentially infected by MV when compared with the TP53 wt cell lines ( P = .046 and P = .045, respectively). Infection of myeloma cells was fully dependent on CD46 expression in both cell lines and primary cells. In the TP53 wt cell lines, but not the TP53 abn cell lines, activation of the p53 pathway with nutlin3a inhibited both CD46 expression and MV infection, while TP53 silencing reciprocally increased CD46 expression and MV infection. We showed using a p53 chromatin immunoprecipitation assay and microRNA assessment that CD46 gene expression was directly and indirectly regulated by p53. Primary myeloma cells overexpressed CD46 as compared with normal cells and were highly infected and killed by MV. CD46 expression and MV infection were inhibited by nutlin3a in primary p53-competent myeloma cells, but not in p53-deficient myeloma cells, and the latter were highly sensitive to MV infection. In summary, myeloma cells were highly sensitive to MV and infection inhibition by the p53 pathway was abrogated in p53-deficient myeloma cells. These results argue for an MV-based clinical trial for patients with p53 deficiency., (© 2018 by The American Society of Hematology.)

E2F1 is the main pro-apoptotic effector of the pRB-regulated tumor suppressor pathway by promoting the transcription of various pro-apoptotic proteins. We report here that E2F1 partly localizes to mitochondria, where it favors mitochondrial outer membrane permeabilization. E2F1 interacts with BCL-xL independently from its BH3 binding interface and induces a stabilization of BCL-xL at mitochondrial membranes. This prevents efficient control of BCL-xL over its binding partners, in particular over BAK resulting in the induction of cell death. We thus identify a new, non-BH3-binding regulator of BCL-xL localization dynamics that influences its anti-apoptotic activity., (© 2017 The Authors.)

In tumours, accumulation of chemoresistant cells that express high levels of anti-apoptotic proteins such as BCL-X L is thought to result from the counter selection of sensitive, low expresser clones during progression and/or initial treatment. We herein show that BCL-X L expression is selectively advantageous to cancer cell populations even in the absence of pro-apoptotic pressure. In transformed human mammary epithelial cells BCL-X L favours full activation of signalling downstream of constitutively active RAS with which it interacts in a BH4-dependent manner. Comparative proteomic analysis and functional assays indicate that this is critical for RAS-induced expression of stemness regulators and maintenance of a cancer initiating cell (CIC) phenotype. Resistant cancer cells thus arise from a positive selection driven by BCL-X L modulation of RAS-induced self-renewal, and during which apoptotic resistance is not necessarily the directly selected trait.

Breast cancer is the most common cancer in women worldwide. Despite the information provided by anatomopathological assessment and molecular markers (such as receptor expression ER, PR, HER2), breast cancer therapies and prognostics depend on the metabolic properties of tumor cells. However, metabolomics have not provided a robust and congruent biomarker yet, likely because individual metabolite contents are insufficient to encapsulate all of the alterations in metabolic fluxes. Here, we took advantage of natural 13 C and 15 N isotope abundance to show there are isotopic differences between healthy and cancer biopsy tissues or between healthy and malignant cultured cell lines. Isotope mass balance further suggests that these differences are mostly related to lipid metabolism, anaplerosis and urea cycle, three pathways known to be impacted in malignant cells. Our results demonstrate that the isotope signature is a good descriptor of metabolism since it integrates modifications in C partitioning and N excretion altogether. Our present study is thus a starting point to possible clinical applications such as patient screening and biopsy characterization in every cancer that is associated with metabolic changes.

