Yusuke Yamamoto

Overcoming platinum-resistant ovarian cancer targeting the activated JAK-STAT pathways via extracellular vesicles

Abstract Platinum-resistant ovarian cancer (PROC) is a clinically severe unresolved issue, and it remains unclearly defined by molecular biology. Extracellular vesicles (EVs) play an essential role in cell-to-cell communication in the tumor microenvironment. This study aimed to investigate the molecular mechanisms of PROC, focusing on the unique ascites environment of ovarian cancer. Multi-transcriptome analyses using clinical samples revealed that PROC exhibited an activated Janus kinase (JAK)/signal transducer and activator of transcription pathway with high JAK1 expression in cancer cells. Immunohistochemistry for patient tissues confirmed the negative association between JAK1 expression and platinum response. JAK inhibitors were effective in PROC cell lines and cell- and patient-derived xenograft models, as well as synergistic with platinum. Furthermore, small RNA sequencing indicated that activated peritoneal mesothelial cell-derived EVs enriched in miR135a-5p increased JAK expression and platinum resistance in cancer cells. Collectively, EVs in ascites regulated platinum sensitivity in ovarian cancer cells, and JAK targeting therapeutic strategy overcomes PROC.

Single‐Nucleus RNA Sequencing and Spatial Transcriptomics for Squamous Cell Carcinoma Arising From Ovarian Mature Teratoma

ABSTRACTSquamous cell carcinoma arising from mature teratoma (SCC‐MT) is a rare ovarian malignancy. The detailed molecular pathology of SCC‐MT is not well understood. Moreover, the prognosis of the patients remains poor because no standard treatment has been established. In this study, we performed single‐nucleus RNA sequencing and spatial transcriptomics using clinical SCC‐MT samples to identify novel therapeutic candidates. snRNA‐seq revealed three epithelial cell clusters, of which one was significantly associated with epidermis and keratinocyte development. Moreover, spatial transcriptomics revealed that the epithelial‐mesenchymal transition was significantly inhibited, and the MYC and E2F targets were significantly activated in cancer spots on specimen sections. We focused on KLF5, which was one of the upregulated genes in cancer cells, and performed a functional analysis using NOSCC‐1, a cell line derived from an SCC‐MT. KLF5 downregulation significantly decreased cell proliferation and increased apoptosis. Furthermore, we previously identified miR‐145‐5p as a downregulated miRNA in SCC‐MT. We demonstrated that miR‐145‐5p overexpression attenuated cell proliferation and decreased KLF5 expression. In conclusion, through multi‐omics analyses, we identified unique gene expression profiles of SCC‐MT and determined a role for KLF5 in SCC‐MT development. Therefore, KLF5‐related factors may be novel therapeutic targets, and further studies are needed to improve the diagnosis and treatment of SCC‐MT.

Circulating serum miRNAs predict response to platinum chemotherapy in high‐grade serous ovarian cancer

Abstract Background Platinum chemotherapy is the cornerstone of treatment for high‐grade serous ovarian cancer (HGSOC); however, validated biomarkers that can accurately predict platinum response are lacking. Based on their roles in the underlying pathophysiology, circulating microRNAs are potential, noninvasive biomarkers in cancer. In the present study, we aimed to evaluate the circulating miRNA profiles of patients with HGSOC and to assess their potential utility as biomarkers to predict platinum response. Methods Pretreatment serum samples collected from patients who received platinum chemotherapy for Stage III–IV HGSOC between 2008 and 2016 were analyzed using miRNA microarray. LASSO logistic regression analysis was used to construct predictive models for treatment‐free interval of platinum (TFIp). Results The median follow‐up was 54.6 (range, 3.5–144.1) months. The comprehensive analysis of 2588 miRNAs was performed in serum samples of 153 eligible patients, and predictive models were constructed using a combination of circulating miRNAs with an area under the receiver operating characteristic curve of 0.944 for TFIp >1 month, 0.637 for TFIp ≥6 months, 0.705 for TFIp ≥12 months, and 0.938 for TFIp ≥36 months. Each predictive model provided a significant TFIp classification ( p  = 0.001 in TFIp >1 month, p  = 0.013 in TFIp ≥6 months, p  < 0.001 in TFIp ≥12 months, and p  < 0.001 in TFIp ≥36 months). Conclusion Circulating miRNA profiles has potential utility in predicting platinum response in patients with HGSOC and can aid clinicians in choosing appropriate treatment strategies.

