RESUMO
Resistance to androgen receptor (AR) inhibitors, including enzalutamide (Enz), as well as bone metastasis, are major challenges for castration-resistant prostate cancer (CRPC) treatment. In this study, we identified that miR26a can restore Enz sensitivity and inhibit bone metastatic CRPC. To achieve the highest combination effect of miR26a and Enz, we developed a cancer-targeted nano-system (Bm@PT/Enz-miR26a) using bone marrow mesenchymal stem cell (BMSC) membrane and T140 peptide to co-deliver Enz and miR26a. The in vitro/in vivo results demonstrated that miR26a can reverse Enz resistance and synergistically shrink tumor growth, invasion, and metastasis (especially secondary metastasis) in both subcutaneous and bone metastatic CRPC mouse models. We also found that the EZH2/SFRP1/WNT5A axis may be involved in this role. These findings open new avenues for treating bone metastatic and Enz-resistant CRPC.
Assuntos
Benzamidas , Feniltioidantoína , Neoplasias de Próstata Resistentes à Castração , Humanos , Masculino , Animais , Camundongos , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Resistencia a Medicamentos Antineoplásicos , Proliferação de Células , Linhagem Celular Tumoral , Nitrilas/farmacologiaRESUMO
Recent studies on CRISPR adaptation revealed that priming is a major pathway of spacer acquisition, at least for the most prevalent type I systems. Priming is guided by a CRISPR RNA which fully/partially matches the invader DNA, but the plasticity of this RNA guide has not yet been characterized. In this study, we extensively modified the two conserved handles of a priming crRNA in Haloarcula hispanica, and altered the size of its central spacer part. Interestingly, priming is insusceptible to the full deletion of 3' handle, which seriously impaired crRNA stability and interference effects. With 3' handle deletion, further truncation of 5' handle revealed that its spacer-proximal 6 nucleotides could provide the least conserved sequence required for priming. Subsequent scanning mutation further identified critical nucleotides within 5' handle. Besides, priming was also shown to tolerate a wider size variation of the spacer part, compared to interference. These data collectively illustrate the high tolerance of priming to extensive structural/size variations of the crRNA guide, which highlights the structural flexibility of the crRNA-effector ribonucleoprotein complex. The observed high priming effectiveness suggests that primed adaptation promotes clearance of the fast-replicating and ever-evolving viral DNA, by rapidly and persistently multiplexing the interference pathway.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Haloarcula/genética , RNA Guia de Cinetoplastídeos , Adaptação Fisiológica , Proteínas Associadas a CRISPR/metabolismo , Primers do DNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Mutação , Plasmídeos/metabolismoRESUMO
Prokaryotes memorize invader information by incorporating alien DNA as spacers into CRISPR arrays. Although the spacer size has been suggested to be predefined by the architecture of the acquisition complex, there is usually an unexpected heterogeneity. Here, we explored the causes of this heterogeneity in Haloarcula hispanica I-B CRISPR. High-throughput sequencing following adaptation assays demonstrated significant size variation among 37 957 new spacers, which appeared to be sequence-dependent. Consistently, the third nucleotide at the spacer 3Î-end (PAM-distal end) showed an evident bias for cytosine and mutating this cytosine in the protospacer sequence could change the final spacer size. In addition, slippage of the 5Î-end (PAM-end), which contributed to most of the observed PAM (protospacer adjacent motif) inaccuracy, also tended to change the spacer size. We propose that both ends of the PAM-protospacer sequence should exhibit nucleotide selectivity (with different stringencies), which fine-tunes the structural ruler, to a certain extent, to specify the spacer size.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Haloarcula/genética , Sequência de Bases , Citosina/metabolismo , DNA Intergênico/genética , DNA Intergênico/metabolismo , Haloarcula/metabolismoRESUMO
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) acquire new spacers to generate adaptive immunity in prokaryotes. During spacer integration, the leader-preceded repeat is always accurately duplicated, leading to speculations of a repeat-length ruler. Here in Haloarcula hispanica, we demonstrate that the accurate duplication of its 30-bp repeat requires two conserved mid-repeat motifs, AACCC and GTGGG. The AACCC motif was essential and needed to be â¼10 bp downstream from the leader-repeat junction site, where duplication consistently started. Interestingly, repeat duplication terminated sequence-independently and usually with a specific distance from the GTGGG motif, which seemingly served as an anchor site for a molecular ruler. Accordingly, altering the spacing between the two motifs led to an aberrant duplication size (29, 31, 32 or 33 bp). We propose the adaptation complex may recognize these mid-repeat elements to enable measuring the repeat DNA for spacer integration.
