Targeting Mitochondrial Cholesterol Efflux via TCF21/ABCA10 Pathway to Enhance Cisplatin Efficacy in Ovarian Cancer.

Cisplatin (DDP) resistance is one of the causes of treatment failure for ovarian cancer (OV). Mitochondrial cholesterol level was reported to be associated with OV chemoresistance. We found that ABCA10, a potential cholesterol transport protein, was highly expressed in ovarian tissues and downregulated in OV tissues. Our study aimed to explore TCF21/ABCA10 axis resistance to DDP therapy in ovarian cancer based on regulating mitochondrial cholesterol efflux. Thirty epithelial ovarian cancer tumors and thirty ovarian tissues from non-cancer patients were collected. Western blot and RT-qPCR were used to measure ABCA10 and TCF21 expression levels in these tissues, as well as in a human ovarian epithelial cell line (IOSE-80), OV cells (A2780 and SKOV3), and DDP-resistant OV cell lines (A2780/DDP and SKOV3/DDP). IOSE-80 cells were also infected with ABCA10 knockdown lentivirus to identify the most effective ABCA10 knockdown plasmid. Lentiviral infection was used to create ABCA10 knockdown, ABCA10 overexpression, and TCF21 overexpression anti-DDP OV cell lines. Cell proliferation was detected by CCK-8 and EDU staining, flow cytometry for apoptosis, MTT for metabolic activity, calcium-induced Cytochrome C release, and mitochondrial matrix swelling for mitochondrial function and Oil Red O staining for lipid accumulation. Cholesterol metabolism was evaluated by measuring mitochondrial cholesterol and cholesterol efflux. Protein concentration was determined using the BCA method. A dual-luciferase reporter assay confirmed TCF21's interaction with ABCA10. ChIP also verified this interaction. The mRNA level (P < 0.01) and protein level (P < 0.001) of ABCA10 were downregulated in cancer tissues of OV patients relative to normal ovarian tissues. Relative to human ovarian epithelial cells, ABCA10 expression was significantly downregulated in OV cells (P < 0.01) and even more significantly downregulated in DDP-resistant OV cells (P < 0.001). Compared to the group treated solely with DDP, the overexpression of ABCA10 significantly inhibited the proliferation of DDP-resistant OV cells (P < 0.01), markedly reduced the staining intensity of EDU in these cells (P < 0.05), and substantially accelerated apoptosis in DDP-resistant OV cells (P < 0.01).Overexpression of ABCA10 further accelerated Cytochrome C expression and mitochondrial matrix swelling in DDP-resistant OV cells compared to the DDP-alone group (P < 0.01). The addition of cholesterol reversed the decrease in lipid accumulation, the decrease in mitochondrial cholesterol levels (P < 0.05), and the increase in cholesterol efflux (P < 0.01) in DDP-resistant OV cells caused by overexpression of ABCA10. The transcription factor TCF21 was bound to the promoter of ABCA10. Overexpression of TCF21 significantly increased ABCA10 expression in DDP-resistant OV cells (P < 0.01) and increased cytochrome C expression in A2780/DDP (P < 0.05) and SKOV3/DDP (P < 0.01) cells, with accelerated mitochondrial matrix swelling in A2780/DDP (P < 0.01) and SKOV3/DDP (P < 0.001) cells, while knockdown of ABCA10 reversed these effects. Our study found that TCF21 boosts ABCA10 expression, which in turn reduces DDP resistance in OV cells by enhancing mitochondrial cholesterol efflux. This mechanism increases the sensitivity of DDP-resistant OV cells to DDP. Our findings will provide new therapeutic targets for the treatment of ovarian cancer.

QL1604 plus paclitaxel-cisplatin/carboplatin in patients with recurrent or metastatic cervical cancer: an open-label, single-arm, phase II trial.

