Cancer Gene Therapy

Has_circ_ASH1L acts as a sponge for miR-1254 to promote the malignant progression of cervical cancer by targeting CD36

Cervical cancer (CC) is a prevalent gynecological malignancy. Increasing evidence suggests that circular RNAs (circRNAs) play a pivotal role in the pathogenesis of CC. However, the regulatory function of circ_ASH1L in CC remains elusive. In this study, we aim to elucidate the precise role and underlying mechanism of circ_ASH1L in the malignant progression of CC. The human CC dataset GSE102686 was extracted from the Gene Expression Omnibus (GEO) database for the analysis of differentially expressed circRNAs. Target gene prediction softwares were utilized to predict the binding of miRNAs to circ_ASH1L sponge. The expression level of circ_ASH1L in CC tissues and cells was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The characteristics of circ_ASH1L were determined by RNase R digestion, actinomycin D, and nucleo-plasmic separation assays. The effects of circ_ASH1L, miR-1254, and CD36 gain-and-loss on the malignant progression of CC were investigated using Cell Counting Kit-8 (CCK-8), colony formation, flow cytometry, wound scratch, transwell, and Western blot assay. The effect of circ_ASH1L on tumorigenicity of CC cells in vivo was evaluated in nude mice through tumor xenograft assay. The targeted regulatory relationship between circ_ASH1L/miR-1254 as well as miR-1254/CD36 was validated by dual-luciferase reporter assay. We screened the differentially expressed circ_ASH1L from the GEO dataset GSE102686 and confirmed its circular structure. Furthermore, we observed a significant upregulation of circ_ASH1L in both CC tissues and cells. Overexpression of circ_ASH1L promotes proliferation, invasion, and migration of CC cells while inhibiting cell apoptosis. However, silencing circ_ASH1L showed opposite results and inhibited tumorigenicity of CC cells in nude mice. Furthermore, we have identified circ_ASH1L as a miR-1254 sponge in CC cells. Notably, our in vitro experiments demonstrated that exogenously modulating the expression of miR-1254 effectively counteracted the impact of circ_ASH1L on the malignant phenotypic characteristics of CC cells. Similarly, modulation of CD36 expression efficiently counteracted the effect of miR-1254 on the malignant biological behavior of CC cells. In conclusion, circ_ASH1L promoted the malignant progression of CC via upregulating CD36 expression through sponging miR-1254.

SOX17, β-catenin and CyclinD1 expression in the endometrioid adenocarcinoma and influence of 5-AZA on expression

The present study discusses the expression and effect of the SOX17 gene in endometrioid adenocarcinoma. MTT assay is performed to determine the growth inhibition ratio of the DNA methyltransferase inhibitor 5-AZA for endometrial carcinoma cells, and the real-time fluorescence quantification PCR (qRT-PCR) was used to detect the mRNA expression of SOX17, β-catenin, and CyclinD1 in endometrial carcinoma tissues before and after using 5-AZA to treat the endometrial carcinoma cell line. There were 30 cases on endometrioid adenocarcinoma tissues and 10 cases on normal endometrial tissues. The results revealed that the expression of SOX17 in endometrioid adenocarcinoma tissues was downregulated (P  0.05; R = -0.463, P < 0.05). The higher the histological grade and FIGO staging were, the lower the expression level of SOX17 was (P < 0.05). After HEC1A cells were treated by 5-AZA, the cell growth inhibition was most obvious (IC

AAV for ovarian cancer gene therapy

Recent advancements in ovarian cancer treatment, particularly with PARP inhibitors, have markedly enhanced the recurrence-free interval, shifting the treatment paradigm and increasing treatment success in patients with BRCA mutations or HRD (homologous recombination deficiency). However, a significant proportion of cases experience relapse, resulting in poorer long-term survival rates when compared to other female cancers, such as breast cancer. This review explores the potential of adeno-associated virus (AAV) vectors for gene therapy in ovarian cancer and examines rational gene therapy strategies by categorizing them based on target cells and target genes to determine the most effective approach for ovarian cancer treatment. Specifically, it examines strategies such as anti-angiogenesis and immune modulation, highlighting the strategy of gene supplementation to hinder ovarian cancer progression. Innovations in AAV capsid design now allow for targeted delivery, focusing on ovarian cancer stem cells (CSCs) identified by specific markers. Additionally, leveraging DNA sequencing technologies enhances the identification and incorporation of therapeutic genes into AAV vectors, promising new avenues for ovarian cancer gene therapy.

