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1.
Proc Natl Acad Sci U S A ; 111(38): 13870-5, 2014 Sep 23.
Article in English | MEDLINE | ID: mdl-25189770

ABSTRACT

Metastatic spread is the leading cause of cancer mortality. Breast cancer (BCa) metastatic recurrence can happen years after removal of the primary tumor. Here we show that Ubc13, an E2 enzyme that catalyzes K63-linked protein polyubiquitination, is largely dispensable for primary mammary tumor growth but is required for metastatic spread and lung colonization by BCa cells. Loss of Ubc13 inhibited BCa growth and survival only at metastatic sites. Ubc13 was dispensable for transforming growth factor ß (TGFß)-induced SMAD activation but was required for activation of non-SMAD signaling via TGFß-activating kinase 1 (TAK1) and p38, whose activity controls expression of numerous metastasis promoting genes. p38 activation restored metastatic activity to Ubc13-deficient cells, and its pharmacological inhibition attenuated BCa metastasis in mice, suggesting it is a therapeutic option for metastatic BCa.


Subject(s)
Breast Neoplasms/enzymology , MAP Kinase Kinase Kinases/metabolism , MAP Kinase Signaling System , Neoplasm Proteins/metabolism , Ubiquitin-Conjugating Enzymes/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , Female , Heterografts , Humans , MAP Kinase Kinase Kinases/genetics , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasm Metastasis , Neoplasm Proteins/genetics , Neoplasm Transplantation , Ubiquitin-Conjugating Enzymes/genetics , p38 Mitogen-Activated Protein Kinases/genetics
2.
Sci Rep ; 10(1): 2688, 2020 02 14.
Article in English | MEDLINE | ID: mdl-32060361

ABSTRACT

B-cell acute lymphoblastic leukemia (B-ALL) accounts for nearly one fifth of all childhood cancers and current challenges in B-ALL treatment include resistance, relapse and late-onset side effects due to the chemotherapy. To overcome these hurdles, novel therapies need to be investigated. One promising target is Polo-like kinase 1 (Plk1), a key regulator of the cell cycle. In this study, the Plk family expression is investigated in primary peripheral blood and bone marrow mononuclear cells from ten pediatric B-ALL patients. For the first time, short interfering RiboNucleic Neutrals (siRNNs) that enter cells without a transfection reagent are used to target Plk1 mRNA in primary cells from pediatric B-ALL patients. Our results show that the expression of Plk1 and Plk4 is significantly higher in pediatric B-ALL patients compared to healthy donors. Moreover, treatment of primary peripheral blood and bone marrow mononuclear cells from pediatric B-ALL patients, cultured ex vivo, with Plk1-targeting siRNNs results in cleavage of Plk1 mRNA. Importantly, the Plk1 knockdown is specific and does not affect other Plk members in contrast to many small molecule Plk1 inhibitors. Thus, Plk1 is a potential therapeutic target in pediatric B-ALL and selective targeting of Plk1 can be achieved by the use of siRNNs.


Subject(s)
Cell Cycle Proteins/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins/genetics , Adolescent , Apoptosis/drug effects , B-Lymphocytes/drug effects , Cell Cycle Proteins/antagonists & inhibitors , Cell Division/drug effects , Cell Line, Tumor , Child , Child, Preschool , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Infant , Male , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , RNA, Messenger , Polo-Like Kinase 1
3.
J Control Release ; 261: 199-206, 2017 09 10.
Article in English | MEDLINE | ID: mdl-28684168

ABSTRACT

Epidemiological studies of childhood leukemia survivors reveal an alarmingly high incidence of chronic health disabilities after treatment, therefore, more specific therapies need to be developed. Polo-like kinase 1 (Plk1) is a key player in mitosis and a target for drug development as it is upregulated in multiple cancer types. Small molecules targeting Plk1 are mainly ATP-competitors and, therefore, are known to elicit side effects due to lack of specificity. RNA interference (RNAi) is known for its high catalytic activity and target selectivity; however, the biggest barrier for its introduction into clinical use is its delivery. RNAi prodrugs are modified, self-delivering short interfering Ribonucleic Neutrals (siRNNs), cleaved by cytoplasmic enzymes into short interfering Ribonucleic Acids (siRNAs) once inside cells. In this study we aimed to investigate the potential of siRNNs as therapeutic tools in T-acute lymphoblastic leukemia (T-ALL) using T-ALL cell lines and patient-derived samples. We demonstrate for the first time that RNAi prodrugs (siRNNs) targeting Plk1, can enter pediatric T-ALL patient cells without a transfection reagent and induce Plk1 knockdown on both protein and mRNA levels resulting in G2/M-arrest and apoptosis. We also show that siRNNs targeting Plk1 generate less toxicity in normal cells compared to the small molecule Plk1 inhibitor, BI6727, suggesting a potentially good therapeutic index.


