Alkenyl oxindole is a novel PROTAC moiety that recruits the CRL4DCAF11 E3 ubiquitin ligase complex for targeted protein degradation.

Alkenyl oxindoles have been characterized as autophagosome-tethering compounds (ATTECs), which can target mutant huntingtin protein (mHTT) for lysosomal degradation. In order to expand the application of alkenyl oxindoles for targeted protein degradation, we designed and synthesized a series of heterobifunctional compounds by conjugating different alkenyl oxindoles with bromodomain-containing protein 4 (BRD4) inhibitor JQ1. Through structure-activity relationship study, we successfully developed JQ1-alkenyl oxindole conjugates that potently degrade BRD4. Unexpectedly, we found that these molecules degrade BRD4 through the ubiquitin-proteasome system, rather than the autophagy-lysosomal pathway. Using pooled CRISPR interference (CRISPRi) screening, we revealed that JQ1-alkenyl oxindole conjugates recruit the E3 ubiquitin ligase complex CRL4DCAF11 for substrate degradation. Furthermore, we validated the most potent heterobifunctional molecule HL435 as a promising drug-like lead compound to exert antitumor activity both in vitro and in a mouse xenograft tumor model. Our research provides new employable proteolysis targeting chimera (PROTAC) moieties for targeted protein degradation, providing new possibilities for drug discovery.

Regulation of cohesin-mediated chromosome folding by PDS5 in mammals.

Cohesin regulates sister chromatid cohesion but also contributes to chromosome folding by promoting the formation of chromatin loops, a process mediated by loop extrusion. Although PDS5 regulates cohesin dynamics on chromatin, the exact function of PDS5 in cohesin-mediated chromatin looping remains unclear. Two paralogs of PDS5 exist in vertebrates, PDS5A and PDS5B. Here we show that PDS5A and PDS5B co-localize with RAD21 and CTCF at loop anchors. Rapid PDS5A or PDS5B degradation in liver cancer cells using an inducible degron system reduces chromatin loops and increases loop size. RAD21 enrichment at loop anchors is decreased upon depletion of PDS5A or PDS5B. PDS5B loss also reduces CTCF signals at loop anchors and has a stronger effect on loop enlargement compared with PDS5A. Co-depletion of PDS5A and PDS5B reduces RAD21 levels at loop anchors although the amount of cohesin on chromatin is increased. Our study provides insight into how PDS5 proteins regulate cohesin-mediated chromatin looping.

Epigenetic regulation of the DNMT1/MT1G/KLF4/CA9 axis synergises the anticancer effects of sorafenib in hepatocellular carcinoma.

Sorafenib, a multikinase inhibitor, has been widely used as a first-line anticancer drug for advanced hepatocellular carcinoma (HCC). However, the development of drug resistance to sorafenib is frequently observed in clinical applications. Potential nonkinase targets of sorafenib have not been well documented and may provide insights into reversing drug resistance and enhancing drug efficacy. Herein, we report that sorafenib exerts its anticancer effects by activating metallothionein 1 G (MT1G) expression. MT1G is a novel marker in HCC that correlates well with patient survival. MT1G overexpression suppressed the cellular proliferation, migration, invasion, and tumour formation of HCC and sensitised cells to sorafenib treatment. However, the disruption of MT1G attenuated the anticancer effects of sorafenib. Mechanistically, sorafenib upregulated MT1G expression via hypomethylation of its promoter region by binding and inhibiting DNA methyltransferase 1 (DNMT1) and increasing its promoter accessibility in HCC cells. Activation of MT1G also inhibited CA9 transcription through the suppression of HIF1A as mediated by KLF4. Our collective data revealed that sorafenib exerts its anticancer effects through epigenetic regulation of the DNMT1/MT1G/KLF4/CA9 axis in HCC and the activation of MT1G might constitute a strategy for enhancing the effect of sorafenib to suppress HCC cells.

The Interaction of Calcium-Sensing Receptor with KIF11 Enhances Cisplatin Resistance in Lung Adenocarcinoma via BRCA1/cyclin B1 pathway.