Reversible downregulation of MYC in a spheroid model of metastatic epithelial ovarian cancer

Upon detachment from the primary tumour, epithelial ovarian cancer cells can form multicellular aggregates, also referred to as spheroids, that have the capacity to establish metastases at distant sites. These structures exhibit numerous adaptations that may facilitate metastatic transit and promote tumorigenic potential. One such adaptation is the acquisition of dormancy, characterized by decreased proliferation and molecular features of quiescence. One of the most frequently dysregulated genes in cancer is MYC, which encodes a transcription factor that promotes cell proliferation. In this study, we demonstrate that MYC protein abundance and associated gene expression is significantly decreased in EOC spheroids compared to adherent cells. This downregulation occurs rapidly upon cell detachment and is proteasome-dependent. Moreover, MYC protein abundance and associated gene expression is restored upon spheroid reattachment to an adherent culture surface. Overall, our findings suggest that suppression of MYC activity is a common feature of EOC spheroids and may contribute to the reversible acquisition of dormancy.

Methylstat sensitizes ovarian cancer cells to PARP-inhibition by targeting the histone demethylases JMJD1B/C

Abstract PARP-inhibitors (PARPi) are an integral part of ovarian cancer treatment. However, overcoming acquired PARPi resistance or increasing the benefit of PARPi in patients without homologous recombination deficiency (HRD) remains an unmet clinical need. We sought to identify genetic modulators of PARPi response, guiding pharmacological PARPi sensitization. CRISPR-Cas9 mediated loss-of-function screen with a focused sgRNA library revealed that DNA-demethylases JMJD1B/JMJD1C, targetable by the small inhibitor methylstat, promote PARPi resistance. Methylstat synergistically interacted with olaparib, and (re-)sensitized ovarian cancer cells to PARPi treatment, surpassing the efficacy of common demethylase inhibitors. Genetic knockout of JMJD1B and/or JMJD1C phenocopied the effect of methylstat in an additive manner. Validation studies revealed methylstat to be a universal PARPi-sensitizing drug, effective, regardless of PARPi resistance status or BRCA1 mutational background. Methylstat modulated clonal cancer dynamics by mitigating positive selection of PARPi-resistant or BRCA1-proficient cells under olaparib treatment. Using a model of PARPi-induced cellular toxicity, we showed that methylstat impairs cellular DNA repair, indicated by an increased susceptibility of ovarian cancer cells to olaparib-induced DNA double strand breaks after methylstat exposure. This study proposes the histone demethylase inhibitor methylstat as an epigenetic drug for overcoming PARPi-resistance or for increasing efficacy of PARPi beyond HRD in ovarian cancer patients.

Long-term severe hypoxia adaptation induces non-canonical EMT and a novel Wilms Tumor 1 (WT1) isoform

Abstract The majority of cancer deaths are caused by solid tumors, where the four most prevalent cancers (breast, lung, colorectal and prostate) account for more than 60% of all cases (1). Tumor cell heterogeneity driven by variable cancer microenvironments, such as hypoxia, is a key determinant of therapeutic outcome. We developed a novel culture protocol, termed the Long-Term Hypoxia (LTHY) time course, to recapitulate the gradual development of severe hypoxia seen in vivo to mimic conditions observed in primary tumors. Cells subjected to LTHY underwent a non-canonical epithelial to mesenchymal transition (EMT) based on miRNA and mRNA signatures as well as displayed EMT-like morphological changes. Concomitant to this, we report production of a novel truncated isoform of WT1 transcription factor (tWt1), a non-canonical EMT driver, with expression driven by a yet undescribed intronic promoter through hypoxia-responsive elements (HREs). We further demonstrated that tWt1 initiates translation from an intron-derived start codon, retains proper subcellular localization and DNA binding. A similar tWt1 is also expressed in LTHY-cultured human cancer cell lines as well as primary cancers and predicts long-term patient survival. Our study not only demonstrates the importance of culture conditions that better mimic those observed in primary cancers, especially with regards to hypoxia, but also identifies a novel isoform of WT1 which correlates with poor long-term survival in ovarian cancer.

