Antimetastatic Properties of Prodigiosin and the BH3-Mimetic Obatoclax (GX15-070) in Melanoma.

Metastasis is the primary cause of death in cancer patients. Many current chemotherapeutic agents only show cytotoxic, but not antimetastatic properties. This leads to a reduction in tumor size, but allows cancer cells to disseminate, which ultimately causes patient death. Therefore, novel anticancer compounds with both effects need to be developed. In this work, we analyze the antimetastatic properties of prodigiosin and obatoclax (GX15-070), anticancer drugs of the Prodiginines (PGs) family. We studied PGs' effects on cellular adhesion and morphology in the human primary and metastatic melanoma cell lines, SK-MEL-28 and SK-MEL-5, and in the murine melanoma cell line, B16F10A. Cell adhesion sharply decreased in the treated cells, and this was accompanied by a reduction in filopodia protrusions and a significant decrease in the number of focal-adhesion structures. Moreover, cell migration was assessed through the wound-healing assay and cell motility was severely inhibited after 24 h of treatment. To elucidate the molecular mechanisms involved, changes in metastasis-related genes were analyzed through a gene-expression array. Key genes related to cellular invasion, migration and chemoresistance were significantly down-regulated. Finally, an in vivo model of melanoma-induced lung metastasis was established and significant differences in lung tumors were observed in the obatoclax-treated mice. Altogether, these results describe, in depth, PGs' cellular antimetastatic effects and identify in vivo antimetastatic properties of Obatoclax.

Pevonedistat (MLN4924): mechanism of cell death induction and therapeutic potential in colorectal cancer.

Pevonedistat (MLN4924), a selective inhibitor of the NEDD8-activating enzyme E1 regulatory subunit (NAE1), has demonstrated significant therapeutic potential in several malignancies. Although multiple mechanisms-of-action have been identified, how MLN4924 induces cell death and its potential as a combinatorial agent with standard-of-care (SoC) chemotherapy in colorectal cancer (CRC) remains largely undefined. In an effort to understand MLN4924-induced cell death in CRC, we identified p53 as an important mediator of the apoptotic response to MLN4924. We also identified roles for the extrinsic (TRAIL-R2/caspase-8) and intrinsic (BAX/BAK) apoptotic pathways in mediating the apoptotic effects of MLN4924 in CRC cells, as well as a role for BID, which modulates a cross-talk between these pathways. Depletion of the anti-apoptotic protein FLIP, which we identify as a novel mediator of resistance to MLN4924, enhanced apoptosis in a p53-, TRAIL-R2/DR5-, and caspase-8-dependent manner. Notably, TRAIL-R2 was involved in potentiating the apoptotic response to MLN4924 in the absence of FLIP, in a ligand-independent manner. Moreoever, when paired with SoC chemotherapies, MLN4924 demonstrated synergy with the irinotecan metabolite SN38. The cell death induced by MLN4924/SN38 combination was dependent on activation of mitochondria through BAX/BAK, but in a p53-independent manner, an important observation given the high frequency of TP53 mutation(s) in advanced CRC. These results uncover mechanisms of cell death induced by MLN4924 and suggest that this second-generation proteostasis-disrupting agent may have its most widespread activity in CRC, in combination with irinotecan-containing treatment regimens.

The SCFSkp2 ubiquitin ligase complex modulates TRAIL-R2-induced apoptosis by regulating FLIP(L).

