Ceramide nanoliposomes augment the efficacy of venetoclax and cytarabine in models of acute myeloid leukemia.

Despite several new therapeutic options for acute myeloid leukemia (AML), disease relapse remains a significant challenge. We have previously demonstrated that augmenting ceramides can counter various drug-resistance mechanisms, leading to enhanced cell death in cancer cells and extended survival in animal models. Using a nanoscale delivery system for ceramide (ceramide nanoliposomes, CNL), we investigated the effect of CNL within a standard of care venetoclax/cytarabine (Ara-C) regimen. We demonstrate that CNL augmented the efficacy of venetoclax/cytarabine in in vitro, ex vivo, and in vivo models of AML. CNL treatment induced non-apoptotic cytotoxicity, and augmented cell death induced by Ara-C and venetoclax. Mechanistically, CNL reduced both venetoclax (Mcl-1) and cytarabine (Chk1) drug-resistant signaling pathways. Moreover, venetoclax and Ara-C augmented the generation of endogenous pro-death ceramide species, which was intensified with CNL. Taken together, CNL has the potential to be utilized as an adjuvant therapy to improve outcomes, potentially extending survival, in patients with AML.

Lysosomal degradation of CD44 mediates ceramide nanoliposome-induced anoikis and diminished extravasation in metastatic carcinoma cells.

The ceramide nanoliposome (CNL) has shown promise in being able to treat a variety of primary tumors. However, its potential for treating metastatic cancer remains unknown. In this study, we demonstrate that CNL increases anoikis while preventing cancer cell extravasation under both static and physiological fluid flow conditions. Mechanistically, CNL limits metastases by decreasing CD44 protein levels in human breast and pancreatic cancer cells via lysosomal degradation of CD44, independent of palmitoylation or proteasome targeting. siRNA down-regulation of CD44 mimics CNL-induced anoikis and diminished extravasation of cancer cells. Taken together, our data indicate that ceramide limits CD44-dependent cancer cell migration, suggesting that CNL could be used to prevent and treat solid tumor metastasis.

Novel type of Ras effector interaction established between tumour suppressor NORE1A and Ras switch II.

A class of putative Ras effectors called Ras association domain family (RASSF) represents non-enzymatic adaptors that were shown to be important in tumour suppression. RASSF5, a member of this family, exists in two splice variants known as NORE1A and RAPL. Both of them are involved in distinct cellular pathways triggered by Ras and Rap, respectively. Here we describe the crystal structure of Ras in complex with the Ras binding domain (RBD) of NORE1A/RAPL. All Ras effectors share a common topology in their RBD creating an interface with the switch I region of Ras, whereas NORE1A/RAPL RBD reveals additional structural elements forming a unique Ras switch II binding site. Consequently, the contact area of NORE1A is extended as compared with other Ras effectors. We demonstrate that the enlarged interface provides a rationale for an exceptionally long lifetime of the complex. This is a specific attribute characterizing the effector function of NORE1A/RAPL as adaptors, in contrast to classical enzymatic effectors such as Raf, RalGDS or PI3K, which are known to form highly dynamic short-lived complexes with Ras.

Growth and tumor suppressor NORE1A is a regulatory node between Ras signaling and microtubule nucleation.

NORE1A is a Ras-binding protein that belongs to a group of tumor suppressors known as the Ras association domain family. Their growth- and tumor-suppressive function is assumed to be dependent on association with the microtubule cytoskeleton. However, a detailed understanding of this interplay is still missing. Here, we show that NORE1A directly interacts with tubulin and is capable of nucleating microtubules. Strikingly, the ability to stimulate nucleation is regulated in a dual specific way either via phosphorylation of NORE1A within the Ras-binding domain by Aurora A kinase or via binding to activated Ras. We also demonstrate that NORE1A mediates a negative effect of activated Ras on microtubule nucleation. On the basis of our results, we propose a novel regulatory network composed of the tumor suppressor NORE1A, the mitotic kinase Aurora A, the small GTPase Ras, and the microtubule cytoskeleton.

Minicells from Highly Genome Reduced Escherichia coli: Cytoplasmic and Surface Expression of Recombinant Proteins and Incorporation in the Minicells.