Hsa_circ_0013561 promotes epithelial-mesenchymal transition and tumor progression by regulating ANXA2 via miR-23b-3p in ovarian cancer

AbstractOur preliminary experiment discovered that hsa_circ_0013561 was aberrantly expressed in OC. However, the underlying mechanism is unclear. The expression of hsa_circ_0013561 in OC cells and tissues was detected by RT-qPCR and fluorescence in situ hybridization. The effects of hsa_circ_0013561 on the proliferation and metastasis of OC were explored by functional experiments such as cell counting kit-8, transwell, and tumor xenograft models. To mechanistically understand the regulatory role of hsa_circ_0013561, bioinformatics analysis, Western blot, luciferase reporter assay, and a series of rescue experiments were applied. We found that the hsa_circ_0013561 expression was elevated in OC cells and tissues, and was correlated with metastasis formation. Downregulation of hsa_circ_0013561 suppressed the proliferation and migration of OC cells both in vitro and in vivo. Regarding the interactions of hsa_circ_0013561, the luciferase reporter assay verified that miR-23b-3p and Annexin A2 (ANXA2) were its downstream targets. MiR-23b-3p inhibition or ANXA2 overexpression reversed OC cell proliferation, migration, and epithelial-mesenchymal transition (EMT) post-hsa_circ_0013561 silencing. Moreover, ANXA2 overexpression also reversed OC cell migration, proliferation, and EMT after miR-23b-3p upregulation. Our data suggest that hsa_circ_0013561 increases the expression of ANXA2 by regulating miR-23b-3p competitively, resulting in EMT and metastasis of OC. Thus, hsa_circ_0013561 may serve as a novel oncogenic biomarker for OC progression.

IGF2BP2-modified circular RNA circCHD7 promotes endometrial cancer progression via stabilizing PDGFRB and activating JAK/STAT signaling pathway

AbstractCircular RNAs (circRNAs) represent a class of covalently closed, single-stranded RNAs and have been linked to cancer progression. N6-methyladenosine (m6A) methylation is a ubiquitous RNA modification in cancer cells. Increasing evidence suggests that m6A can mediate the effects of circRNAs in cancer biology. In contrast, the post-transcriptional systems of m6A and circRNA in the progression of endometrial cancer (EC) remain obscure. The current study identified a novel circRNA with m6A modification, hsa_circ_0084582 (circCHD7), which was upregulated in EC tissues. Functionally, circCHD7 was found to promote the proliferation of EC cells. Mechanistically, circCHD7 interacted with insulin-like growth factor 2 mRNA-binding protein (IGF2BP2) to amplify its enrichment. Moreover, circCHD7 increased the mRNA stability of platelet-derived growth factor receptor beta (PDGFRB) in an m6A-dependent manner, thereby enhancing its expression. In addition, the circCHD7/IGF2BP2/PDGFRB axis activated the JAK/STAT signaling pathway and promoted EC cell proliferation. In conclusion, these findings provide new insights into the regulation of circRNA-mediated m6A modification, and the new “circCHD7-PDGFRB” model of regulation offers new perspectives on circCHD7 as a potential target for EC therapy.

The involvement of lncRNA EMSLR in the disulfidptosis and progression of endometrial carcinoma

The incidence of endometrial cancer (EC) continues to rise. Disulfidptosis, a novel form of cell death, may represent a potential therapeutic target in EC. Through bioinformatic analysis of The Cancer Genome Atlas (TCGA) database, E2F1 mRNA-stabilizing lncRNA (EMSLR) was identified as a lncRNA related to disulfidptosis in EC. Functional assays, including cell proliferation and xenograft assays, demonstrated that knockdown of EMSLR significantly impeded EC cell proliferation, whereas overexpression of EMSLR promoted cell viability. Additionally, EMSLR was found to be associated with glucose uptake and NADPH production in glucose-restricted culture conditions. Moreover, downregulation of EMSLR markedly increased cell death and induced cytoskeletal collapse under glucose deprivation, as evidenced by F-actin and cell death staining. Notably, we observed a strong correlation between EMSLR and the c-MYC-GLUT1 pathway. Mechanistically, EMSLR was found to mediate the expression and nuclear translocation of c-MYC, thereby regulating the progression of EC and its associated disulfidptosis. In conclusion, EMSLR is identified as a disulfidptosis-related gene in endometrial cancer. Elucidating the function and molecular mechanisms of EMSLR in EC presents a promising avenue for therapeutic intervention in patients.