Hsp90 is an ATP-dependent chaperone known to be overexpressed in many cancers. This way, Hsp90 is an important target for drug discovery. Novobiocin, an aminocoumarin antibiotic, was reported to inhibit Hsp90 targeting C-terminal domain, and showed anti-proliferative properties, leading to the development of new and more active compounds. Consequently, a new set of novobiocin analogs derived from 1,6-naphthyridin-2(1H)-one scaffold was designed, synthesized and evaluated against two breast cancer cell lines. Subsequently, cell cycle progression and apoptosis were conducted on best candidates, finally Western Blot analysis was performed to measure their ability to induce degradation of Hsp90 client proteins., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Background: ADAM8 (a disintegrin and metalloproteinase 8) protein promotes the invasive and metastatic phenotype of triple-negative breast cancer (TNBC) cells. High ADAM8 expression in breast cancer patients is an independent predictor of poor prognosis. Here, we investigated whether ADAM8 regulates specific miRNAs, their roles in aggressive phenotype, and potential use as biomarkers of disease., Methods: Microarray analysis was performed on RNA from MDA-MB-231 cells after transient ADAM8 knockdown using TaqMan miRNA cards. Changes in miRNA levels were confirmed using two ADAM8 siRNAs in TNBC cell lines. Kinase inhibitors, β1-integrin antagonist antibody, and different forms of ADAM8 were employed to elucidate the signaling pathway required for miR-720 expression. miR-720 levels were modulated using a specific antagomiR or a mimic, and effects on aggressive phenotype of TNBC cells were determined using Boyden chamber and 3D-Matrigel outgrowth assays. Plasma was isolated from mice before and after implantation of MDA-MB-231 cells and analyzed for miR-720 levels. Serum samples of TNBC patients were evaluated for their ADAM8 and miR-720 levels., Results: We identified 68 miRNAs differentially regulated upon ADAM8 knockdown, including decreased levels of secreted miR-720. Ectopic overexpression of wild-type ADAM8 or forms that lack metalloproteinase activity similarly induced miR-720 levels. The disintegrin and cysteine-rich domains of ADAM8 were shown to induce miR-720 via activation of a β1-integrin to ERK signaling cascade. Knockdown of miR-720 led to a significant decrease in migratory and invasive abilities of TNBC cells. Conversely, miR-720 overexpression rescued these properties. A profound increase in plasma levels of miR-720 was detected 7 days after TNBC cell inoculation into mouse mammary fat pads when tumors were barely palpable. Concordantly, miR-720 levels were found to be significantly higher in serum samples of TNBC patients with high ADAM8 expression., Conclusions: We have shown for the first time that miR-720 is induced by ADAM8 signaling via ERK and plays an essential role in promoting the aggressive phenotype of TNBCs. miR-720 is elevated in serum of patients with ADAM8-high TNBC and, in a group with other miRNAs downstream of ADAM8, holds promise as a biomarker for early detection of or treatment response of ADAM8-positive TNBCs.

Terminal fucosylated motifs of glycoproteins and glycolipid chains are often altered in cancer cells. We investigated the link between fucosylation changes and critical steps in cancer progression: epithelial-to-mesenchymal transition (EMT) and lymph node metastasis.Using mammary cell lines, we demonstrate that during EMT, expression of some fucosylated antigens (e.g.: Lewis Y) is decreased as a result of repression of the fucosyltransferase genes FUT1 and FUT3. Moreover, we identify the fucose-binding bacterial lectin BC2L-C-Nt as a specific probe for the epithelial state.Prolectin (CLEC17A), a human lectin found on lymph node B cells, shares ligand specificities with BC2L-C-Nt. It binds preferentially to epithelial rather than to mesenchymal cells, and microfluidic experiments showed that prolectin behaves as a cell adhesion molecule for epithelial cells. Comparison of paired primary tumors/lymph node metastases revealed an increase of prolectin staining in metastasis and high FUT1 and FUT3 mRNA expression was associated with poor prognosis. Our data suggest that tumor cells invading the lymph nodes and expressing fucosylated motifs associated with the epithelial state could use prolectin as a colonization factor.

Survivin overexpression, frequently found in breast cancers and others, is associated with poor prognosis. Its dual regulation of cell division and apoptosis makes it an attractive therapeutic target but its exact functions that are required for tumor maintenance are still elusive. Survivin protects cancer cells from genotoxic agents and this ability is generally assigned to a universal anti-apoptotic function. However, a specific role in cancer cell protection from DNA damage has been overlooked so far. We assessed DNA damage occurrence in Survivin-depleted breast cancer cells using γH2AX staining and comete assay. QPCR data and a gene conversion assay indicated that homologous recombination (HR) was impaired upon Survivin depletion. We conducted the analysis of Survivin and HR genes' expression in breast tumors. We revealed BRCAness phenotype of Survivin-depleted cells using cell death assays combined to PARP targeting. Survivin silencing leads to DNA double-strand breaks in breast cancer cells and functionally reduces HR. Survivin depletion decreases the transcription of a set of genes involved in HR, decreases RAD51 protein expression and impairs the endonuclease complex MUS81/EME1 involved in the resolution of Holliday junctions. Clinically, EME1, RAD51, EXO1, BLM expressions correlate with that of BIRC5 (coding for Survivin) and are of prognostic value. Functionally, Survivin depletion triggers p53 activation and sensitizes cancer cells to of PARP inhibition. We defined Survivin as a constitutive actor of HR in breast cancers, and implies that its inhibition would enhance cell vulnerability upon PARP inhibition.