TRAIL-R2 (DR5) is a clinically-relevant therapeutic target and a key target for immune effector cells. Herein, we identify a novel interaction between TRAIL-R2 and the Skp1-Cullin-1-F-box (SCF) Cullin-Ring E3 Ubiquitin Ligase complex containing Skp2 (SCFSkp2). We find that SCFSkp2 can interact with both TRAIL-R2's pre-ligand association complex (PLAC) and ligand-activated death-inducing signalling complex (DISC). Moreover, Cullin-1 interacts with TRAIL-R2 in its active NEDDylated form. Inhibiting Cullin-1's DISC recruitment using the NEDDylation inhibitor MLN4924 (Pevonedistat) or siRNA increased apoptosis induction in response to TRAIL. This correlated with enhanced levels of the caspase-8 regulator FLIP at the TRAIL-R2 DISC, particularly the long splice form, FLIP(L). We subsequently found that FLIP(L) (but not FLIP(S), caspase-8, nor the other core DISC component FADD) interacts with Cullin-1 and Skp2. Importantly, this interaction is enhanced when FLIP(L) is in its DISC-associated, C-terminally truncated p43-form. Prevention of FLIP(L) processing to its p43-form stabilises the protein, suggesting that by enhancing its interaction with SCFSkp2, cleavage to the p43-form is a critical step in FLIP(L) turnover. In support of this, we found that silencing any of the components of the SCFSkp2 complex inhibits FLIP ubiquitination, while overexpressing Cullin-1/Skp2 enhances its ubiquitination in a NEDDylation-dependent manner. DISC recruitment of TRAF2, previously identified as an E3 ligase for caspase-8 at the DISC, was also enhanced when Cullin-1's recruitment was inhibited, although its interaction with Cullin-1 was found to be mediated indirectly via FLIP(L). Notably, the interaction of p43-FLIP(L) with Cullin-1 disrupts its ability to interact with FADD, caspase-8 and TRAF2. Collectively, our results suggest that processing of FLIP(L) to p43-FLIP(L) at the TRAIL-R2 DISC enhances its interaction with co-localised SCFSkp2, leading to disruption of p43-FLIP(L)'s interactions with other DISC components and promoting its ubiquitination and degradation, thereby modulating TRAIL-R2-mediated apoptosis.

FLIP as a therapeutic target in cancer.

One of the classic hallmarks of cancer is disruption of cell death signalling. Inhibition of cell death promotes tumour growth and metastasis, causes resistance to chemo- and radiotherapies as well as targeted agents, and is frequently due to overexpression of antiapoptotic proteins rather than loss of pro-apoptotic effectors. FLIP is a major apoptosis-regulatory protein frequently overexpressed in solid and haematological cancers, in which its high expression is often correlated with poor prognosis. FLIP, which is expressed as long (FLIP(L)) and short (FLIP(S)) splice forms, achieves its cell death regulatory functions by binding to FADD, a critical adaptor protein which links FLIP to the apical caspase in the extrinsic apoptotic pathway, caspase-8, in a number of cell death regulating complexes, such as the death-inducing signalling complexes (DISCs) formed by death receptors. FLIP also plays a key role (together with caspase-8) in regulating another form of cell death termed programmed necrosis or 'necroptosis', as well as in other key cellular processes that impact cell survival, including autophagy. In addition, FLIP impacts activation of the intrinsic mitochondrial-mediated apoptotic pathway by regulating caspase-8-mediated activation of the pro-apoptotic Bcl-2 family member Bid. It has been demonstrated that FLIP can not only inhibit death receptor-mediated apoptosis, but also cell death induced by a range of clinically relevant chemotherapeutic and targeted agents as well as ionizing radiation. More recently, key roles for FLIP in promoting the survival of immunosuppressive tumour-promoting immune cells have been discovered. Thus, FLIP is of significant interest as an anticancer therapeutic target. In this article, we review FLIP's biology and potential ways of targeting this important tumour and immune cell death regulator.

MED12 and HMGA2 mutations: two independent genetic events in uterine leiomyoma and leiomyosarcoma.

Recent identification of somatic MED12 mutations in most uterine leiomyomas brings a new venue for the study of the tumorigenesis of leiomyomas. We are particularly interested in the correlation of MED12 and HMGA2 gene products in leiomyomas and leiomyosarcomas with and without MED12 mutations. To address these issues, in this study we examined MED12 mutations in a large cohort of usual type leiomyomas (178 cases) and uterine leiomyosarcomas (32 cases). We found that 74.7% (133/178) of leiomyomas had MED12 mutations, which was consistent with several independent studies. In contrast, only 9.7% (3/32) of leiomyosarcomas harbored MED12 mutations. Expression analysis by western blot and immunohistochemistry revealed that those leiomyomas with complex MED12 mutations had significantly lower protein products than the matched myometrium. Interestingly, most leiomyosarcomas without MED12 mutations also had very low levels of MED12 expression in comparison to the matched myometrium. These findings suggest a potential functional role of MED12 in both benign and malignant uterine smooth muscle tumors. When we further examined HMGA2 expression in all leiomyomas and leiomyosarcomas, we found that HMGA2 overexpression was exclusively present in those leiomyomas with no MED12 mutation, accounting for 10.1% (18/178) of total leiomyomas and 40% (18/45) of non-MED12 mutant leiomyomas. Twenty-five percent (8/32) of leiomyosarcomas had HMGA2 overexpression, and no MED12 mutations were found in HMGA2 positive leiomyosarcoma. These findings strongly suggest that MED12 mutations and HMGA2 overexpression are independent genetic events that occur in leiomyomas, and they may act differently in the tumorigenesis of uterine leiomyomas.