OBJECTIVE: QL1604 is a highly selective, humanized monoclonal antibody against programmed death protein 1. We assessed the efficacy and safety of QL1604 plus chemotherapy as first-line treatment in patients with advanced cervical cancer. METHODS: This was a multicenter, open-label, single-arm, phase II study. Patients with advanced cervical cancer and not previously treated with systemic chemotherapy were enrolled to receive QL1604 plus paclitaxel and cisplatin/carboplatin on day 1 of each 21-day cycle for up to 6 cycles, followed by QL1604 maintenance treatment. RESULTS: Forty-six patients were enrolled and the median follow-up duration was 16.5 months. An 84.8% of patients had recurrent disease and 13.0% had stage IVB disease. The objective response rate (ORR) per Response Evaluation Criteria in Advanced Solid Tumors (RECIST) v1.1 was 58.7% (27/46). The immune ORR per immune RECIST was 60.9% (28/46). The median duration of response was 9.6 months (95% confidence interval [CI]=5.5-not estimable). The median progression-free survival was 8.1 months (95% CI=5.7-14.0). Forty-five (97.8%) patients experienced treatment-related adverse events (TRAEs). The most common grade≥3 TRAEs (>30%) were neutrophil count decrease (50.0%), anemia (32.6%), and white blood cell count decrease (30.4%). CONCLUSION: QL1604 plus paclitaxel-cisplatin/carboplatin showed promising antitumor activity and manageable safety profile as first-line treatment in patients with advanced cervical cancer. Programmed cell death protein 1 inhibitor plus chemotherapy may be a potential treatment option for the patient population who have contraindications or can't tolerate bevacizumab, which needs to be further verified in phase III confirmatory study. Trial RegistrationClinicalTrials.gov Identifier: NCT04864782.

Reversal of the immunosuppressive tumor microenvironment via platinum-based neoadjuvant chemotherapy in cervical cancer.

Background: Immunotherapy favors patients with tumors; however, only 3-26.3% of patients with cervical cancer benefit from single-agent immune checkpoint inhibitors. Combined immunotherapy and chemotherapy has been explored against tumor; however, the combination remains controversial. This study aimed to investigate the tumor immune microenvironment (TIME) and the effects of platinum-based neoadjuvant chemotherapy (NACT) in cervical cancer to identify the clinical value of combining chemotherapy with immunotherapy. Methods: Multiplex immunohistochemistry (IHC) with 11 markers (cluster of differentiation [CD]3, CD8, CD4, CD11c, CD68, forkhead box P3 [Foxp3], programmed cell death 1 [PD-1], programmed cell death 1 ligand 1 [PD-L1], indoleamine 2,3-dioxygenase [IDO], cyclin-dependent kinase inhibitor 2A [p16], and cytokeratin [CK]) was performed to evaluate TIME from 108 matched pre- and post-NACT cervical cancer samples. The mechanism of antitumor immunity triggered by NACT was explored using RNA sequencing (RNA-seq) from four paired samples and subsequently verified in 41 samples using IHC. Results: The infiltration rate of the CD8+ T cells in treatment-naive cervical cancer was 0.73%, and those of Foxp3+ regulatory T cells (Tregs) and IDO+ cells were 0.87% and 17.15%, respectively. Moreover, immunoreactive T cells, dendritic cells, and macrophages were more in the stromal than the intratumor region. NACT increased dendritic, CD3+ T, CD8+ T, and CD4+ T cells and decreased Tregs. The aforementioned alterations occurred predominantly in the stromal region and were primarily in responders. Non-responders primarily showed decreased Tregs and no increase in CD8+ T or dendritic cell infiltration. Furthermore, dendritic cells interacted more closely with CD3+ T cells after NACT, an effect primarily observed in responders. RNA-seq data revealed activation of the antigen receptor-mediated signaling pathway and upregulation of major histocompatibility complex (MHC) I and MHC II after chemotherapy, validated using IHC. Conclusions: NACT can reduce Tregs, and when tumor cells are effectively killed, antigen presentation is enhanced, subsequently activating antitumor immunity finitely. Our study provides the molecular characteristics and theoretical basis for the simultaneous or sequential combination of platinum-based NACT and immunotherapy for cervical cancer.

SHR-1701, a Bifunctional Fusion Protein Targeting PD-L1 and TGFß, for Recurrent or Metastatic Cervical Cancer: A Clinical Expansion Cohort of a Phase I Study.