Subject(s)
Cell Cycle Proteins/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/therapy , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins/genetics , RNA Interference , RNA, Small Interfering/administration & dosage , Apoptosis/genetics , Cell Line, Tumor , Child , Drug Delivery Systems , G2 Phase Cell Cycle Checkpoints/genetics , Gene Knockdown Techniques , Gene Silencing , Humans , M Phase Cell Cycle Checkpoints/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Prodrugs , Pteridines/pharmacology , Pteridines/toxicity , RNA, Messenger/genetics , RNA, Small Interfering/toxicity , Polo-Like Kinase 1
4.
Nucleic Acid Ther ; 27(5): 260-271, 2017 Oct.
Article in English | MEDLINE | ID: mdl-28933656

ABSTRACT

Small double-stranded, left-handed hairpin (LHP) RNAs containing a 5'-guide-loop-passenger-3' structure induce RNAi responses by a poorly understood mechanism. To explore LHPs, we synthesized fully 2'-modified LHP RNAs targeting multiple genes and found all to induce robust RNAi responses. Deletion of the loop and nucleotides at the 5'-end of the equivalent passenger strand resulted in a smaller LHP that still induced strong RNAi responses. Surprisingly, progressive deletion of up to 10 nucleotides from the 3'-end of the guide strand resulted in a 32mer LHP capable of inducing robust RNAi responses. However, further guide strand deletion inhibited LHP activity, thereby defining the minimal length guide targeting length to 13 nucleotides. To dissect LHP processing, we examined LHP species that coimmunoprecipitated with Argonaute 2 (Ago2), the catalytic core of RNA-induced silencing complex, and found that the Ago2-associated processed LHP species was of a length that correlated with Ago2 cleavage of the passenger strand. Placement of a blocking 2'-OMe blocking modification at the LHP predicted Ago2 cleavage site resulted in an intact LHP loaded into Ago2 and no RNAi response. Taken together, these data argue that in the absence of a substantial loop, this novel class of small LHP RNAs enters the RNAi pathway by a Dicer-independent mechanism that involves Ago2 cleavage and results in an extended guide strand. This work establishes LHPs as an alternative RNAi trigger that can be produced from a single synthesis for potential use as an RNAi therapeutic.


Subject(s)
Argonaute Proteins/metabolism , RNA Interference/drug effects , RNA, Double-Stranded/therapeutic use , RNA, Small Interfering/therapeutic use , RNA-Induced Silencing Complex/metabolism , RNAi Therapeutics/methods , Argonaute Proteins/genetics , Cell Line, Tumor , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Humans , RNA, Double-Stranded/chemical synthesis , RNA, Double-Stranded/genetics , RNA, Double-Stranded/metabolism , RNA, Small Interfering/chemical synthesis , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA-Induced Silencing Complex/genetics , Ribonuclease III/genetics , Ribonuclease III/metabolism , Sequence Deletion
5.
Methods Mol Biol ; 1364: 1-9, 2016.
Article in English | MEDLINE | ID: mdl-26472437

ABSTRACT

Due to their high potency (EC50 ~1 pM) and exquisite target selectivity for all expressed mRNAs, small interfering RNA (siRNA)-induced RNAi responses hold significant promise as a therapeutic modality. However, the size and high negative charge of siRNAs render them unable to enter cells without assistance from a delivery agent. Most current methods of siRNA delivery rely on encasing siRNA molecules in large nanoparticles or cationic liposomes. However, these approaches suffer from a number of problems, including a poor diffusion coefficient, cytotoxicity, and poor pharmacokinetics. To address the problem of siRNA in vivo delivery, we developed monomeric neutral RNAi prodrugs, termed siRibonucleic neutrals (siRNNs), that directly neutralize the phosphate backbone negative charge by synthesis with bioreversible phosphotriester groups that are enzymatically cleaved off in the cytoplasm of cells. Here we describe the synthesis and purification of siRNN conjugates that induce in vivo target gene knockdown following systemic delivery into mice.


