Preclinical efficacy of CBR-5884 against epithelial ovarian cancer cells by targeting the serine synthesis pathway.

Reprogramming of the serine synthesis pathway (SSP) is intricately linked to the progression of epithelial ovarian cancer (EOC). CBR-5884, a selective small-molecule inhibitor targeting phosphoglycerate dehydrogenase (PHGDH), effectively impedes the de novo synthesis of serine within cancer cells. This study aimed to evaluate the inhibitory effect of CBR-5884 on EOC cells and delineate its specific mechanism, thereby proposing a novel therapeutic approach for treating EOC. The suppression of serine biosynthesis after CBR-5884 treatment was evaluated using RNA sequencing and a serine assay kit, and the results showed that CBR-5884 effectively downregulated serine biosynthesis in EOC cells, particularly those expressing high levels of PHGDH. In vitro studies revealed that CBR-5884 demonstrated significant antitumor effects and suppressed migration and invasion of EOC cells through down-regulation of the integrin subunit beta 4 (ITGB4)/extracellular signal-regulated kinase (ERK)/epithelial-mesenchymal transition signal axis. Additionally, CBR-5884 mitigated the stemness of EOC cells and heightened their sensitivity to chemotherapy. Moreover, in vivo studies revealed that CBR-5884 significantly delayed tumor growth, with histological analysis indicating the safety profile of CBR-5884. Finally, the patient-derived organoid (PDO) models were utilized to explore the preclinical efficacy of CBR-5884 against EOC cells, and the results unveiled that CBR-5884 impeded proliferation and downregulated the expression of ITGB4 in EOC PDO models. Our findings supports the anticancer properties of CBR-5884 in EOC cells exhibiting high PHGDH expression, manifesting through the suppression of proliferation, migration, and invasion, while enhancing chemotherapy sensitivity, suggesting that CBR-5884 holds promise as an efficacious strategy for the treatment of EOC.

ZSWIM4 inhibition improves chemosensitivity in epithelial ovarian cancer cells by suppressing intracellular glycine biosynthesis.

BACKGROUND: Zinc finger SWIM-type containing 4 (ZSWIM4) induces drug resistance in breast cancer cells. However, its role in epithelial ovarian cancer (EOC) remains unknown. In this study, we aimed to investigate the clinical significance of ZSWIM4 expression in EOC and develop new clinical therapeutic strategies for EOC. METHODS: ZSWIM4 expression in control and EOC tumor tissues was examined using immunohistochemistry. Lentiviral transduction, Cell Counting Kit-8 assay, tumorsphere formation assay, flow cytometry, western blotting, and animal xenograft model were used to assess the role of ZSWIM4 in chemotherapy. Cleavage Under Targets and Tagmentation (CUT&Tag) assays, chromatin immunoprecipitation assays, and luciferase reporter assays were used to confirm FOXK1-mediated upregulation of ZSWIM4 expression. The mechanism by which ZSWIM4 inhibition improves chemosensitivity was evaluated using RNA-sequencing. A ZSWIM4-targeting inhibitor was explored by virtual screening and surface plasmon resonance analysis. Patient-derived organoid (PDO) models were constructed from EOC tumor tissues with ZSWIM4 expression. RESULTS: ZSWIM4 was overexpressed in EOC tumor tissues and impaired patient prognoses. Its expression correlated positively with EOC recurrence. ZSWIM4 expression was upregulated following carboplatin treatment, which, in turn, contributed to chemoresistance. Silencing ZSWIM4 expression sensitized EOC cells to carboplatin treatment in vitro and in vivo. FOXK1 could bind to the GTAAACA sequence of the ZSWIM4 promoter region to upregulate ZSWIM4 transcriptional activity and FOXK1 expression increased following carboplatin treatment, leading to an increase in ZSWIM4 expression. Mechanistically, ZSWIM4 knockdown downregulated the expression of several rate-limiting enzymes involved in glycine synthesis, causing a decrease in intracellular glycine levels, thus enhancing intracellular reactive oxygen species production induced by carboplatin treatment. Compound IPN60090 directly bound to ZSWIM4 protein and exerted a significant chemosensitizing effect in both EOC cells and PDO models. CONCLUSIONS: ZSWIM4 inhibition enhanced EOC cell chemosensitivity by ameliorating intracellular glycine metabolism reprogramming, thus providing a new potential therapeutic strategy for EOC.