PURPOSE: Patients with recurrent or metastatic cervical cancer have limited treatment options after platinum-containing treatment. We initiated a phase I study to assess SHR-1701, a novel bifunctional fusion protein composed of a mAb against programmed death ligand 1 (PD-L1) fused with the extracellular domain of TGFß receptor II, in solid tumors (NCT03774979). Here, results from the cervical cancer cohort are presented. PATIENTS AND METHODS: Patients with recurrent or metastatic cervical cancer who progressed during or after platinum-based therapy were enrolled to receive SHR-1701 at 30 mg/kg every 3 weeks. Primary endpoint was objective response rate (ORR) per RECIST v1.1. RESULTS: In total, 32 patients were recruited. ORR was 15.6% [95% confidence interval (CI), 5.3-32.8], and disease control rate was 50.0% (95% CI, 31.9-68.1). Responses were still ongoing in 80.0% of the responders; 6-month duration of response rate was 80.0% (95% CI, 20.4-96.9). Median progression-free survival (PFS) was 2.7 months (95% CI, 1.4-4.1). Of note, as assessed by immune-modified RECIST, median PFS was 4.1 months (95% CI, 1.6-4.3). Overall survival rate at 12 months was 54.6% (95% CI, 31.8-72.7). Treatment-related adverse events of grade 3 or 4 were reported in 11 (34.4%) patients. No treatment-related deaths occurred. No difference in ORR was found between patients with PD-L1 combined positive score ≥1 or <1; patients with high phosphorylated SMAD2 level in immune cells or tumor cells had numerically higher ORR. CONCLUSIONS: SHR-1701 exhibits encouraging antitumor activity and controllable safety in patients with recurrent or metastatic cervical cancer after platinum-based regimens, and therefore might provide another treatment option for this population. See related commentary by Miller and Friedman, p. 5238.

Identification and characterization of a subpopulation of CD133+ cancer stem-like cells derived from SK-UT-1 cells.

BACKGROUND: Uterine leiomyosarcoma (ULMS) is a malignant tumor found in the smooth muscle lining the walls of the uterus. Cancer stem cells (CSCs) are responsible for metastasis, drug resistance, and relapse of cancer, resulting in treatment failure. However, little is known about CSCs and their associated-markers in ULMS. We aimed to characterize and identify a subpopulation of CD133+ cancer stem-like cells derived from SK-UT-1 cell line. METHODS: SK-UT-1 cells were sphere-forming cultured in vitro. We also sorted the CD133+ cells derived from SK-UT-1 cell line by immunomagnetic beads. CD133+ subpopulation and apoptotic cells were detected by flow cytometry. Self-renewal and anchorage-independent growth capabilities were examined using sphere and colony formation assays. The tumorigenicity of the fourth-passage spheres and parental SK-UT-1 cells was used by mouse xenograft model in vivo. Cell proliferation ability and sensitivity to doxorubicin (DXR) were assessed by CCK-8 assay. Cell migration and invasion were tested by wound healing assay or Transwell migration and invasion assays. Expressions of CSC-related marker were analyzed by Western blotting. RESULTS: The fourth-passage spheres were defined as a CD133+ cell population, which was accompanied by increase of sphere and colony forming rate, migration and invasion abilities, as well as drug-resistant properties in vitro. Moreover, the fourth-passage spheres showed a stronger tumorigenic potential in vivo. CD133+ cell population sorted from SK-UT-1 line showed an increased ability in sphere and colony formation, proliferation, migration, invasion, resistance to apoptosis after treatment with doxorubicin (DXR) compared with CD133- cell population. The expression levels of CSCs-related markers (e.g., CD44, ALDH1,BMI1, and Nanog), were significantly elevated in CD133+ cells compared with those in CD133- cells. CONCLUSIONS: Collectively, our findings indicated that CD133 may be a significant marker for cancer stem-like cells, and it may be a potential therapeutic target for human ULMS.

Long non-coding RNA: A recently accentuated molecule in chemoresistance in cancer.

