Systemic Delivery of Tumor-Targeted Bax-Derived Membrane-Active Peptides for the Treatment of Melanoma Tumors in a Humanized SCID Mouse Model.

Melanoma is a highly metastatic and deadly form of cancer. Invasive melanoma cells overexpress integrin αvß3, which is a well-known target for Arg-Gly-Asp-based (RGD) peptides. We developed a sophisticated method to synthetize milligram amounts of a targeted vector that allows the RGD-mediated targeting, internalization, and release of a mitochondria-disruptive peptide derived from the pro-apoptotic Bax protein. We found that 2.5 µM Bax[109-127] was sufficient to destabilize the mitochondria in ten different tumor cell lines, even in the presence of the anti-apoptotic Bcl2 protein, which is often involved in tumor resistance. This pore-forming peptide displayed antitumor activity when it was covalently linked by a disulfide bridge to the tetrameric RAFT-c[RGD]4-platform and after intravenous injection in a human melanoma tumor model established in humanized immuno-competent mice. In addition to its direct toxic effect, treatment with this combination induced the release of the immuno-stimulating factor monocyte chimoattractant protein 1 (MCP1) in the blood and a decrease in the level of the pro-angiogenic factor FGF2. Our novel multifunctional, apoptosis-inducing agent could be further customized and assayed for potential use in tumor-targeted therapy.

Bax-derived membrane-active peptides act as potent and direct inducers of apoptosis in cancer cells.

Although many cancer cells are primed for apoptosis, they usually develop resistance to cell death at several levels. Permeabilization of the outer mitochondrial membrane, which is mediated by proapoptotic Bcl-2 family members such as Bax, is considered as a point of no return for initiating apoptotic cell death. This crucial role has placed Bcl-2 family proteins as recurrent targets for anticancer drug development. Here, we propose and demonstrate a new concept based on minimal active versions of Bax to induce cell death independently of endogenous Bcl-2 proteins. We show that membrane-active segments of Bax can directly induce the release of mitochondria-residing apoptogenic factors and commit tumor cells promptly and irreversibly to caspase-dependent apoptosis. On this basis, we designed a peptide encompassing part of the Bax pore-forming domain, which can target mitochondria, induce cytochrome c release and trigger caspase-dependent apoptosis. Moreover, this Bax-derived 'poropeptide' produced effective tumor regression after peritumoral injection in a nude mouse xenograft model. Thus, peptides derived from proteins that form pores in the mitochondrial outer membrane represent novel templates for anticancer agents.

Defective ubiquitin-mediated degradation of antiapoptotic Bfl-1 predisposes to lymphoma.

The antiapoptotic Bcl-2 family member Bfl-1 is up-regulated in many human tumors in which nuclear factor-kappaB (NF-kappaB) is implicated and contributes significantly to tumor cell survival and chemoresistance. We previously found that NF-kappaB induces transcription of bfl-1 and that the Bfl-1 protein is also regulated by ubiquitin-mediated proteasomal degradation. However, the role that dysregulation of Bfl-1 turnover plays in cancer is not known. Here we show that ubiquitination-resistant mutants of Bfl-1 display increased stability and greatly accelerated tumor formation in a mouse model of leukemia/lymphoma. We also show that tyrosine kinase Lck is up-regulated and activated in these tumors and leads to activation of the IkappaB kinase, Akt, and extracellular signal-regulated protein kinase signaling pathways, which are key mediators in cancer. Coexpression of Bfl-1 and constitutively active Lck promoted tumor formation, whereas Lck knockdown in tumor-derived cells suppressed leukemia/lymphomagenesis. These data demonstrate that ubiquitination is a critical tumor suppression mechanism regulating Bfl-1 function and suggest that mutations in bfl-1 or in the signaling pathways that control its ubiquitination may predispose one to cancer. Furthermore, because bfl-1 is up-regulated in many human hematopoietic tumors, this finding suggests that strategies to promote Bfl-1 ubiquitination may improve therapy.

TRIM17 and TRIM28 antagonistically regulate the ubiquitination and anti-apoptotic activity of BCL2A1.