The Bcl-2 family includes 26 proteins involved in apoptosis. Cancer cells can develop the ability to avoid apoptosis through the upregulation and/or down regulation of such proteins Bax, Bcl-xL or Mcl-1, especially during chemoresistance progress. These proteins engaged in a network of dynamic interactions that control apoptosis triggering have become attractive therapeutic targets in cancers including melanoma. Among them, the Bax/Bcl-xL interaction appears critical in maintaining mitochondria integrity. Therefore a series of mixed polyphenol-heterocyclic molecules, were rationally designed by molecular docking as Bax/Bcl-xL inhibitors. It has been screened against B16-F10 melanoma cancer cells for a preliminary investigation of their cytotoxicity. All these compounds exhibited a significant cytotoxicity against these cancer cells, in the 0.3-6 .M range. A pyrazole-type molecule, which had a submicromolar IC50 value with an excellent selectivity index (14), is the most promising derivative for further development.

Specific overexpression in cancer cells and evidence of oncogenic functions make Survivin an attractive target in cancer therapy. The small molecule compound YM155 has been described as the first "Survivin suppressant" but molecular mechanisms involved in its biological activity and its clinical potential remain obscure. We herein show that YM155 exerts single agent toxicity on primary breast cancer cells grown in an ex vivo assay preserving tumor microenvironment. In vitro assays indicate that YM155 more efficiently triggers cell death in breast cancer cells (including these with stem-cell like properties) than in non tumorigenic mammary cells. YM155-induced cell death is critically dependent on autophagy and NF-kB but independent of p53 and it coïncides with DNA damage and a DNA damage response in p53-proficient cells. Our results point out a crosstalk between NF-kB and autophagy controlling YM155-induced death in breast cancer cells and argue for the potential use of YM155 as a genotoxic agent in breast cancer therapy.

Inhibition of Bcl-2 family protein-protein interactions (PPI) is a very promising direction in cancer chemotherapy. Hence over the last decade, many medicinal chemistry studies endeavoured to discover drug candidates, and a wealth of chemical scaffolds with striking chemical diversity was reported as Bcl-xL inhibitors. This raises the question of whether all these molecules could occupy a unique binding site, or rather discrete pockets of the protein surface. To test if small and chemically diverse Bcl-xL inhibitors are likely to bind a single pocket, and to identify which pocket, we used a battery of computational and modeling approaches. We first checked that the large dataset of Bcl-xL inhibitors we built can actually fit to a universal pharmacophore. Then we defined the probable binding hot spots of interaction through comparison of crystal structures, as well as virtual fragment screening. Finally, new analogues of small polyphenol derivatives were synthesized to precisely probe a hydrogen bond suggested by docking experiments. Bcl-xL inhibition potency of these products confirmed the predicted binding mode. This combination of X-ray structure exploration, molecular modeling studies and medicinal chemistry supports that all these small Bcl-xL inhibitors occupy the same hot spot of interaction. The identification of this binding site should help the design and optimization of small PPI Bcl-xL inhibitors., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

We describe the synthesis of a series of new molecules containing phenol and triazoles moieties, compounds which have been evaluated for their ability to inhibit Bax/Bcl-xL interactions in cancer cells, by using BRET assays, and to induce cell death. Several derivatives exhibit a very promising activity, being more potent than the reference compounds acylpyrogallol A and ABT-737. These preliminary results demonstrate that derivatives of this family can be attractive to develop new molecules with potent anticancer activity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

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.