Chemotherapy is one of the important and effective options for cancer treatment in the past decades. Although the response rate of initial chemotherapy is considerably high in certain types of cancers, such as ovarian cancer and lung cancer, the patients frequently suffer from chemoresistance and recurrence of disease. Recent genome-wide studies have shown that the large number of long non-coding RNAs (lncRNAs) are transcribed from the human genome and involved in many biological processes including carcinogenesis. They aberrantly regulate variety of cell functions, such as cell cycle, apoptosis, autophagy, and metabolisms, which are associated with chemosensitivity. Therefore, understanding the biological and clinical impacts of lncRNAs on tumor behavior and its potential as a predictive biomarker for chemotherapy effectiveness is highly desired. In this review, we classify the major mechanisms of lncRNA-related chemoresistance and provide theoretical evidences for targeting lncRNAs in certain types of cancers that may open up new therapeutic paradigm for cancer treatment.

Label-free detection of miRNA cancer markers based on terminal deoxynucleotidyl transferase-induced copper nanoclusters.

The variations in microRNA (miRNA) expression levels can be useful biomarkers for the diagnosis of different cancers. In this work, a label-free and sensitive fluorescent method for detection of miRNA-21 is described based on duplex-specific nuclease (DSN) assist target recycling and terminal deoxynucleotidyl transferase (TdT) induced copper nanoclusters (CuNCs). In the absence of target, the 3'-phosphorylated probe DNA cannot be hydrolyzed by DSN and extended by TdT, and failed to synthesizing fluorescent CuNCs. However, the target miRNA-21 can caused the digestion of probe DNA with DSN, releasing primer DNA with 3'-OH. After that, the primer DNA can forms long poly T with the assistance of TdT, leading to synthesize high fluorescent CuNCs. The fluorescence change of CuNCs can be used to identify the concentration of target miRNA-21. Under optimal experimental conditions, this strategy could quantitatively detect miRNA-21 down to 18.7 pM. We have also demonstrated the practical application of our proposed method for monitoring miRNA-21 expression levels in cancer cells. Moreover, this method show good specificity for miRNA-21 detection due to the strong preference of DSN for cutting perfectly matched DNA/RNA duplex, which holds great potential for highly specific quantification of biomarkers in bioanalysis and clinical diagnosis.

MiR-218 produces anti-tumor effects on cervical cancer cells in vitro.

BACKGROUND: As indoleamine-2,3-dioxygenase 1 (IDO1) is critical in tumor immune escape, we determined to study the regulatory mechanism of miR-218 on IDO1 in cervical cancer. METHODS: Real-time PCR (RT-qPCR) was carried out to measure the expression of miR-218. RT-qPCR and Western blot were performed to detect the expression of IDO1 in cervical cancer. Dual-luciferase reporter assay was used to determine the binding of miR-218 on the IDO1 3'UTR. Cell viability, apoptosis, and related factors were determined using cell counting kit-8 (CCK-8), Annexin-V/PI (propidium) assay, enzyme-linked immunosorbnent assay (ELISA), RT-qPCR, and Western blot assays after miR-218 mimics has been transfected to HeLa cervical cancer cells. RESULTS: MiR-218 was downregulated in cervical cancer. The expression of miR-218 was negatively correlated with IDO1 in cervical cancer tissues and cells. IDO1 is a direct target of miR-218. MiR-218 overexpression was found to inhibit cell viability and promoted apoptosis via activating the expression of Cleaved-Caspase-3 and to inhibit the expression of Survivin, immune factors (TGF-ß, VEGF, IL-6, PGE2, COX-2), and JAK2/STAT3 pathway. CONCLUSION: MiR-218 inhibits immune escape of cervical cancer cells by direct downregulating IDO1.

Isolation, Culture and Identification of Choriocarcinoma Stem-Like Cells from the Human Choriocarcinoma Cell-Line JEG-3.