Doublecortin-like kinase 2 promotes breast cancer cell invasion and metastasis.

BACKGROUND: Doublecortin-like kinase 2 (DCLK2) is a microtubule-associated protein kinase that participates in neural development and maturation; however, whether it is involved in tumour progression remains unclear. METHODS: DCLK2 overexpression and knockdown clones were established by lentivirus transfection. Western blot, PCR assays and bioinformatics analyses were conducted to observe the expression of DCLK2. CCK8, colony formation, scratch migration and Transwell assays were used to detect cell proliferation, migration and invasion, respectively. Tumour metastasis was evaluated in vivo using a tail vein metastasis model. Bioinformatics analyses were performed to analyse the expression correlation between DCLK2 and TCF4, or EMT markers in breast cancer. RESULTS: Our data indicate that DCLK2 is highly expressed in breast cancer cells and is associated with poor prognosis. Silencing DCLK2 does not affect the proliferation rate of tumour cells, but significantly suppresses migration and invasion as well as lung metastasis processes. Overexpression of DCLK2 can enhance the migratory and invasive abilities of normal breast epithelial cells. Moreover, TCF4/ß-catenin inhibitor LF3 downregulates the expression of DCLK2 and inhibits the migration and invasion of breast cancer cells. Furthermore, we found that the downregulation of DCLK2 blocks the epithelial-mesenchymal transition (EMT) process. CONCLUSION: Our study indicates that DCLK2 plays an important role in EMT, cell invasion and metastasis, suggesting that DCLK2 is a potential target for the treatment of metastatic breast cancer.

Development of a RPA-CRISPR-Cas12a Assay for Rapid, Simple, and Sensitive Detection of Mycoplasma hominis.

Mycoplasma hominis, which is difficult to culture and identify by ordinary methods, is one of the smallest pathogens in the human genitourinary tract causing urogenital infections. A CRISPR-Cas12a-based detection system might provide a novel application for M. hominis nucleic acid detection in molecular diagnostics. A plasmid containing the glyceraldehyde-3-phosphate dehydrogenase gene of M. hominis (ATCC_27545) as the positive control was constructed by homologous recombination. The active Cas12a protein was purified by affinity chromatography. The primers for recombinase polymerase amplification (RPA), the CRISPR RNA (crRNA), and the ratio of Cas12a to crRNA were further optimized. Finally, the sensitivity, specificity, and clinical effectiveness of the Cas12a detection system were confirmed. We successfully constructed and optimized a novel nucleic acid detection system for M. hominis based on RPA-CRISPR-Cas12a, and the whole process takes only 1 h. The limit of detection for the gap gene of M. hominis was 3 copies/µl and no cross-reactivity with other urogenital pathogens appeared. In the evaluation of 111 clinical samples, the sensitivity and specificity were both 1.000 and the area under the curve of the receiver operating characteristic was 1.000 (p < 0.001), indicating that the RPA-Cas12a-fluorescent assay was fully comparable to the traditional culture method. Finally, the RPA-Cas12a detection system can also be combined with lateral flow strips (LFS) to achieve visual detection. We successfully developed a low-cost and rapid detection method of M. hominis based on RPA-Cas12a technology. This method realized by fluorescence value readout and visual detection by LFS could be applied in population screening and resource-limited conditions.

Preclinical efficacy and involvement of mTOR signaling in the mechanism of Orf virus against nasopharyngeal carcinoma cells.

AIMS: Orf virus (ORFV) is a parapoxvirus causing contagious ecthyma in sheep and goats. With inhibitory role of ORFV reported by previous studies, ORFV can be a candidate of oncolytic virus. However, few studies reported the application and mechanism of ORFV in nasopharyngeal carcinoma (NPC). We aimed to elucidate the anti-tumor mechanism of ORFV against NPC cells. MATERIALS AND METHODS: The anti-tumor effect of ORFV in NPC cells was confirmed by cell counting kit 8 (CCK-8) assay, flow cytometry and Western blot. In vitro and in vivo experiments were adopted to evaluate the inhibitory effect of ORFV in NPC cells. Western blot was used to determine the down-regulation of rapamycin (mTOR) signaling and autophagy enhancement induced by ORFV. To explore the mechanism of ORFV on NPC cells, mTOR signaling agonist and autophagy inhibitors were used to rescue the effects of ORFV. KEY FINDINGS: The results indicated that ORFV replicates in NPC cells, thus induces the apoptosis of NPC cells. Moreover, ORFV can effectively inhibit NPC cell growth in vivo. ORFV infection in NPC cells leads to the mTOR signaling inhibition and up-regulated autophagy, which might be the specific mechanism of ORFV in killing tumor cells. As to safety confirmation, normal nasopharyngeal epithelial cells NP69 are insensitive to ORFV. More importantly, ORFV would not cause organ damage in vivo. SIGNIFICANCES: Our data clarified that ORFV induces autophagy of NPC cells via inhibiting mTOR signaling, thus further inducing apoptosis. The anti-tumor role of ORFV might provide a preclinical strategy for NPC treatment.