Embelin is a natural product, inhibitor of XIAP (X-chromosome-linked Inhibitor of APoptosis) with strong proapoptotic properties on cancer cells. In order to clarify the role of two OH groups on benzoquinone core, we have prepared by hemisynthesis close analogs of embelin, where these OH groups have been replaced in a systematic manner by OMe and OAc groups. Proapoptotic activities of six embelin derivatives have been studied as single agent, or in combination with TRAIL, and their abilities to interact with XIAP have been evaluated by Surface Plasmon Biacore. Our results show that these new embelin analogs have good proapoptotic properties against selected cancer cells, often higher than the natural product itself. Further, this activity is not directly mediated by XIAP. Altogether these preliminary results demonstrate that for active embelin analogs, the two OH groups are not absolutely required for anticancer activity, opening new possibilities for the design of proapoptotic derivatives in these series.

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 © 2012 John Wiley & Sons, Ltd.)

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.

Attacking cancer cell survival defense by targeting B-Cell Lymphoma 2 (BCL2) family of anti-apoptotic proteins may provide a powerful means to improve chemotherapy efficiency. This could be particularly relevant to anti-mitotic-based therapy, where tumor response relates to a competing network between mitotic cell death signaling and mitotic slippage as an adaptative response to a leaky mitotic checkpoint. In this review, we focus on recent findings that point out the major role played by BCL2 family members in response to anti-mitotic agents, which reveal dependence of cancer cell survival on BCL2 homologs during mitotic arrest and after mitotic slippage. Finally, we discuss pre-clinical data combining anti-mitotic agents with BCL2 inhibitors.

Introduction: Inappropriate 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., Methods: By 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., Results: We 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., Conclusions: We 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.

Background: Notch signaling pathway controls key functions in vascular and endothelial cells (ECs) where Notch4 plays a major role. However, little is known about the contribution of other Notch receptors. This study investigated regulation of Notch2 and further examined its implication in EC dysfunction., Methodology/principal Findings: Here, we provide evidence for a novel link between Notch and TNF signaling, where Notch2 is upregulated and activated in response to TNF. Forced expression of Notch2 intracellular domain in cultured ECs promotes apoptosis and allows the significant downregulation of several cell-death-related transcripts in a dose-dependent manner. In particular, activation of Notch2 led to a rapid decrease in survivin mRNA and protein expression, while survivin upregulation was obtained by the selective knockdown of Notch2 in ECs, indicating that survivin expression is controlled at the Notch level. Moreover, Notch2 silencing and ectopic expression of survivin, but not XIAP or Bcl2, rescued ECs from TNF and Notch2-mediated apoptosis, respectively., Conclusions/significance: In conclusion, TNF signaling activates Notch2 that sensitizes ECs to apoptosis via modulation of the key apoptosis regulator survivin. Overall, our findings also indicate that specific Notch receptors control distinct functions in vascular cells and inflammatory cytokines contribute to this specificity.

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.

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.

Discovered 10 years ago, survivin has a dual role in the smooth progress of mitosis and in apoptosis resistance. Survivin plays an important physiological role in development, but is absent in differentiated adult tissues. In contrast, aberrant survivin expression is found in most human cancers because of the activation of various signalling pathways. A complex survivin network appears to intersect multiple pathways in cell biology, related to several molecular partners and fine subcellular localizations. Based on its pro-oncogenic properties, basic and translational studies have shown a growing interest in survivin that has led to consider survivin as a prognostic marker and a promising target for anti-tumoral therapies.

Purpose: Intrinsic activation of nuclear factor kappaB (NF-kappaB) characterizes various hematologic malignancies. In this study, we specifically address the role of NF-kappaB blockade in mediated antimyeloma activity using the IkappaB 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-kappaB 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 IkappaB 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-kappaB 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-kappaB pathway in myeloma and clearly encourage clinical evaluation of novel therapies based on targeting NF-kappaB, especially in combination with TRAIL.

The aim of this review is to integrate non-exhaustive relevant data on the phenotype of human plasma cells (PC), including normal, reactive and malignant (multiple myeloma, MM) PC. This review focuses on (i) a universal marker of both normal and malignant plasma cells, CD138; (ii) markers related to malignancy i.e., CD19, CD27, CD28, and CD56; (iii) markers associated with signaling and severity of MM (CD45, CD221). Finally, this review presents data from normal PC up to human myeloma cell lines in order to: (i) define different entities of MM based on expression of CD19, CD20, CD27 and CD117; and (ii) identify new therapeutic targets.

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-1alpha and MIP-1beta 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.