Assuntos
DNA Arqueal/genética , Haloarcula/genética , Sequência de Bases , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Replicação do DNA , Duplicação Gênica , Genes Arqueais , Motivos de Nucleotídeos , Análise de Sequência de DNARESUMO
Cancer-associated fibroblasts (CAFs) play a significant role in tumor development and treatment failure, yet the precise mechanisms underlying their contribution to renal cell carcinoma (RCC) remains underexplored. This study explored the interaction between CAFs and tumor cells, and related mechanisms. CAFs isolated from tumor tissues promoted the tumor progression and drugs resistance both in vivo and in vitro. Mechanistically, chemokine (C-X-C motif) ligand (CXCL) 3 secreted from CAFs mediated its effects. CXCL3 activated its receptor CXCR2 to active the downstream ERK1/2 signaling pathway, subsequently promoting epithelial-mesenchymal transition and cell stemness. Blocking the crosstalk between CAFs and tumor cells by CXCR2 inhibitor SB225002 attenuated the functions of CAFs. Furthermore, Renca cells facilitated the transformation of normal interstitial fibroblasts (NFs) into CAFs and the expression of CXCL3 through TGF-ß-Smad2/3 signaling pathway. In turn, transformed NFs promoted the tumor progression and drug resistance of RCC. These findings may constitute potential therapeutic strategies for RCC treatment.
RESUMO
Introduction: Metastatic castration-resistant prostate cancer (mCRPC) patients face challenges due to limited treatment options. About 50% of patients with mCRPC have a functional loss of phosphatase and tensin homology deleted on chromosome 10 (PTEN), leading to tumor progression, metastasis, and immune suppression. Moreover, elevated IL-23 produced by myeloid-derived suppressor cells (MDSCs) is found in CRPC patients, driving tumor progression. Therefore, a combination strategy based on PTEN restoration and IL-23 inhibition may block CRPC progression and metastasis. Methods: The antitumor effect of restoring PTEN expression combined with the IL-23 inhibitor Apilimod was studied in a mouse model of bone metastasis CRPC and mouse prostate cancer RM-1 cells. To verify the targeting ability of PTEN DNA coated with lipid nanoparticles (LNP@PTEN) in vitro and in vivo. In addition, RT-qPCR and flow cytometry were used to investigate the related mechanisms of the antitumor effect of LNP@PTEN combined with Apilimod. Results: LNPs exhibited significant tumor-targeting and tumor accumulation capabilities both in vitro and in vivo, enhancing PTEN expression and therapeutic efficacy. Additionally, the combination of LNP@PTEN with the IL-23 inhibitor Apilimod demonstrated enhanced inhibition of tumor growth, invasion, and metastasis (particularly secondary organ metastasis) compared to other groups, and extended the survival of mice to 41 days, providing a degree of bone protection. These effects may be attributed to the PTEN function restoration combined with IL-23 inhibition, which help reverse immune suppression in the tumor microenvironment by reducing MDSCs recruitment and increasing the CD8+/CD4+ T cell ratio. Discussion: In summary, these findings highlight the potential of LNPs for delivering gene therapeutic agents. And the combination of LNP@PTEN with Apilimod could achieve anti-tumor effects and improve tumor microenvironment. This combinational strategy opens new avenues for the treatment of mCRPC.
