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Proceedings of the National Academy of... Jul 2023The MYC proto-oncogene contributes to the pathogenesis of more than half of human cancers. Malignant transformation by MYC transcriptionally up-regulates the core...
The MYC proto-oncogene contributes to the pathogenesis of more than half of human cancers. Malignant transformation by MYC transcriptionally up-regulates the core pre-mRNA splicing machinery and causes misregulation of alternative splicing. However, our understanding of how splicing changes are directed by MYC is limited. We performed a signaling pathway-guided splicing analysis to identify MYC-dependent splicing events. These included an HRAS cassette exon repressed by MYC across multiple tumor types. To molecularly dissect the regulation of this HRAS exon, we used antisense oligonucleotide tiling to identify splicing enhancers and silencers in its flanking introns. RNA-binding motif prediction indicated multiple binding sites for hnRNP H and hnRNP F within these cis-regulatory elements. Using siRNA knockdown and cDNA expression, we found that both hnRNP H and F activate the HRAS cassette exon. Mutagenesis and targeted RNA immunoprecipitation implicate two downstream G-rich elements in this splicing activation. Analyses of ENCODE RNA-seq datasets confirmed hnRNP H regulation of HRAS splicing. Analyses of RNA-seq datasets across multiple cancers showed a negative correlation of HNRNPH gene expression with MYC hallmark enrichment, consistent with the effect of hnRNP H on HRAS splicing. Interestingly, HNRNPF expression showed a positive correlation with MYC hallmarks and thus was not consistent with the observed effects of hnRNP F. Loss of hnRNP H/F altered cell cycle progression and induced apoptosis in the PC3 prostate cancer cell line. Collectively, our results reveal mechanisms for MYC-dependent regulation of splicing and point to possible therapeutic targets in prostate cancers.
Topics: Male; Humans; Heterogeneous-Nuclear Ribonucleoprotein Group F-H; RNA Precursors; RNA Splicing; RNA-Binding Proteins; Exons; Alternative Splicing; Prostatic Neoplasms; Proto-Oncogene Proteins p21(ras)
PubMed: 37399401
DOI: 10.1073/pnas.2220190120 -
Journal of Experimental & Clinical... Nov 2023Cancer-associated fibroblasts (CAFs) play pivotal roles in chemoresistance of pancreatic ductal adenocarcinoma (PDAC). However, the underlying mechanisms are poorly...
BACKGROUND
Cancer-associated fibroblasts (CAFs) play pivotal roles in chemoresistance of pancreatic ductal adenocarcinoma (PDAC). However, the underlying mechanisms are poorly understood. Revealing the cross-talk network between tumor stroma and pancreatic cancer and developing effective strategies against oxaliplatin resistance are highly desired in the clinic.
METHODS
High-throughput sequence was used to screened the key circRNAs transmitted by extracellular vesicles (EVs) from CAFs to pancreatic cancer cells. The associations between EV-packaged circBIRC6 and chemotherapy responsiveness were validated in a cohort of 82 cases of advanced PDAC patients. Then, the effects of EV-packaged circBIRC6 on CAF-induced oxaliplatin resistance were investigated by flow cytometry, colony formation, viability of pancreatic cancer organoids in vitro and by xenograft models in vivo. RNA pulldown, RNA immunoprecipitation, and sites mutation assays were used to reveal the underlying mechanism.
RESULTS
We identified a circRNA, circBIRC6, is significantly upregulated in CAF-derived EVs and is positively associated with oxaliplatin-based chemoresistance. In vitro and in vivo functional assays showed that CAF-derived EV-packaged circBIRC6 enhance oxaliplatin resistance of pancreatic cancer cells and organoids via regulating the non-homologous end joining (NHEJ) dependent DNA repair. Mechanistically, circBIRC6 directly binds with XRCC4 and enhanced the interaction of XRCC4 with SUMO1 at the lysine 115 residue, which facilitated XRCC4 chromatin localization. XRCC4 mutation dramatically abrogated the EV-packaged circBIRC6 induced effect. Moreover, combination of antisense oligonucleotide inhibitors against circBIRC6 with Olaparib dramatically suppressed chemoresistance in patient-derived xenograft models.
CONCLUSIONS
Our study revealed that EV-packaged circBIRC6 confer oxaliplatin resistance in PDAC by mediating SUMOylation of XRCC4, introducing a promising predictive and therapeutic target for PDAC on oxaliplatin resistance.