BCL2A1 is an anti-apoptotic member of the BCL-2 family that contributes to chemoresistance in a subset of tumors. BCL2A1 has a short half-life due to its constitutive processing by the ubiquitin-proteasome system. This constitutes a major tumor-suppressor mechanism regulating BCL2A1 function. However, the enzymes involved in the regulation of BCL2A1 protein stability are currently unknown. Here, we provide the first insight into the regulation of BCL2A1 ubiquitination. We present evidence that TRIM28 is an E3 ubiquitin-ligase for BCL2A1. Indeed, endogenous TRIM28 and BCL2A1 bind to each other at the mitochondria and TRIM28 knock-down decreases BCL2A1 ubiquitination. We also show that TRIM17 stabilizes BCL2A1 by blocking TRIM28 from binding and ubiquitinating BCL2A1, and that GSK3 is involved in the phosphorylation-mediated inhibition of BCL2A1 degradation. BCL2A1 and its close relative MCL1 are thus regulated by common factors but with opposite outcome. Finally, overexpression of TRIM28 or knock-out of TRIM17 reduced BCLA1 protein levels and restored sensitivity of melanoma cells to BRAF-targeted therapy. Therefore, our data describe a molecular rheostat in which two proteins of the TRIM family antagonistically regulate BCL2A1 stability and modulate cell death.

Unexpected cross-reactivity of anti-cathepsin B antibodies leads to uncertainties regarding the mechanism of action of anti-CD20 monoclonal antibody GA101.

GA101, also known as obinutuzumab or Gazyva (Gazyvaro), is a glycoengineered type II humanized antibody that targets the CD20 antigen expressed at the surface of B-cells. This novel anti-CD20 antibody is currently assessed in clinical trials with promising results as a single agent or as part of therapeutic combinations for the treatment of B-cell malignancies. Detailed understanding of the mechanisms of GA101-induced cell death is needed to get insight into possible resistance mechanisms occurring in patients. Although multiple in vitro and in vivo mechanisms have been suggested to describe the effects of GA101 on B-cells, currently available data are ambiguous. The aim of our study was to clarify the cellular mechanisms involved in GA101-induced cell death in vitro, and more particularly the respective roles played by lysosomal and mitochondrial membrane permeabilization. Our results confirm previous reports suggesting that GA101 triggers homotypic adhesion and caspase-independent cell death, two processes that are dependent on actin remodeling and involve the production of reactive oxygen species. With respect to lysosomal membrane permeabilization (LMP), our data suggest that lack of specificity of available antibodies directed against cathepsin B may have confounded previously published results, possibly challenging current LMP-driven model of GA101 action mode.

To be, or not to be: NF-kappaB is the answer--role of Rel/NF-kappaB in the regulation of apoptosis.

During their lifetime, cells encounter many life or death situations that challenge their very own existence. Their survival depends on the interplay within a complex yet precisely orchestrated network of proteins. The Rel/NF-kappaB signaling pathway and the transcription factors that it activates have emerged as critical regulators of the apoptotic response. These proteins are best known for the key roles that they play in normal immune and inflammatory responses, but they are also implicated in the control of cell proliferation, differentiation, apoptosis and oncogenesis. In recent years, there has been remarkable progress in understanding the pathways that activate the Rel/NF-kappaB factors and their role in the cell's decision to either fight or surrender to apoptotic challenge. Whereas NF-kappaB is most commonly involved in suppressing apoptosis by transactivating the expression of antiapoptotic genes, it can promote programmed cell death in response to certain death-inducing signals and in certain cell types. This review surveys our current understanding of the role of NF-kappaB in the apoptotic response and focuses on many developments since this topic was last reviewed in Oncogene 4 years ago. These recent findings shed new light on the activity of NF-kappaB as a critical regulator of apoptosis in the immune, hepatic, epidermal and nervous systems, on the mechanisms through which it operates and on its role in tissue development, homoeostasis and cancer.

Differential mechanisms of asparaginase resistance in B-type acute lymphoblastic leukemia and malignant natural killer cell lines.

Bacterial L-asparaginase (ASNase), hydrolyzing L-asparagine (Asn), is an important drug for treating patients with acute lymphoblastic leukaemia (ALL) and natural killer (NK) cell lymphoma. Although different native or pegylated ASNase-based chemotherapy are efficient, disease relapse is frequently observed, especially in adult patients. The neo-synthesis of Asn by asparagine synthetase (AsnS) following ASNase treatment, which involves the amino acid response and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways, is believed to be the basis of ASNase-resistance mechanisms. However, AsnS expression has not emerged as an accurate predictive factor for ASNase susceptibility. The aim of this study was to identify possible ASNase sensitivity/resistance-related genes or pathways using a new asparaginase, namely a pegylated r-crisantaspase, with a focus on classic Asn-compensatory responses and cell death under conditions of Asn/L-glutamine limitation. We show that, for B-ALL cell lines, changes in the expression of apoptosis-regulatory genes (especially NFκB-related genes) are associated with ASNase susceptibility. The response of malignant NK cell lines to ASNase may depend on Asn-compensatory mechanisms and other cellular processes such as cleavage of BCL2A1, a prosurvival member of the Bcl-2 protein family. These results suggest that according to cellular context, factors other than AsnS can influence ASNase susceptibility.