NrCAM secreted by endometrial stromal cells enhances the progestin sensitivity of endometrial cancer cells through epigenetic modulation of PRB

AbstractProgestin is one of the main hormone treatment regimens for early-stage estrogen receptor- and progesterone receptor (PR)-positive endometrial cancer (EC). However, the response rate of EC to progestins is unsatisfactory. Investigating the mechanisms related to progestin treatment could help improve treatment efficacy. Studies have demonstrated that normal endometrial stromal cells (ESCs) increase the inhibitory effect of progestin on EC cell proliferation via paracrine signaling, but the mechanisms involved remain unclear. In this study, we found that ESCs had different morphological features between progestin-sensitive and -insensitive EC tissues. ESCs presented typical decidualization changes in progestin-sensitive cases, while they remained slim in progestin-insensitive EC lesions, indicating no response. Furthermore, ESCs enhanced the inhibitory effect of medroxyprogesterone acetate (MPA) on EC cell proliferation by secreting neuron cell adhesion molecule (NrCAM). MPA treatment enhanced NrCAM secretion by ESCs. EC xenografts in BALB/C nude mice demonstrated that MPA combined with NrCAM had an increased tumor inhibitory effect compared with MPA or NrCAM alone. Mechanistically, MPA upregulated NrCAM expression in ESCs through PR. Specifically, NrCAM increased PR expression in EC cells through TET1-induced hydroxymethylation of the PRB gene promoter region. These findings indicate that NrCAM or NrCAM combined with progestins could be a new EC treatment.

IER3: exploring its dual function as an oncogene and tumor suppressor

Abstract The IER3 gene has a complex role in cancer biology, acting either as a tumor suppressor or an oncogene, depending on the cancer type. This duality underscores the complexity and importance of molecular pathways in modulating cancer behavior. Despite its significance in cancer development, there is a dearth of studies elucidating the exact mechanisms underlying IER3’s involvement in modulating cancer behavior. Here, utilizing cervical carcinoma and neuroblastoma (NB) cell lines as model systems we characterized the pathways that mediate the functional switch between the oncogenic and tumor suppressor roles of IER3. In HeLa cells, IER3 expression promotes an oncogenic program that includes immediate early response pathway genes such as EGR2, FOS, and JUN. However, in NB cells, IER3 suppresses the EGR2-dependent oncogenic program. This differential regulation of EGR2 by IER3 involves epigenetic modulation of the EGR2 promoter. IER3 dependent tumor suppressor pathway in NB cells relies on ADAM19 gene. Thus, our findings uncover the molecular pathways that dictate the context-dependent roles of IER3 in cancer, providing insights into its dual functionality in different cancer types.

CASC4/GOLM2 drives high grade serous carcinoma anoikis resistance through the recycling of EGFR

AbstractOvarian cancer is the deadliest gynecological malignancy, and accounts for over 150,000 deaths per year worldwide. The high grade serous ovarian carcinoma (HGSC) subtype accounts for almost 70% of ovarian cancers and is the deadliest. HGSC originates in the fimbria of the fallopian tube and disseminates through the peritoneal cavity. HGSC survival in peritoneal fluid requires cells to resist anoikis (anchorage-independent apoptosis). Most anoikis resistant mechanisms are dependent on microenvironment interactions with cell surface-associated proteins, such as integrins and receptor tyrosine kinases (RTKs). We previously identified the gene CASC4 as a driver of anoikis resistance. CASC4 is predicted to be a Golgi-associated protein that may regulate protein trafficking to the plasma membrane, but CASC4 is largely uncharacterized in literature; thus, we sought to determine how CASC4 confers anoikis resistance to HGSC cells. Mining of publicly available ovarian cancer datasets (TCGA) showed that CASC4 is associated with worse overall survival and increased resistance to platinum-based chemotherapies. For experiments, we cultured three human HGSC cell lines (PEO1, CaOV3, OVCAR3), and a murine HGSC cell line, (ID8) with shRNA-mediated CASC4 knockdowns (CASC4 KD) in suspension, to recapitulate the peritoneal fluid environment in vitro. CASC4 KD significantly inhibited cell proliferation and colony formation ability, and increased apoptosis. A Reverse Phase Protein Assay (RPPA) showed that CASC4 KD resulted in a broad re-programming of membrane-associated proteins. Specifically, CASC4 KD led to decreased protein levels of the RTK Epidermal Growth Factor Receptor (EGFR), an initiator of several oncogenic signaling pathways, leading us to hypothesize that CASC4 drives HGSC survival through mediating recycling and trafficking of EGFR. Indeed, loss of CASC4 led to a decrease in both EGFR membrane localization, reduced turnover of EGFR, and increased EGFR ubiquitination. Moreover, a syngeneic ID8 murine model of ovarian cancer showed that knocking down CASC4 leads to decreased tumor burden and dissemination.