Topics: Humans; Platinum; Cancer-Associated Fibroblasts; Oxaliplatin; Sumoylation; Pancreatic Neoplasms; Carcinoma, Pancreatic Ductal; Extracellular Vesicles; RNA
PubMed: 38012734
DOI: 10.1186/s13046-023-02854-3 -
Advanced Science (Weinheim,... Sep 2023Endocrine therapy is the frontline treatment for estrogen receptor (ER) positive breast cancer patients. However, the primary and acquired resistance to endocrine...
Endocrine therapy is the frontline treatment for estrogen receptor (ER) positive breast cancer patients. However, the primary and acquired resistance to endocrine therapy drugs remain as a major challenge in the clinic. Here, this work identifies an estrogen-induced lncRNA, LINC02568, which is highly expressed in ER-positive breast cancer and functional important in cell growth in vitro and tumorigenesis in vivo as well as endocrine therapy drug resistance. Mechanically, this work demonstrates that LINC02568 regulates estrogen/ERα-induced gene transcriptional activation in trans by stabilizing ESR1 mRNA through sponging miR-1233-5p in the cytoplasm. Meanwhile, LINC02568 contributes to tumor-specific pH homeostasis by regulating carbonic anhydrase CA12 in cis in the nucleus. The dual functions of LINC02568 together contribute to breast cancer cell growth and tumorigenesis as well as endocrine therapy drug resistance. Antisense oligonucleotides (ASO) targeting LINC02568 significantly inhibits ER-positive breast cancer cell growth in vitro and tumorigenesis in vivo. Furthermore, combination treatment with ASO targeting LINC02568 and endocrine therapy drugs or CA12 inhibitor U-104 exhibits synergistic effects on tumor growth. Taken together, the findings reveal the dual mechanisms of LINC02568 in regulating ERα signaling and pH homeostasis in ER-positive breast cancer, and indicated that targeting LINC02568 might represent a potential therapeutic avenue in the clinic.
Topics: Humans; Female; Breast Neoplasms; Estrogen Receptor alpha; Receptors, Estrogen; RNA, Long Noncoding; Cell Line, Tumor; Estrogens; Drug Resistance, Neoplasm; Carcinogenesis
PubMed: 37404090
DOI: 10.1002/advs.202206663 -
Molecular Therapy. Nucleic Acids Mar 2024Pharmacokinetics (PK) of antisense oligonucleotides (ASOs) is characterized by rapid distribution from plasma to tissue and slow terminal plasma elimination driven by...
Pharmacokinetics (PK) of antisense oligonucleotides (ASOs) is characterized by rapid distribution from plasma to tissue and slow terminal plasma elimination driven by re-distribution from tissue. Quantitative understanding of tissue PK and RNA knockdown for various ASO chemistries, conjugations, and administration routes is critical for successful drug discovery. Here, we report concentration-time and RNA knockdown profiles for a gapmer ASO with locked nucleic acid ribose chemistry in mouse liver, kidney, heart, and lung after subcutaneous and intratracheal administration. Additionally, the same ASO with liver targeting conjugation (galactosamine--acetyl) is evaluated for subcutaneous administration. Data indicate that exposure and knockdown differ between tissues and strongly depend on administration route and conjugation. In a second study, we show that tissue PK is similar between the three different ribose chemistries locked nucleic acid, constrained ethyl and 2'--methoxyethyl, both after subcutaneous and intratracheal administration. Further, we show that the half-life in mouse liver may vary with ASO sequence. Finally, we report less than dose-proportional increase in liver concentration in the dose range of 3-30 μmol/kg. Overall, our studies contribute pivotal data to support design and interpretation of ASO studies, thereby increasing the probability of delivering novel ASO therapies to patients.
PubMed: 38419941
DOI: 10.1016/j.omtn.2024.102133 -
Molecular Therapy. Nucleic Acids Dec 2023Myotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by a CTG repeat expansion in the gene that generates toxic RNA with a myriad of downstream...
Myotonic dystrophy type 1 (DM1) is a rare neuromuscular disease caused by a CTG repeat expansion in the gene that generates toxic RNA with a myriad of downstream alterations in RNA metabolism. A key consequence is the sequestration of alternative splicing regulatory proteins MBNL1/2 by expanded transcripts in the affected tissues. MBNL1/2 depletion interferes with a developmental alternative splicing switch that causes the expression of fetal isoforms in adults. Boosting the endogenous expression of MBNL proteins by inhibiting the natural translational repressors miR-23b and miR-218 has previously been shown to be a promising therapeutic approach. We designed antimiRs against both miRNAs with a phosphorodiamidate morpholino oligonucleotide (PMO) chemistry conjugated to cell-penetrating peptides (CPPs) to improve delivery to affected tissues. In DM1 cells, CPP-PMOs significantly increased MBNL1 levels. In some candidates, this was achieved using concentrations less than two orders of magnitude below the median toxic concentration, with up to 5.38-fold better therapeutic window than previous antagomiRs. In mice, intravenous injections of CPP-PMOs improve molecular, histopathological, and functional phenotypes, without signs of toxicity. Our findings place CPP-PMOs as promising antimiR candidates to overcome the treatment delivery challenge in DM1 therapy.