µ-Calpain conversion of antiapoptotic Bfl-1 (BCL2A1) into a prodeath factor reveals two distinct alpha-helices inducing mitochondria-mediated apoptosis.

Anti-apoptotic Bfl-1 and pro-apoptotic Bax, two members of the Bcl-2 family sharing a similar structural fold, are classically viewed as antagonist regulators of apoptosis. However, both proteins were reported to be death inducers following cleavage by the cysteine protease µ-calpain. Here we demonstrate that calpain-mediated cleavage of full-length Bfl-1 induces the release of C-terminal membrane active α-helices that are responsible for its conversion into a pro-apoptotic factor. A careful comparison of the different membrane-active regions present in the Bfl-1 truncated fragments with homologous domains of Bax show that helix α5, but not α6, of Bfl-1 induces cell death and cytochrome c release from purified mitochondria through a Bax/Bak-dependent mechanism. In contrast, both helices α5 and α6 of Bax permeabilize mitochondria regardless of the presence of Bax or Bak. Moreover, we provide evidence that the α9 helix of Bfl-1 promotes cytochrome c release and apoptosis through a unique membrane-destabilizing action whereas Bax-α9 does not display such activities. Hence, despite a common 3D-structure, C-terminal toxic domains present on Bfl-1 and Bax function in a dissimilar manner to permeabilize mitochondria and induce apoptosis. These findings provide insights for designing therapeutic approaches that could exploit the cleavage of endogenous Bcl-2 family proteins or the use of Bfl-1/Bax-derived peptides to promote tumor cell clearance.

R-Cadherin expression inhibits myogenesis and induces myoblast transformation via Rac1 GTPase.

Cadherins are transmembrane glycoproteins that mediate Ca(2+)-dependent homophilic cell-cell adhesion and play a crucial role in proliferation, differentiation, and cell transformation. The goal of this study was to understand why R-cadherin is found in rhabdomyosarcomas (RMS), tumors of skeletal muscle origin, whereas it is absent in normal myoblasts. We show that R-cadherin expression in C2C12 myoblasts causes inhibition of myogenesis induction and impairment of cell cycle exit when cells are cultured in differentiation medium. Furthermore, R-cadherin expression elicits myoblast transformation, as shown by anchorage-independent growth in soft agar in vivo tumor formation assays and increased cell motility. In contrast, inhibition of R-cadherin expression using RNA interference hinders growth of RD cell line in soft agar and its tumorigenicity in mice. The analysis of the nature of R-cadherin-mediated signals shows that R-cadherin-dependent adhesion increases Rac1 activity. Dominant-negative forms of Rac1 inhibit R-cadherin-mediated signaling and transformation. In addition, expression of R-cadherin results in perturbed function of endogenous N-cadherin and M-cadherin. Together, these data suggest that R-cadherin expression inhibits myogenesis and induces myoblast transformation through Rac1 activation. Therefore, the properties of R-cadherin make it an attractive target for therapeutic intervention in RMS.

C-terminal heptapeptide of gastrin inhibits astrocytomas motility by interacting with a new gastrin binding site.

It is well known that the amidated C-terminal part of gastrin is crucial for its interaction with the classical seven transmembrane domain receptors CCK-1 or CCK-2. Nevertheless, over the past 10 years, several groups have characterized new binding sites using peptides related to gastrin (particularly glycine-extended forms of gastrin) on various tumoral and nontumoral cell lines. In the present study, we focused on the human astrocytic tumoral cell line U373. Although it has been described that gastrin was able to inhibit the motility of these cells, we were unable to detect any classical CCK/gastrin receptor. On the other hand, by using the radiolabeled C-terminal heptapeptide of gastrin ((125)I-G-7), we evidenced a new binding site that possessed a pharmacological profile different from the classical CCK/gastrin receptors. This new gastrin binding site seemed to be coupled to G proteins and be implicated in c-Fos transcription gene. Moreover, we showed that G-7 was able to induce a strong inhibition of U373 cell migration, a crucial biological effect when we know that astrocytoma cells' migration in brain parenchyma constitutes a major feature of malignancy in astrocytic tumors.