Minicells, small cells lacking a chromosome, produced by bacteria with mutated min genes, which control cell division septum placement, have many potential uses. Minicells have contributed to basic bacterial physiology studies and can enable new biotechnological applications, including drug delivery and vaccines. Genome-reduced bacteria are another informative area of investigation. Investigators identified that with even almost 30% of the E. coli genome deleted, the bacteria still live. In biotechnology and synthetic biology, genome-reduced bacteria offer certain advantages. With genome-reduced bacteria, more recombinant genes can be placed into genome-reduced chromosomes and fewer cell resources are devoted to purposes apart from biotechnological goals. Here, we show that these two technologies can be combined: min mutants can be made in genome-reduced E. coli. The minC minD mutant genome-reduced E. coli produce minicells that concentrate engineered recombinant proteins within these spherical delivery systems. We expressed recombinant GFP protein in the cytoplasm of genome-reduced bacteria and showed that it is concentrated within the minicells. We also expressed proteins on the surfaces of minicells made from genome-reduced bacteria using a recombinant Gram-negative AIDA-I autotransporter expression cassette. Some autotransporters, like AIDA-I, are concentrated at the bacterial poles, where minicells bud. Recombinant proteins expressed on surfaces of the genome-reduced bacteria are concentrated on the minicells. Minicells made from genome-reduced bacteria may enable useful biotechnological innovations, such as drug delivery vehicles and vaccine immunogens.

Identification of a novel Ras-regulated proapoptotic pathway.

BACKGROUND: The Ras-GTPase controls cell fate decisions through the binding of an array of effector molecules, such as Raf and PI 3-kinase, in a GTP-dependent manner. NORE1, a noncatalytic polypeptide, binds specifically to Ras-GTP and to several other Ras-like GTPases. NORE is homologous to the putative tumor suppressor RASSF1 and to the Caenorhabditis elegans polypeptide T24F1.3. RESULTS: We find that all three NORE-related polypeptides bind selectively to the proapoptotic protein kinase MST1, a member of the Group II GC kinases. Endogenous NORE and MST1 occur in a constitutive complex in vivo that associates with endogenous Ras after serum stimulation. Targeting recombinant MST1 to the membrane, either through NORE or myristoylation, augments the apoptotic efficacy of MST1. Overexpression of constitutively active Ki-RasG12V promotes apoptosis in a variety of cell lines; Ha-RasG12V is a much less potent proapoptotic agent; however, a Ha-RasG12V effector loop mutant (E37G) that binds NORE, but not Raf or PI 3-kinase, exhibits proapoptotic efficacy approaching that of Ki-RasG12V. The apoptotic action of both Ki-RasG12V and Ha-RasG12V, E37G is suppressed by overexpression of the MST1 carboxy-terminal noncatalytic segment or by the NORE segment that binds MST1. CONCLUSIONS: MST1 is a phylogenetically conserved partner of the NORE/RASSF polypeptide family, and the NORE-MST1 complex is a novel Ras effector unit that mediates the apoptotic effect of Ki-RasG12V.

The putative tumor suppressor RASSF1A homodimerizes and heterodimerizes with the Ras-GTP binding protein Nore1.

Nore and RASSF1A are noncatalytic proteins that share 50% identity over their carboxyterminal 300 AA, a segment that encompasses a putative Ras-Rap association (RA) domain. RASSF1 is expressed as several splice variants, each of which contain an RA domain, however the 340 AA RASSF1A, but not the shorter RASSF1C variant, is a putative tumor suppressor. Nore binds to Ras and several Ras-like GTPases in a GTP dependent fashion however neither RASSF1 (A or C) or the C. elegans Nore/RASSF1 homolog, T24F1.3 exhibit any interaction with Ras or six other Ras-like GTPases in a yeast two-hybrid expression assay. A low recovery of RASSF1A (but not RASSF1C) in association with RasG12V is observed however on transient expression in COS cells. Nore and RASSF1A can each efficiently homodimerize and heterodimerize with each other through their nonhomologous aminoterminal segments. Recombinant RASSF1C exhibits a much weaker ability to homodimerize or heterodimerize; thus the binding of RASSF1C to Nore is very much less than the binding of RASSF1A to Nore. The association of RASSF1A with RasG12V in COS cells appears to reflect the heterodimerization of RASSF1A with Nore, inasmuch the recovery of RASSF1A with RasG12V is increased by concurrent expression of full length Nore, and abolished by expression of Nore deleted of its RA domain. The preferential ability of RASSF1A to heterodimerize with Nore and thereby associate with Ras-like GTPases may be relevant to its putative tumor suppressor function.