Cisplatin (DDP) is commonly used in the treatment of non-small cell lung cancer (NSCLC), including lung adenocarcinoma (LUAD), and the primary cause for its clinical inefficacy is chemoresistance. Here, we aimed to investigate a novel mechanism of chemoresistance in LUAD cells, focusing on the calcium-sensing receptor (CaSR). In this study, high CaSR expression was detected in DDP-resistant LUAD cells, and elevated CaSR expression is strongly correlated with poor prognosis in LUAD patients receiving chemotherapy. LUAD cells with high CaSR expression exhibited decreased sensitivity to cisplatin, and the growth of DDP-resistant LUAD cells was inhibited by cisplatin treatment in combination with CaSR suppression, accompanied by changes in BRCA1 and cyclin B1 protein expression both in vitro and in vivo. Additionally, an interaction between CaSR and KIF11 was identified. Importantly, suppressing KIF11 resulted in decreased protein levels of BRCA1 and cyclin B1, enhancing the sensitivity of DDP-resistant LUAD cells to cisplatin with no obvious decrease in CaSR. Here, our findings established the critical role of CaSR in promoting cisplatin resistance in LUAD cells by modulating cyclin B1 and BRCA1 and identified KIF11 as a mediator, highlighting the potential therapeutic value of targeting CaSR to overcome chemoresistance in LUAD.

Myeloid-like tumor hybrid cells in bone marrow promote progression of prostate cancer bone metastasis.

BACKGROUND: Bone metastasis is the leading cause of death in patients with prostate cancer (PCa) and currently has no effective treatment. Disseminated tumor cells in bone marrow often obtain new characteristics to cause therapy resistance and tumor recurrence. Thus, understanding the status of disseminated prostate cancer cells in bone marrow is crucial for developing a new treatment. METHODS: We analyzed the transcriptome of disseminated tumor cells from a single cell RNA-sequencing data of PCa bone metastases. We built a bone metastasis model through caudal artery injection of tumor cells, and sorted the tumor hybrid cells by flow cytometry. We performed multi-omics analysis, including transcriptomic, proteomic and phosphoproteomic analysis, to compare the difference between the tumor hybrid cells and parental cells. In vivo experiments were performed to analyze the tumor growth rate, metastatic and tumorigenic potential, drug and radiation sensitivity in hybrid cells. Single cell RNA-sequencing and CyTOF were performed to analyze the impact of hybrid cells on tumor microenvironment. RESULTS: Here, we identified a unique cluster of cancer cells in PCa bone metastases, which expressed myeloid cell markers and showed a significant change in pathways related to immune regulation and tumor progression. We found that cell fusion between disseminated tumor cells and bone marrow cells can be source of these myeloid-like tumor cells. Multi-omics showed the pathways related to cell adhesion and proliferation, such as focal adhesion, tight junction, DNA replication, and cell cycle, were most significantly changed in these hybrid cells. In vivo experiment showed hybrid cells had a significantly increased proliferative rate, and metastatic potential. Single cell RNA-sequencing and CyTOF showed tumor-associated neutrophils/monocytes/macrophages were highly enriched in hybrid cells-induced tumor microenvironment with a higher immunosuppressive capacity. Otherwise, the hybrid cells showed an enhanced EMT phenotype with higher tumorigenicity, and were resistant to docetaxel and ferroptosis, but sensitive to radiotherapy. CONCLUSION: Taken together, our data demonstrate that spontaneous cell fusion in bone marrow can generate myeloid-like tumor hybrid cells that promote the progression of bone metastasis, and these unique population of disseminated tumor cells can provide a potential therapeutic target for PCa bone metastasis.

Downregulation of SEPTIN5 inhibits prostate cancer progression by increasing CD8+ T cell infiltration.

Background: Prostate cancer (PCa) is one of the most common carcinomas in men, and aberrant expression of SEPTIN5 (SEPT5) has been detected in PCa tissues. However, the role of SEPT5 in PCa is still unclear. In this study, we attempted to explore the expression changes, clinical relevance and immunomodulatory function of SEPT5 in PCa. Methods: The expression and clinical significance of SEPT5 were evaluated based on bioinformatic analysis and were verified by western blotting, real time PCR and IHC. Allograft mouse models were used to assess the role of SEPT5 on PCa tumour formation and immunomodulatory function. Mass cytometry and IHC were used to determine the effects of SEPT5 on immune cell infiltration, especially CD8+ T cell infiltration. Correlations between SEPT5 expression and cytokine gene expression were analyzed based on TCGA and DKFZ datasets. RNA-seq and chemokine array were performed to confirm the effects of SEPT5 on cytokine production. Results: High SEPT5 expression was found in PCa and was associated with PCa prognosis. Importantly, downregulation of SEPT5 inhibited PCa progression in vivo. In addition, SEPT5 expression was negatively correlated with immune infiltrating cell levels, chemokine and cytokine gene expression associated with CD8+ T cell infiltration and activation. Downregulation of SEPT5 increased the proportion of immune cells, especially CD8+ T cells, in tumour tissue. Both the expression of CCL5, CXCL5, CXCL9, CXCL10 and INFGR1 were elevated in mRNA and protein levels after SEPT5 knockdown. Conclusions: In summary, downregulation of SEPT5 inhibited PCa progression, which may be mediated by increasing immune cell infiltration levels, especially CD8+ T cells, by promoting the production of IFNG-inducible chemokines and cytokines expression associated with immune cell infiltration. Our findings suggest that SEPT5 may serve as a prognostic biomarker of PCa and may be a target molecule to enhance the efficacy of immunotherapy for PCa in the future.