Small Extracellular Vesicles from adipose-derived stem cells suppress cell proliferation by delivering the let-7 family of microRNAs in ovarian cancer

Ovarian cancer is the leading cause of death among women with gynecological cancer, and novel treatment options are urgently needed. Extracellular vesicles (EVs), including exosomes, may be one of the most promising therapeutic tools for various diseases. In this study, we aimed to investigate the therapeutic effects of adipose-derived stem cell-derived EVs (ADSC-EVs) on ovarian cancer cell lines. ADSCs and the ovarian cancer cell lines SKOV3 and OV90 were used for analysis. ADSC-EVs were isolated through ultracentrifugation and validated using a cryotransmission electron microscope, nanoparticle tracking analysis, and western blotting. Then, the effect of ADSC-EVs on ovarian cancer cells was investigated using IncuCyte and microRNA sequencing. Moreover, the potential functions of miRNAs were evaluated by gain-of function analysis and in silico analysis. ADSC-EVs suppressed SKOV3 and OV90 cell proliferation. In particular, small EVs (sEVs) from ADSCs exhibited a stronger antitumor effect than ADSC-medium/large EVs (m/lEVs). Comparison of the miRNA profiles between ADSC-sEVs and ADSC-m/lEVs, along with downstream pathway analysis, suggested the involvement of the let-7 family. Overexpression of hsa-let-7b-5p and hsa-let-7e-5p significantly suppressed the proliferation of SKOV3 cells. In silico analysis revealed that four potential target genes of hsa-let-7b-5p and hsa-let-7e-5p were significantly associated with the prognoses of the patients. ADSC-sEVs had a stronger antitumor effect than ADSC-m/lEVs. Hsa-let-7b-5p and hsa-let-7e-5p, which are highly abundant in ADSC-sEVs, suppressed cell proliferation. These findings may open up new possibilities for therapeutic approaches using ADSC-sEVs.

In‐Tumor CRISPR ‐Cas9 Knockout Screening and Novel Therapy Development for Malignant Transformation of Ovarian Teratoma

ABSTRACT Malignant transformation of mature cystic teratoma (MTMCT) of the ovary is a rare but aggressive malignancy for which no standardized chemotherapy or effective targeted therapies currently exist. To identify therapeutic vulnerabilities in MTMCT, we performed a genome‐wide CRISPR‐Cas9 knockout screen using the MTMCT‐derived NOSCC1 cell line. Two parallel selective pressures were applied: in vivo tumorigenicity in immunodeficient mice and cisplatin exposure in vitro. From this screen, 67 negatively selected genes were identified, among which SOD1 and NDUFB4 emerged as top candidates based on high basal expression levels and clinical relevance. Integration with spatial transcriptomic data from three independent MTMCT patient tumors further supported the prioritization of these targets. SOD1 was selected for further investigation due to the availability of known pharmacological inhibitors. Both siRNA‐mediated knockdown and small‐molecule inhibition of SOD1 using LCS‐1 significantly suppressed MTMCT cell proliferation in vitro by inducing oxidative stress and impairing cell cycle progression. This antiproliferative effect was reversed by co‐treatment with N ‐acetylcysteine, a reactive oxygen species scavenger. In vivo validation using patient‐derived xenograft models demonstrated that oral administration of LCS‐1 led to significant tumor growth suppression and increased expression of apoptotic and DNA damage markers, including cleaved caspase‐3 and γH2AX. These findings establish SOD1 as a critical vulnerability in MTMCT and provide preclinical evidence supporting redox modulation as a therapeutic strategy for this highly chemoresistant and understudied ovarian cancer subtype. Our integrative approach combining functional genomics, spatial transcriptomics, and pharmacologic validation offers a framework for the discovery of novel targets in rare gynecologic malignancies.