Targeting SOD1 via RNAi with PEGylated graphene oxide nanoparticles in platinum-resistant ovarian cancer

AbstractAcquired platinum resistance poses a significant therapeutic impediment to ovarian cancer patient care, accounting for more than 200,000 deaths annually worldwide. We previously identified that overexpression of the antioxidant superoxide dismutase 1 (SOD1) in ovarian cancer is associated with a platinum-resistant phenotype via conferring oxidative stress resistance against platinum compounds. We further demonstrated that enzymatic inhibition using small-molecule inhibitors or silencing of SOD1 via RNA interference (RNAi) increased cisplatin sensitivity and potency in vitro. We launched this study to explore the potential therapeutic applications of SOD1 silencing in vivo in order to reverse cisplatin resistance using a graphene-based siRNA delivery platform. PEGylated graphene oxide (GO) polyethyleneimine (GOPEI-mPEG) nanoparticle was complexed with SOD1 siRNA. GOPEI-mPEG-siSOD1 exhibited high biocompatibility, siRNA loading capacity, and serum stability, and showed potent downregulation of SOD1 mRNA and protein levels. We further observed that cisplatin and PEI elicited mitochondrial dysfunction and transcriptionally activated the mitochondrial unfolded protein response (UPRmt) used as a reporter for their respective cytotoxicities. SOD1 silencing was found to augment cisplatin-induced cytotoxicity resulting in considerable tumour growth inhibition in cisplatin-sensitive A2780 and cisplatin-resistant A2780DDP subcutaneous mouse xenografts. Our study highlights the potential therapeutic applicability of RNAi-mediated targeting of SOD1 as a chemosensitizer for platinum-resistant ovarian cancers.

Modified hTERT promoters-driven purine nucleoside phosphorylase-gene therapy in association with chemo- and targeted therapy in the context of ovarian cancer

Ovarian cancer has the highest mortality-to-incidence ratio among gynecologic cancers worldwide. E.coli Purine Nucleoside Phosphorylase-based gene-directed enzyme prodrug therapy (PNP-GDEPT) offers a promising alternative for the treatment of solid tumors. This study proposes an original ovarian cancer treatment through the use of two recently developed modified hTERT promoters: the mutated hTERT (hTERTm) and the chimeric hTERT-CMV. Four plasmids were engineered to investigate the effects of cancer-specific PNP gene expression: pCMV-PNP, phTERT-PNP, phTERTm-PNP, and phTERT-CMV-PNP. The cationic lipid formulation BSV163/DOPE was employed to transfect PNP-coding plasmids into cisplatin-sensitive ovarian cancer cells and their resistant counterparts. hTERT-driven PNP-GDEPT selectively reduced cancer cell viability while sparing primary human fibroblasts. In addition, combining PNP-GDEPT with either cisplatin or olaparib further enhanced anticancer effects on cell viability and apoptosis. However, no combined effects were observed for the concurrent use of cisplatin and olaparib, with or without PNP-GDEPT. Our results demonstrate that targeted PNP-GDEPT has the potential to enhance the efficacy of chemotherapy and targeted therapy against ovarian cancer while minimizing side effects on healthy cells. This treatment is effective irrespective of cisplatin resistance status and warrants further investigation.