SWIM domain protein ZSWIM4 is required for JAK2 inhibition resistance in breast cancer.

AIMS: Janus kinase 2 (JAK2)/signal transducer and activator of transcription (STAT) signaling plays a critical role in the progression of breast cancer. However, a small part of tumor cells survived from the killing effect of JAK2 inhibitor. We aimed to find out the mechanism of drug resistance in breast cancer cells and develop new therapeutic strategies. MATERIALS AND METHODS: The anti-tumor effect of TG101209 in breast cancer cells was confirmed by cell counting kit 8 and flow cytometry. Western blotting was used to determine the up-regulation of zinc finger SWIM-type containing 4 (ZSWIM4) induced by TG101209. In vitro and in vivo experiments were performed to evaluate the role of ZSWIM4 in the resistance of breast cancer cells to TG101209. Through the determination and analysis of 50% inhibiting concentration (IC50) curves, the effect of combination therapy was confirmed. KEY FINDINGS: Our data indicate that the elevated expression of ZSWIM4 contributes to JAK2 inhibition resistance, as knockdown of ZSWIM4 significantly enhances the sensitivity of breast cancer cells to TG101209 and over-expression of this gene mitigates the killing effect. Furthermore, the expression of vitamin D receptor (VDR) and utilization of 1α,25-(OH)2VD3 is decreased in ZSWIM4-knockdown breast cancer cells. VDR-silencing or GW0742-mediated blockade of VDR activity can partially reverse the JAK2 inhibition resistance. SIGNIFICANCE: Our data implicated that ZSWIM4 might be an inducible resistance gene of JAK2 inhibition in breast cancer cells. The combination of JAK2 inhibitor and VDR inhibitor may achieve better coordinated therapeutic effect in breast cancer.

Enhanced JunD/RSK3 signalling due to loss of BRD4/FOXD3/miR-548d-3p axis determines BET inhibition resistance.

BET bromodomain inhibitors (BETi), such as JQ1, have been demonstrated to effectively kill multiple types of cancer cells. However, the underlying mechanisms for BETi resistance remain largely unknown. Our evidences show that JQ1 treatment evicts BRD4 from the FOXD3-localized MIR548D1 gene promoter, leading to repression of miR-548d-3p. The loss of miRNA restores JunD expression and subsequent JunD-dependent transcription of RPS6KA2 gene. ERK1/2/5 kinases phosphorylate RSK3 (RPS6KA2), resulting in the enrichment of activated RSK3 and blockade of JQ1 killing effect. Dual inhibition of MEKs/ERKs or single EGFR inhibition are able to mimic the effect of JunD/RSK3-knockdown to reverse BETi resistance. Collectively, our study indicates that loss of BRD4/FOXD3/miR-548d-3p axis enhances JunD/RSK3 signalling and determines BET inhibition resistance, which can be reversed by targeting EGFR-MEK1/2/5-ERK1/2/5 signalling.

PA28α/ß Promote Breast Cancer Cell Invasion and Metastasis via Down-Regulation of CDK15.

PA28α/ß activated immunoproteasome frequently participates in MHC class I antigen processing, however, whether it is involved in breast tumor progression remains largely unclear. Here, our evidences show that PA28α/ß proteins are responsible for breast cancer cell migration, invasion, and metastasis. Knockdown of immunoproteasome core subunit ß5i also robustly suppresses the tumor cell migration and invasion. Interestingly, silencing of PA28α/ß and ß5i up-regulates the protein expression of cyclin-dependent kinase 15 (CDK15). Our data further indicate that the loss of CDK15 is important for breast tumor cell invasion and metastasis. Taken together, this study implicates that targeting of PA28α/ß represents a potential way for treatment of metastatic breast cancer.