RESUMO
Sunitinib is the first-line drug for renal cell carcinoma (RCC) treatment. However, patients who received sunitinib treatment will ultimately develop drug resistance after 6-15 months, creating a huge obstacle to the current treatment of renal cell carcinoma. Therefore, it is urgent to clarify the mechanisms of sunitinib resistance and develop new strategies to overcome it. In this review, the mechanisms of sunitinib resistance in renal cell carcinoma have been summarized based on five topics: activation of bypass or alternative pathway, inadequate drug accumulation, tumour microenvironment, metabolic reprogramming and epigenetic regulation. Furthermore, present and potential biomarkers, as well as potential treatment strategies for overcoming sunitinib resistance in renal cell carcinoma, are also covered.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Humanos , Carcinoma de Células Renais/tratamento farmacológico , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Sunitinibe/uso terapêutico , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/genética , Neoplasias Renais/patologia , Epigênese Genética , Resistencia a Medicamentos Antineoplásicos , Linhagem Celular Tumoral , Pirróis , Microambiente TumoralRESUMO
The type I system is the most widely distributed CRISPR-Cas system identified so far. Recently, we have revealed the natural reprogramming of the type I CRISPR effector for gene regulation with a crRNA-resembling RNA in halophilic archaea. Here, we conducted a comprehensive study of the impact of redesigned crRNAs with different spacer lengths on gene regulation with the native type I-B CRISPR system in Haloarcula hispanica. When the spacer targeting the chromosomal gene was shortened from 36 to 28 bp, transformation efficiencies of the spacer-encoding plasmids were improved by over three orders of magnitude, indicating a significant loss of interference. However, by conducting whole-genome sequencing and measuring the growth curves of the hosts, we still detected DNA cleavage and its influence on cell growth. Intriguingly, when the spacer was shortened to 24 bp, the transcription of the target gene was downregulated to 10.80%, while both interference and primed adaptation disappeared. By modifying the lengths of the spacers, the expression of the target gene could be suppressed to varying degrees. Significantly, by designing crRNAs with different spacer lengths and targeting different genes, we achieved simultaneous gene editing (cdc6E) and gene regulation (crtB) for the first time with the endogenous type I CRISPR-Cas system.
RESUMO
CRISPR-Cas systems provide RNA-guided adaptive immunity in prokaryotes. We report that the multisubunit CRISPR effector Cascade transcriptionally regulates a toxin-antitoxin RNA pair, CreTA. CreT (Cascade-repressed toxin) is a bacteriostatic RNA that sequesters the rare arginine tRNAUCU (transfer RNA with anticodon UCU). CreA is a CRISPR RNA-resembling antitoxin RNA, which requires Cas6 for maturation. The partial complementarity between CreA and the creT promoter directs Cascade to repress toxin transcription. Thus, CreA becomes antitoxic only in the presence of Cascade. In CreTA-deleted cells, cascade genes become susceptible to disruption by transposable elements. We uncover several CreTA analogs associated with diverse archaeal and bacterial CRISPR-cas loci. Thus, toxin-antitoxin RNA pairs can safeguard CRISPR immunity by making cells addicted to CRISPR-Cas, which highlights the multifunctionality of Cas proteins and the intricate mechanisms of CRISPR-Cas regulation.
Assuntos
Proteínas Associadas a CRISPR/fisiologia , Sistemas CRISPR-Cas/fisiologia , Haloarcula/fisiologia , RNA Arqueal/fisiologia , Sistemas Toxina-Antitoxina/fisiologia , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas/genética , Análise Mutacional de DNA , Regulação da Expressão Gênica em Archaea , Haloarcula/genética , Óperon , RNA de Transferência de Arginina/metabolismo , Sistemas Toxina-Antitoxina/genéticaRESUMO
Research on CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated protein) systems has led to the revolutionary CRISPR/Cas9 genome editing technique. However, for most archaea and half of bacteria, exploitation of their native CRISPR-Cas machineries may be more straightforward and convenient. In this study, we harnessed the native type I-B CRISPR-Cas system for precise genome editing in the polyploid haloarchaeon Haloarcula hispanica. After testing different designs, the editing tool was optimized to be a single plasmid that carries both the self-targeting mini-CRISPR and a 600-800 bp donor. Significantly, chromosomal modifications, such as gene deletion, gene tagging or single nucleotide substitution, were precisely introduced into the vast majority of the transformants. Moreover, we showed that simultaneous editing of two genomic loci could also be readily achieved by one step. In summary, our data demonstrate that the haloarchaeal CRISPR-Cas system can be harnessed for genome editing in this polyploid archaeon, and highlight the convenience and efficiency of the native CRISPR-based genome editing strategy.