BACKGROUND/AIMS: Cancer stem cells (CSCs) exhibit enhanced proliferative capacity and resistance to chemotherapy; however, choriocarcinoma CSCs have not yet been reported. In this study the human choriocarcinoma cell line JEG-3 was cultured in serum free media, and the characteristics of suspension and parental adherent JEG-3 cells were compared. METHODS: Cell proliferation, colony-formation, soft agar clonogenicity, and transwell invasion assays were performed in vitro, and tumor xenografts in BALB/c nude mice were used to evaluate stem cell properties. RESULTS: In serum-supplemented medium (SSM), JEG-3 cells were 4.51 ± 1.71% CD44+, 7.67 ± 2.67% CD133+, and 13.85 ± 2.95% ABCG2+. In serum-free medium (SFM), the expression of these markers increased to 53.08 ± 3.15%, 47.40 ± 2.67%, and 78.70 ± 7.16%, respectively. Moreover, suspension JEG-3 cells exhibited enhanced colony-formation capability as well as invasive and proliferative ability in vitro, alongside enhanced tumorigenic properties in vivo. Suspension JEG-3 cells also exhibited resistance to the chemotherapeutic drugs methotrexate, fluorouracil and etoposide. When seeded in serum supplemented medium, suspension JEG-3 cells readopted an adherent phenotype and continued to differentiate with no significant difference in the morphology between suspension and parent cells. CONCLUSION: In this study, choriocarcinoma stem-like cells (CSLCs) were isolated from the human choriocarcinoma JEG-3 cell line by SFM culture and characterized.

Isolation and comparison of mesenchymal stem cell­like cells derived from human gastric cancer tissues and corresponding ovarian metastases.

Mesenchymal stem cell (MSC)-like cells have been isolated from various types of tumor. It has previously been reported that MSCs are involved in tumorigenesis and its prognosis. The aim of the present study was to isolate and compare MSC-like cells from human gastric cancer (GC) and its metastatic deposits in ovarian tissue. MSC-like cells were isolated from human gastric cancer (hGC-MSCs) and the corresponding ovarian metastatic tissues (hGCOM-MSCs) from 40 patients. The characteristics of hGC-MSCs and hGCOM-MSCs, including their morphology, surface antigens, specific gene expression and differentiation potential, were similar to those of MSCs derived from human bone marrow (hBM-MSCs) but different from GC cells. In conclusion, the present study demonstrated that MSC-like cells could be isolated from GC tissue and its ovarian metastatic tissues. The existence of MSC-like cells in GC tissues and its ovarian metastatic tissues suggests that they may be a potential target for cancer therapy, and provides an experimental foundation for investigating their role in the initiation and progression of ovarian metastasis of GC.

Diagnosis and multimodal therapy for extragastrointestinal stromal tumor of the prostate: A case report.

Extragastrointestinal stromal tumors (EGISTs), which are neoplasms outside the digestive tract, are predominantly observed in the greater omentum and retroperitoneum. The clinicopathological and molecular characteristics of EGISTs are similar to those of gastrointestinal stromal tumors (GISTs). EGISTs originating from the prostate are extremely rare. In this study, we report a case of a prostatic EGIST in a 39-year-old male, who presented with frequency, urgency, dysuria and a prostatic mass. A 10-core transrectal ultrasound-guided prostate biopsy was performed, and the histological and immunohistochemical results confirmed the diagnosis of EGIST. The patient received a radical prostatectomy, followed by targeted therapy with imatinib (400 mg, daily) for 1 year. Neither recurrence nor metastasis was detected at a 24-month follow-up.

[Analysis of the security, pregnancy outcomes, and the tumor recurrence related factors of young patients with cervical cancer treated with different radical trachelectomy].