The growth and tumor suppressors NORE1A and RASSF1A are targets for calpain-mediated proteolysis.

BACKGROUND: NORE1A and RASSF1A are growth and tumour suppressors inactivated in a variety of cancers. Methylation of NORE1A and RASSF1A promoters is the predominant mechanism for downregulation of these proteins; however, other mechanisms are likely to exist. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a proteolysis of NORE1A and RASSF1A by calpains as alternative mechanism of their downregulation. Extracts of H358 cell line, a human bronchoalveolar carcinoma, and H460, a large cell carcinoma, were capable of proteolysis of NORE1A protein in the calpain-dependent manner. Likewise, RASSF1A tumor suppressor was proteolyzed by the H358 cell extract. Addition of calpain inhibitor to H358 and H460 cells growing in tissue culture resulted in re-expression of endogenous NORE1A. A survey of 10 human lung tumours revealed that three of them contain an activity capable of inducing NORE1A degradation. CONCLUSIONS/SIGNIFICANCE: Thus, degradation by calpains is a novel mechanism for downregulation of NORE1A and RASSF1A proteins and might be the mechanism allowing cancer cells to escape growth suppression.

Interaction of the growth and tumour suppressor NORE1A with microtubules is not required for its growth-suppressive function.

BACKGROUND: The NORE1 protein was identified in a yeast two-hybrid screen as a Ras effector that binds Ras protein in a GTP-dependent manner. NORE1A is a growth and tumour suppressor that is inactivated in a variety of cancers. In transformed human cells, both full-length NORE1A protein and its effector domain alone (amino acids 191-363) are localized to microtubules and centrosomes. However, the mechanism by which NORE1A associates with these cytoskeletal elements is not known; furthermore, whether centrosomally-associated or microtubule-associated NORE1A suppresses tumour cell growth has not been yet established. FINDINGS: We have shown that purified NORE1A fails to bind to microtubules in vitro suggesting that other protein(s) mediate NORE1A-microtubule association. Using mass-spectrometry, we identified the Microtubule-Associated Protein 1B (MAP1B) and its homologue C19ORF5 as NORE1A interaction partners. Suppression of C19ORF5 expression by RNA interference (RNAi) and immunodepletion of C19ORF5 protein from cell extracts showed that binding of NORE1A to microtubules is not dependent on C19ORF5. Conversely, RNAi suppression of MAP1B revealed that MAP1B is required for association of NORE1A with microtubules. RNAi-mediated depletion of C19ORF5 or MAP1B did not prevent centrosomal localization of NORE1A. Moreover, the depletion of C19ORF5 or MAP1B did not prevent NORE1A's ability to suppress tumour cell growth. CONCLUSION: The interaction of NORE1A with microtubules is mediated by MAP1B, but not C19ORF5 protein. Interaction of NORE1A with centrosomes is not dependent on C19ORF5 or MAP1B, and appears to involve a different mechanism independent of binding to microtubules. The NORE1A microtubular localization is not required for growth suppression.

The growth and tumor suppressor NORE1A is a cytoskeletal protein that suppresses growth by inhibition of the ERK pathway.

NORE1A is a growth and tumor suppressor that is inactivated in a variety of cancers. NORE1A has been shown to bind to the active Ras oncogene product. However, the mechanism of NORE1A-induced growth arrest and tumor suppression remains unknown. Using anchorage-independent growth assays, we mapped the NORE1A effector domain (the minimal region of the protein responsible for its growth-suppressive effects) to the fragment containing the central and Ras association domains of NORE1A (amino acids 191-363). Expression of the NORE1A effector domain in A549 lung adenocarcinoma cells resulted in the selective inhibition of signal transduction through the ERK pathway. The full-length NORE1A (416 amino acids) and its fragments capable of growth suppression were localized to centrosomes and microtubules in normal and transformed human cells in a Ras-independent manner. A mutant that was deficient in binding to centrosomes and microtubules was also deficient in inducing cell cycle arrest. This suggests that cytoskeletal localization is required for growth-suppressive effects of NORE1A. Ras binding function was required for growth-suppressive effects of the full-length NORE1A but not for the growth-suppressive effects of the effector domain. Our studies suggest that association of NORE1A with cytoskeletal elements is essential for NORE1A-induced growth suppression and that the ERK pathway is a target for NORE1A growth-suppressive activities.

Characterization of mitogen-activated protein kinase (MAPK) dimers.