Even during this past year, further advances have been made in understanding the molecular genetics of the disease, the mechanisms involved in the generation of myeloma-associated bone disease and elucidation of critical signaling pathways as therapeutic targets. New agents (thalidomide, Revimid, Velcade) providing effective salvage therapy for end-stage myeloma, have broadened the therapeutic armamentarium markedly. As evidenced in Section I by Drs. Kuehl and Bergsagel, five recurrent primary translocations resulting from errors in IgH switch recombination during B-cell development in germinal centers involve 11q13 (cyclin D1), 4p16.3 (FGFR3 and MMSET), 6p21 (cyclin D3), 16q23 (c-maf), and 20q11 (mafB), which account for about 40% of all myeloma tumors. Based on gene expression profiling data from two laboratories, the authors propose 5 multiple myeloma (MM) subtypes defined by the expression of translocation oncogenes and cyclins (TC molecular classification of MM) with different prognostic implications. In Section II, Drs. Barillé-Nion and Bataille review new insights into osteoclast activation through the RANK Ligand/OPG and MIP-1 chemokine axes and osteoblast inactivation in the context of recent data on DKK1. The observation that myeloma cells enhance the formation of osteoclasts whose activity or products, in turn, are essential for the survival and growth of myeloma cells forms the basis for a new treatment paradigm aimed at reducing the RANKL/OPG ratio by treatment with RANKL inhibitors and/or MIP inhibitors. In Section III, Dr. Fenton reviews apoptotic pathways as they relate to MM therapy. Defects in the mitochrondrial intrinsic pathway result from imbalances in expression levels of Bcl-2, Bcl-XL and Mcl-1. Mcl-1 is a candidate target gene for rapid induction of apoptosis by flavoperidol. Antisense oglionucleotides (ASO) lead to the rapid induction of caspace activity and apoptosis, which was potentiated by dexamethasone. Similar clinical trials with Bcl-2 ASO molecules alone and in combination with doxorubicin and dexamethasone or thalidomide showed promising results. The extrinsic pathway can be activated upon binding of the ligand TRAIL. OPG, released by osteoblasts and other stromal cells, can act as a decoy receptor for TRAIL, thereby blocking its apoptosis-inducing activity. MM cells inhibit OPG release by stromal cells, thereby promoting osteoclast activation and lytic bone disease (by enhancing RANKL availability) while at the same time exposing themselves to higher levels of ambient TRAIL. Thus, as a recurring theme, the relative levels of pro- versus anti-apoptotic molecules that act in a cell autonomous manner or in the milieu of the bone marrow microenvironment determine the outcome of potentially lethal signals. In Section IV, Dr. Barlogie and colleagues review data on single and tandem autotransplants for newly diagnosed myeloma. CR rates of 60%-70% can be reached with tandem transplants extending median survival to approximately 7 years. Dose adjustments of melphalan in the setting of renal failure and age > 70 may be required to reduce mucositis and other toxicities in such patients, especially in the context of amyloidosis with cardiac involvement. In Total Therapy II the Arkansas group is evaluating the role of added thalidomide in a randomized trial design. While data are still blinded as to the contribution of thalidomide, the overriding adverse importance of cytogenetic abnormalities, previously reported for Total Therapy I, also pertain to this successor trial. In these two-thirds of patients without cytogenetic abnormalities, Total Therapy II effected a doubling of the 4-year EFS estimate from 37% to 75% (P <.0001) and increased the 4-year OS estimate from 63% to 84% (P =.0009). The well-documented graft-vs-MM effect of allotransplants can be more safely examined in the context of non-myeloablative regimens, applied as consolidation after a single autologous transplant with melphalan 200 mg/m(2), have been found to be much better tolerated than standard myeloablative conditioning rege conditioning regimens and yielding promising results even in the high-risk entity of MM with cytogenetic abnormalities. For previously treated patients, the thalidomide congener Revimid and the proteasome inhibitor Velcade both are active in advanced and refractory MM (approximately 30% PR). Gene expression profiling (GEP) has unraveled distinct MM subtypes with different response and survival expectations, can distinguish the presence of or future development of bone disease, and, through serial investigations, can elucidate mechanisms of actions of new agents also in the context of the bone marrow microenvironment. By providing prognostically relevant distinction of MM subgroups, GEP should aid in the development of individualized treatment for MM.