OBJECTIVE: To explore the security, pregnancy outcomes, and the tumor recurrence related factors of young patients with cervical cancer treated with different radical trachelectomy (RT). METHODS: Thirty-two young patients < 40 years of age with early cervical cancer from May 2004 to July 2012 admitted in Tumor Hospital Xiangya School of Medicine of Central South University were divided into two groups based on different operation methods: vaginal radical trachelectomy (RVT) group and abdominal radical trachelectomy (RAT) group.The clinical data were analyzed by One-way Anova and multivariate Cox stepwise regression analysis. RESULTS: The operation duration, number of lymph node dissection, the height of the cervical resection, postoperative hospitalization time, incidence of vascular injury and incidence of postoperative lymphocele were respectively (250 ± 82) min, 15 ± 6, (2.31 ± 0.21) cm, (9.2 ± 2.9) d, 1/18 and 1/18 in RVT group, while (263 ± 60) min,16 ± 8, (2.32 ± 0.26) cm, (10.3 ± 3.5) d,0 and 1/14 in RAT group. There was no statistically significant difference between the two groups (all P > 0.05). The blood loss (281 ± 201) ml in RVT group was significantly lower than that in the RAT group (492 ± 320) ml (P < 0.05). The length of Vaginal hysterectomy [(2.61 ± 0.50) cm] and the width of parametrial resection [ (2.38 ± 0.36) cm] in RVT group were significantly less than those [(2.95 ± 0.10), (2.81 ± 0.22) cm] in the RAT group (all P < 0.05).The pregnancy rate between RVT group (3/18) and RAT group (2/14) were no significant difference (P > 0.05).One-way Anova analysis showed that the recurrence of early cervical cancer was related to tumor size in diameter (F = 4.911, P = 0.047), while there were no correlation with age, clinical stage, histological type and surgical approach (all P > 0.05).Multivariate analysis showed that tumor diameter size was an independent risk factor for tumor recurrence (ß = 0.259, P = 0.031). CONCLUSIONS: RT for young patients with early cervical cancer is feasible.Pregnancy outcomes after RT need to be study in the future. Tumor size in diameter is the major risk factor for tumor recurrence.

[Comparative study of different chemotherapy approaches for advanced epithelial ovarian cancer].

OBJECTIVE: To probe into the advantages and disadvantages of intravenous chemotherapy and intraperitoneal chemotherapy for advanced epithelial ovarian cancer. METHODS: All of the 226 patients with advanced epithelial ovarian cancer were treated by maximum cytoreductive surgery or non-effective cytoreductive surgery and received 6 - 8 courses of postoperative regular chemotherapy (chemotherapy regimens, TP: taxol and cis-platinum or carboplatinum; PC: cis-platinum and cyclophosphamide; PAC: cis-platinum and adriamycin and cyclophosphamide) during Jan 1998 - Jan 2006. We systematically compared the characteristics of patients in intraperitoneal chemotherapy (IPC) group and intravenous chemotherapy (IVC) group. We measured the incidence rate of the response, side-effects, the recurrence time of intraperitoneal tumor and survival time of the two groups respectively. RESULTS: For the first phase after operation (three courses of treatment), the response rate of two groups were 75.8% and 52.8% respectively. For the response rate of IPC was higher than that of IVC (P < 0.01). The second phase after operation (all courses finished), the response rate of two groups were 93.9% and 87.7%, respectively (P > 0.05). After maximum cytoreductive surgery, the recurrence rate of IPC and IVC were 47.0% and 59.4%, respectively (P > 0.05). After non-effective cytoreductive surgery of IPC and IVC groups, the recurrence rates were 84.8% and 86.2%, respectively (P > 0.05). The recurrence time of intraperitoneal tumor of IPC and IVC groups were 24 and 18 months, respectively (P = 0.001). The overall survival time of groups IPC and IVC were 32 and 30 months (P = 0.188). There were some differences in the side-effect between IPC and IVC. The rates of chemotherapeutic phlebitis of IPC and IVC were 34.0% and 10.8% respectively (P < 0.01). The rates of serious gastrointestinal reaction of IPC and IVC were 33.8% and 25.8%, respectively (P = 0.236). There was no significant difference in bone marrow depression, intestinal adhesion and intestinal obstruction. CONCLUSIONS: IPC can extend the disease progression free survival than IVC, without increasing overall survival period. IPC can also reduce the side-effect of chemotherapeutic phlebitis. However, IPC is used limitedly, and can not substitute for IVC. Combination of IPC with IVC may enhance their effectiveness and reduce the side-effects.