Down-regulation of miR-29b is essential for pathogenesis of uterine leiomyoma.

Uterine leiomyomata (LMs) are the most common tumor affecting the female reproductive organs. The most notable pathophysiologic feature of this tumor is the excessive accumulation of rigid extracellular matrix (ECM) composed mainly of collagen types I and III. It is believed that the rigidity of the collagen-rich ECM causes symptoms such as abnormal bleeding and pelvic pain/pressure. However, the molecular pathogenesis for this ECM-rich tumor has yet to be elucidated. We have established that miR-29b was consistently down-regulated in LM compared with myometrium (MM). Hence, the function of miR-29b in LM was examined in vivo using adult female ovariectomized NOD-scid IL2Rγ(null) mice for subrenal xenograft models. In LM xenografts, restoring miR-29b inhibited the accumulation of ECM and the development of solid tumors. Although the miR-29b knockdown in MM cells increased the expression of collagens, it did not transform MM cells into tumorigenic, indicating that the down-regulation of miR-29b is essential but not sufficient for LM tumorigenesis. In addition, 17ß-estradiol and progesterone down-regulated miR-29b and up-regulated mRNAs for multiple collagens in LM xenografts. Thus, we conclude that ECM production in LMs is regulated by steroid hormones via down-regulation of miR-29b, which is one of the mechanisms underlying the excessive accumulation of ECM.

Molecular interactions of prodiginines with the BH3 domain of anti-apoptotic Bcl-2 family members.

Prodigiosin and obatoclax, members of the prodiginines family, are small molecules with anti-cancer properties that are currently under preclinical and clinical trials. The molecular target(s) of these agents, however, is an open question. Combining experimental and computational techniques we find that prodigiosin binds to the BH3 domain in some BCL-2 protein families, which play an important role in the apoptotic programmed cell death. In particular, our results indicate a large affinity of prodigiosin for MCL-1, an anti-apoptotic member of the BCL-2 family. In melanoma cells, we demonstrate that prodigiosin activates the mitochondrial apoptotic pathway by disrupting MCL-1/BAK complexes. Computer simulations with the PELE software allow the description of the induced fit process, obtaining a detailed atomic view of the molecular interactions. These results provide new data to understand the mechanism of action of these molecules, and assist in the development of more specific inhibitors of anti-apoptotic BCL-2 proteins.

Bcl-2 family proteins and cytoskeleton changes involved in DM-1 cytotoxic effect on melanoma cells.

Melanoma is one of the most aggressive types of skin cancer and its incidence rate is still increasing. All existing treatments are minimally effective. Consequently, new therapeutic agents for melanoma treatment should be developed. The DM-1 compound is a curcumin analog that possesses several curcumin characteristics, such as antiproliferative, antitumor, and anti-metastatic properties. The aim of this study was to evaluate the different signaling pathways involved in the cytotoxic effect of DM-1 on melanoma cells. The apoptotic process and cytoskeletal changes were evaluated by immunoblotting and immunofluorescence, respectively, in melanoma cells. After DM-1 treatment, SK-MEL-5 melanoma cells showed actin filament disorganization with spicule formation throughout the cytoskeleton and significant reduction of focal adhesion as well as they were present only at cell extremities, conferring a poor connection between the cell and the substrate. Besides this, there was significant filopodium retraction and loss of typical cytoskeleton scaffold. These modifications contributed to cell detachment followed by cell death. Furthermore, DM-1-induced apoptosis was triggered by multiple Bcl-2 proteins involved in both the extrinsic and the intrinsic apoptotic pathways. SK-MEL-5 cells showed a death mechanism mainly by Bcl-2/Bax ratio decrease, whereas A375 cells presented apoptosis induction by Mcl-1 and Bcl-xL downregulation. In SK-MEL-5 and A375 melanoma cells, there was a significant increase in the active form of caspase 9, and the inactive form of the effector caspase 3 was decreased in both cell lines. Expression of cleaved poly ADP ribose polymerase was increased after DM-1 treatment in these melanoma cell lines, demonstrating that the apoptotic process occurred. Altogether, these data elucidate the cellular and molecular mechanisms involved in the cytotoxicity induced by the antitumor agent DM-1 in melanoma cells.