PubMed: 37744174
DOI: 10.1016/j.omtn.2023.09.001 -
Molecular Therapy. Nucleic Acids Dec 2023Exon-skipping therapy mediated by antisense oligonucleotides is expected to provide a therapeutic option for Duchenne muscular dystrophy. Antisense oligonucleotides for...
Exon-skipping therapy mediated by antisense oligonucleotides is expected to provide a therapeutic option for Duchenne muscular dystrophy. Antisense oligonucleotides for exon skipping reported so far target a single continuous sequence in or around the target exon. In the present study, we investigated antisense oligonucleotides for exon 44 skipping (applicable to approximately 6% of all Duchenne muscular dystrophy patients) to improve activity by using a novel antisense oligonucleotide design incorporating two connected sequences. Phosphorodiamidate morpholino oligomers targeting two separate sequences in exon 44 were created to target two splicing regulators in exon 44 simultaneously, and their exon 44 skipping was measured. NS-089/NCNP-02 showed the highest skipping activity among the oligomers. NS-089/NCNP-02 also induced exon 44 skipping and dystrophin protein expression in cells from a Duchenne muscular dystrophy patient to whom exon 44 skipping is applicable. We also assessed the activity of NS-089/NCNP-02 by intravenous administration to cynomolgus monkeys. NS-089/NCNP-02 induced exon 44 skipping in skeletal and cardiac muscle of cynomolgus monkeys. In conclusion, NS-089/NCNP-02, an antisense oligonucleotide with a novel connected-sequence design, showed highly efficient exon skipping both and .
PubMed: 37854955
DOI: 10.1016/j.omtn.2023.102034 -
Molecular Therapy : the Journal of the... Jan 2024Fibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer caused by a dominant recurrent fusion of the heat shock protein (DNAJB1) and the catalytic subunit of...
Fibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer caused by a dominant recurrent fusion of the heat shock protein (DNAJB1) and the catalytic subunit of protein kinase A (PRKACA). Current therapies such as chemotherapy and radiation have limited efficacy, and new treatment options are needed urgently. We have previously shown that FLC tumors are dependent on the fusion kinase DNAJB1::PRKACA, making the oncokinase an ideal drug target. mRNA degrading modalities such as antisense oligonucleotides or small interfering RNAs (siRNAs) provide an opportunity to specifically target the fusion junction. Here, we identify a potent and specific siRNA that inhibits DNAJB1::PRKACA expression. We found expression of the asialoglycoprotein receptor in FLC to be maintained at sufficient levels to effectively deliver siRNA conjugated to the GalNAc ligand. We observe productive uptake and siRNA activity in FLC patient-derived xenografts (PDX) models in vitro and in vivo. Knockdown of DNAJB1::PRKACA results in durable growth inhibition of FLC PDX in vivo with no detectable toxicities. Our results suggest that this approach could be a treatment option for FLC patients.
Topics: Humans; Carcinoma, Hepatocellular; Liver Neoplasms; RNA, Small Interfering; Cyclic AMP-Dependent Protein Kinases; RNA, Double-Stranded; HSP40 Heat-Shock Proteins; Cyclic AMP-Dependent Protein Kinase Catalytic Subunits
PubMed: 37980543
DOI: 10.1016/j.ymthe.2023.11.012 -
Molecular Cancer Jan 2024Cisplatin (CDDP) is the first-line chemotherapeutic strategy to treat patients with ovarian cancer (OC). The development of CDDP resistance remains an unsurmountable...
BACKGROUND
Cisplatin (CDDP) is the first-line chemotherapeutic strategy to treat patients with ovarian cancer (OC). The development of CDDP resistance remains an unsurmountable obstacle in OC treatment and frequently induces tumor recurrence. Circular RNAs (circRNAs) are noncoding RNAs with important functions in cancer progression. Whether circRNAs function in CDDP resistance of OC is unclear.
METHODS
Platinum-resistant circRNAs were screened via circRNA deep sequencing and examined using in situ hybridization (ISH) in OC. The role of circPLPP4 in CDDP resistance was assessed by clone formation and Annexin V assays in vitro, and by OC patient-derived xenografts and intraperitoneal tumor models in vivo. The mechanism underlying circPLPP4-mediated activation of miR-136/PIK3R1 signaling was examined by luciferase reporter assay, RNA pull-down, RIP, MeRIP and ISH.