Phosphorylated ERK2 has an increased capacity to form homodimers relative to unphosphorylated ERK2. We have characterized the nature of the ERK2 dimer and have mutated residues in the crystal dimer interface to examine the impact of dimerization on ERK2 activity. Analysis of the mutants by gel filtration indicates that at least five residues must be mutated simultaneously to produce an ERK2 mutant that is predominantly monomeric. Mutants, whether monomers or dimers, have specific protein kinase activities under fixed assay conditions that are roughly equivalent to wild-type ERK2. The ratio of dimers to monomers is increased as the salt concentration increases, consistent with a strong hydrophobic contribution to the energy of dimer formation. ERK2 dimerization also requires divalent cations. Sedimentation analysis indicates that the related c-Jun N-terminal kinase SAPKalphaI/JNK2 also forms dimers, but dimerization displays no dependence on phosphorylation; the unphosphorylated and phosphorylated forms of the kinase behave similarly, with low micromolar dimer dissociation constants.

Death-associated protein 4 binds MST1 and augments MST1-induced apoptosis.

The protein kinase MST1 is proapoptotic when overexpressed in an active form, however, its physiologic regulation and cellular targets are unknown. An overexpressed inactive MST1 mutant associates in COS-7 cells with an endogenous 761-amino acid polypeptide known as "death-associated protein 4" (DAP4). The DAPs are a functionally heterogeneous array of polypeptides previously isolated by Kimchi and colleagues (Kimchi, A. (1998) Biochim. Biophys. Acta 1377, F13-F33 in a screen for elements involved in the interferon gamma-induced apoptosis of HeLa cells. DAP4, which is encoded by a member of a vertebrate-only gene family, contains no identifiable domains, but is identical over its amino-terminal 488 amino acids to p52(rIPK), a putative modulator of protein kinase R. DAP4 is a widely expressed, constitutively nuclear polypeptide that homodimerizes through its amino terminus and binds MST1 through its carboxyl-terminal segment. MST1 is predominantly cytoplasmic, but cycles continuously through the nucleus, as evidenced by its rapid accumulation in the nucleus after addition of the Crm1 inhibitor, leptomycin B. Overexpression of DAP4 does not cause apoptosis, however, coexpression of DAP4 with a submaximal amount of MST1 enhances MST1-induced apoptosis in a dose-dependent fashion. DAP4 is not significantly phosphorylated by MST1 nor does it alter MST1 kinase activity in vivo or in vitro. MST1-induced apoptosis is suppressed by a dominant interfering mutant of p53. MST1 is unable to directly phosphorylate p53, however, DAP4 binds endogenous and recombinant p53. DAP4 may promote MST1-induced apoptosis by enabling colocalization of MST with p53.

The scaffold protein CNK1 interacts with the tumor suppressor RASSF1A and augments RASSF1A-induced cell death.

The connector enhancer of KSR (CNK) is a multidomain scaffold protein discovered in Drosophila, where it is necessary for Ras activation of the Raf kinase. Recent studies have shown that CNK1 also interacts with RalA and Rho and participates in some aspects of signaling by these GTPases. Herein we demonstrate a novel aspect of CNK1 function, i.e. reexpression of CNK1 suppresses tumor cell growth and promotes apoptosis. As shown previously for apoptosis induced by Ki-Ras(G12V), CNK1-induced apoptosis is suppressed by a dominant inhibitor of the mammalian sterile 20 kinases 1 and (MST1/MST2). Immunoprecipitates of MST1 endogenous to LoVo colon cancer cells contain endogenous CNK1; however, no association of these two polypeptides can be detected in a yeast two-hybrid assay. CNK1 does, however, bind directly to the RASSF1A and RASSF1C polypeptides, constitutive binding partners of the MST1/2 kinases. Deletion of the MST1 carboxyl-terminal segment that mediates its binding to RASSF1A/C eliminates the association of MST1 with CNK1. Coexpression of CNK1 with the tumor suppressive isoform, RASSF1A, greatly augments CNK1-induced apoptosis, whereas the nonsuppressive RASSF1C isoform is without effect on CNK1-induced apoptosis. Overexpression of CNK1-(1-282), a fragment that binds RASSF1A but is not proapoptotic, blocks the apoptosis induced by CNK1 and by Ki-Ras(G12V). Thus, in addition to its positive role in the proliferative outputs of active Ras, the CNK1 scaffold protein, through its binding of a RASSF1A.MST complex, also participates in the proapoptotic signaling initiated by active Ras.