Subject(s)
Oligonucleotides/chemistry , Prodrugs/chemistry , Prodrugs/chemical synthesis , RNA, Small Interfering/chemistry , Acetylgalactosamine/chemistry , Animals , Chemistry Techniques, Synthetic , Gene Knockdown Techniques , Mice , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism
6.
Sci Rep ; 6: 32301, 2016 09 08.
Article in English | MEDLINE | ID: mdl-27604151

ABSTRACT

Bioactive macromolecular peptides and oligonucleotides have significant therapeutic potential. However, due to their size, they have no ability to enter the cytoplasm of cells. Peptide/Protein transduction domains (PTDs), also called cell-penetrating peptides (CPPs), can promote uptake of macromolecules via endocytosis. However, overcoming the rate-limiting step of endosomal escape into the cytoplasm remains a major challenge. Hydrophobic amino acid R groups are known to play a vital role in viral escape from endosomes. Here we utilize a real-time, quantitative live cell split-GFP fluorescence complementation phenotypic assay to systematically analyze and optimize a series of synthetic endosomal escape domains (EEDs). By conjugating EEDs to a TAT-PTD/CPP spilt-GFP peptide complementation assay, we were able to quantitatively measure endosomal escape into the cytoplasm of live cells via restoration of GFP fluorescence by intracellular molecular complementation. We found that EEDs containing two aromatic indole rings or one indole ring and two aromatic phenyl groups at a fixed distance of six polyethylene glycol (PEG) units from the TAT-PTD-cargo significantly enhanced cytoplasmic delivery in the absence of cytotoxicity. EEDs address the critical rate-limiting step of endosomal escape in delivery of macromolecular biologic peptide, protein and siRNA therapeutics into cells.


Subject(s)
Biological Products/metabolism , Cell-Penetrating Peptides/metabolism , Endosomes/metabolism , Macromolecular Substances/metabolism , Binding Sites/genetics , Biological Products/administration & dosage , Cell Line , Cell Line, Tumor , Cell-Penetrating Peptides/genetics , Drug Delivery Systems/methods , Endocytosis , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Humans , MCF-7 Cells , Macromolecular Substances/administration & dosage , Microscopy, Fluorescence , Peptides/administration & dosage , Peptides/genetics , Peptides/metabolism , RNA, Small Interfering/administration & dosage , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Time-Lapse Imaging/methods
7.
Nat Biotechnol ; 32(12): 1256-61, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25402614

ABSTRACT

RNA interference (RNAi) has great potential to treat human disease. However, in vivo delivery of short interfering RNAs (siRNAs), which are negatively charged double-stranded RNA macromolecules, remains a major hurdle. Current siRNA delivery has begun to move away from large lipid and synthetic nanoparticles to more defined molecular conjugates. Here we address this issue by synthesis of short interfering ribonucleic neutrals (siRNNs) whose phosphate backbone contains neutral phosphotriester groups, allowing for delivery into cells. Once inside cells, siRNNs are converted by cytoplasmic thioesterases into native, charged phosphodiester-backbone siRNAs, which induce robust RNAi responses. siRNNs have favorable drug-like properties, including high synthetic yields, serum stability and absence of innate immune responses. Unlike siRNAs, siRNNs avidly bind serum albumin to positively influence pharmacokinetic properties. Systemic delivery of siRNNs conjugated to a hepatocyte-specific targeting domain induced extended dose-dependent in vivo RNAi responses in mice. We believe that siRNNs represent a technology that will open new avenues for development of RNAi therapeutics.


Subject(s)
Drug Delivery Systems , Prodrugs/therapeutic use , RNA, Small Interfering/therapeutic use , Animals , Humans , Mice , Nanoparticles/chemistry , Nanoparticles/therapeutic use , Prodrugs/chemistry , RNA, Small Interfering/chemistry , RNA, Small Interfering/genetics , Serum Albumin/chemistry
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