RESULTS
circPLPP4 was remarkably upregulated in platinum resistant OC. circPLPP4 overexpression significantly enhanced, whereas circPLPP4 silencing reduced, OC cell chemoresistance. Mechanistically, circPLPP4 acts as a microRNA sponge to sequester miR-136, thus competitively upregulating PIK3R1 expression and conferring CDDP resistance. The increased circPLPP4 level in CDDP-resistant cells was caused by increased RNA stability, mediated by increased N6-methyladenosine (mA) modification of circPLPP4. In vivo delivery of an antisense oligonucleotide targeting circPLPP4 significantly enhanced CDDP efficacy in a tumor model.
CONCLUSIONS
Our study reveals a plausible mechanism by which the mA -induced circPLPP4/ miR-136/ PIK3R1 axis mediated CDDP resistance in OC, suggesting that circPLPP4 may serve as a promising therapeutic target against CDDP resistant OC. A circPLPP4-targeted drug in combination with CDDP might represent a rational regimen in OC.
Topics: Humans; Female; Cisplatin; Up-Regulation; RNA, Circular; Neoplasm Recurrence, Local; Ovarian Neoplasms; MicroRNAs; Adenosine; Class Ia Phosphatidylinositol 3-Kinase
PubMed: 38184597
DOI: 10.1186/s12943-023-01917-5 -
Nutrition, Metabolism, and... Oct 2023In view of the consolidating evidence on the causal role of Lp(a) in cardiovascular disease, the Italian Society for the Study of Atherosclerosis (SISA) has assembled a...
AIMS
In view of the consolidating evidence on the causal role of Lp(a) in cardiovascular disease, the Italian Society for the Study of Atherosclerosis (SISA) has assembled a consensus on Lp(a) genetics and epidemiology, together with recommendations for its measurement and current and emerging therapeutic approaches to reduce its plasma levels. Data on the Italian population are also provided.
DATA SYNTHESIS
Lp(a) is constituted by one apo(a) molecule and a lipoprotein closely resembling to a low-density lipoprotein (LDL). Its similarity with an LDL, together with its ability to carry oxidized phospholipids are considered the two main features making Lp(a) harmful for cardiovascular health. Plasma Lp(a) concentrations vary over about 1000 folds in humans and are genetically determined, thus they are quite stable in any individual. Mendelian Randomization studies have suggested a causal role of Lp(a) in atherosclerotic cardiovascular disease (ASCVD) and aortic valve stenosis and observational studies indicate a linear direct correlation between cardiovascular disease and Lp(a) plasma levels. Lp(a) measurement is strongly recommended once in a patient's lifetime, particularly in FH subjects, but also as part of the initial lipid screening to assess cardiovascular risk. The apo(a) size polymorphism represents a challenge for Lp(a) measurement in plasma, but new strategies are overcoming these difficulties. A reduction of Lp(a) levels can be currently attained only by plasma apheresis and, moderately, with PCSK9 inhibitor treatment.
CONCLUSIONS
Awaiting the approval of selective Lp(a)-lowering drugs, an intensive management of the other risk factors for individuals with elevated Lp(a) levels is strongly recommended.
Topics: Humans; Lipoprotein(a); Proprotein Convertase 9; Consensus; Atherosclerosis; Aortic Valve Stenosis
PubMed: 37586921
DOI: 10.1016/j.numecd.2023.07.019 -
Antibiotics (Basel, Switzerland) Sep 2023The treatment of skin and soft tissue infections faces several challenges, such as the increased incidence of antibiotic-resistant strains and the fact that the... (Review)
Review
The treatment of skin and soft tissue infections faces several challenges, such as the increased incidence of antibiotic-resistant strains and the fact that the antibiotics available to treat methicillin-resistant present low bioavailability, are not easily metabolized, and cause severe secondary effects. Moreover, besides the susceptibility pattern of the isolates detected in vitro, during patient treatment, the antibiotics may never encounter the bacteria because hides within biofilms or inside eukaryotic cells. In addition, vascular compromise as well as other comorbidities of the patient may impede proper arrival to the skin when the antibiotic is given parenterally. In this manuscript, we revise some of the more promising strategies to improve antibiotic sensitivity, bioavailability, and delivery, including the combination of antibiotics with bactericidal nanomaterials, chemical inhibitors, antisense oligonucleotides, and lytic enzymes, among others. In addition, alternative non-antibiotic-based experimental therapies, including the delivery of antimicrobial peptides, bioactive glass nanoparticles or nanocrystalline cellulose, phototherapies, and hyperthermia, are also reviewed.
PubMed: 37887178
DOI: 10.3390/antibiotics12101477