IgG4+ plasma cell infiltration is correlated with the development of inflammatory bowel disease and can be regulated by TLR-4.

Immunoglobulin 4 (IgG4) is commonly considered a hallmark of autoimmune pancreatitis (AIP). Inflammatory bowel disease (IBD) is believed to play a substantial role in the setting of AIP. Toll-like receptor 4 (TLR4) plays an important role in inflammation. The relationship between IgG4 and TLR4 in the process of IBD is incompletely explored. Our study aimed to assess the expression of IgG4 and TLR4 in IBD patients and to find the role of IgG4 and TLR4 in the IBD process. A cohort of 68 IBD patients was enrolled in our study, and 20 healthy persons served as a control group. Intestinal IgG4 positive (IgG4+) plasma cell infiltration was measured by immunohistochemistry. Serum IgG4 and TLR4 levels were measured by ELISA. Fifteen additional features from the patients' general medical information and lab data were also collected to assess the risk factors of IBD activity by logistical analysis. BALB/c mice were used to build a rat IBD model with dextran sulfate sodium (DSS). The TLR4 inhibitor TAK242 was used to regulate the expression of TLR4. The expression of IgG4 and TLR4 in serum was detected by ELISA. The expression of IgG4 and TLR4 in the intestines were assayed with western blot. Our results revealed that the infiltration of IgG4+ plasma cells was higher in IBD patients (14/68 vs 0/20, P<0.05). The incidence of IgG4+ plasma cells in the IBD group (48.5%) was higher than in the control group (33/68 vs 0/20, P<0.05). Serum IgG4 and TLR4 levels in the IBD group were significantly higher compared with the control group (P<0.05). Based on our logistical analysis, three variables: IgG4+ plasma cell infiltration, CRP, and HB were identified as independent risk factors with odds ratios of 10.917, 1.031, and 0.923, respectively (P<0.05). After the TLR4 was suppressed, the infiltration of IgG4+ plasma cells in the intestines decreased significantly, and expression of IgG4 in the serum and intestines was suppressed. This study demonstrated that intestinal IgG4+ plasma cell infiltration was higher in IBD patients than in the control group. IgG4+ cell infiltration is significantly enhanced in ulcerative colitis patients. IgG4+ plasma cell infiltration can be regulated by TLR4, and an increase of IgG4+ plasma cell infiltration, CRP, and anemia are correlated with an increased risk of active IBD.

[Construction of a microfluidic chip-based 72 plex single nucleotide polymorphisms ancestry inference system].

The aim was to develop an autosomal single nucleotide polymorphism (SNP) multiplex detection chip system for the inference of the ancestry of unknown individuals. On the basis of the 74-ancestry informative SNP (AISNP) panel screened by our group, a multiplex SNP amplification and detection system, based on a microfluidic chip of competitive allele-specific polymerase chain reaction (PCR), was constructed. The allele-specific primers of each SNP were positioned into a single well on the microfluidic chip and each chip included 112 wells. For detection of SNPs, the chip was placed on the plate thermocycler to perform PCR. The fluorescence signal of the reaction in each well was detected by a confocal laser scanning instrument to evaluate the SNP genotypes of each sample. Fifty-two samples were used for SNP typing. The accuracy of typing results was 100%. Taking 3628 samples of 57 populations as the reference population database, the ancestry of 20 samples was inferred. The results were consistent with the actual sources of the samples. The autosomal 72-SNP microfluidic chip system established can accurately detect SNPs. The ancestry of tested individuals can be accurately inferred from the reference database.