ECT2 promotes malignant phenotypes through the activation of the AKT/mTOR pathway and cisplatin resistance in cervical cancer

Epithelial cell transforming sequence 2 (ECT2) is expressed at high levels in various malignancies and contributes to malignant phenotypes in cancers. However, ECT2 is still not fully understood regarding its function and carcinogenic mechanism in cervical cancer. This research indicated that ECT2 expression was elevated in cervical cancer based on bioinformatics analysis and clinical specimens. Experiments in vitro and in vivo confirmed that ECT2 knockdown could suppress the proliferation and metastasis of cervical carcinoma cells. In addition, we found that silencing ECT2 could enhance the sensitivity to cisplatin and promote cell apoptosis. Mechanistically, we observed that ECT2 knockdown could inhibit the AKT/mTOR pathway and activate apoptosis, while ECT2 overexpression induced the opposite effect. The relationship between ECT2 and AKT was further confirmed by immunoprecipitation and rescue experiments. We found that the ECT2 and AKT could interact to form a complex, and knockdown AKT could offset all of the effects induced by ECT2. Our study emphasized the key point of ECT2 in the reversal of cisplatin resistance, and ECT2 could become a potential therapeutic target in cervical cancer.

MiR-181c sensitizes ovarian cancer cells to paclitaxel by targeting GRP78 through the PI3K/Akt pathway

Primary cytoreductive surgery with platinum-taxane-based chemotherapy is the standard treatment for ovarian cancer (OC) patients; however, resistance to chemotherapy is a contributing factor to OC mortality. Paclitaxel (PTX), the most widely used taxane, has become the first-line drug against OC. The molecular mechanism of PTX resistance is different from that of platinum-based agents and is still not completely elucidated. Our previous study showed that glucose-regulated protein 78 (GRP78) is involved in the resistance of OC cells to PTX. However, little is known regarding endogenous inhibitors of this gene. MicroRNAs (miRNAs) play critical roles in the regulation of gene expression; therefore, we sought to identify miRNA(s) with potential to target GRP78 under the hypothesis that miRNA(s) could serve as potential therapeutic targets. Here, we show that miR-181c, predicted to target GRP78, was downregulated in PTX-resistant OC cells and tissues. MiR-181c downregulated GRP78 expression and induced apoptosis by directly targeting its 3'-untranslated region (UTR). Overexpression of miR-181c sensitized resistant OC to PTX by inhibiting the PI3K/Akt pathway in vitro and in vivo. Taken together, our findings indicate that the delivery of miR-181c can efficiently suppress GRP78 expression and GRP78-mediated PTX resistance in OC and suggest that this strategy has therapeutic potential.

LncRNA MAFG-AS1 promotes the malignant phenotype of ovarian cancer by upregulating NFKB1-dependent IGF1

Long non-coding RNAs (lncRNAs) were recently recognized to vitally function in a variety of cancer cellular events, including epithelial-mesenchymal transition (EMT), invasion, and migration, particularly in ovarian cancer (OC). Herein, we sought to investigate the potential role of MAFG-AS1 in the malignant behaviors of OC cells. The binding affinity between MAFG-AS1, miR-339-5p, NFKB1 or IGF1 was characterized so as to identify the underlying mechanism of corresponding their interactions. We conducted MAFG-AS1 overexpression or knockdown along with NFKB1 and IGF1 silencing to examine their effects on the EMT, migration, and invasion of OC cells. Tumors were xenografted in nude mice to validate the in vitro findings. Our data showed significantly high expression pattern of MAFG-AS1 in the OC tissues and cells. Further mechanistic investigations revealed that MAFG-AS1 upregulated the IGF1 expression pattern through recruitment of NFKB1, whereas MAFG-AS1 upregulated the NFKB1 expression pattern through binding to miR-339-5p. Thus, MAFG-AS1 overexpression accelerated the EMT, invasion, and migration of OC cells, which could be annulled by silencing of IGF1 or NFKB1. Besides, our in vitro findings were successfully recapitulated in the xenograft mice. These results determined that MAFG-AS1 stimulated the OC malignant progression by upregulating the NFKB1-mediated IGF1 via miR-339-5p, thus highlighting a novel potential therapeutic target against OC.

Proof of principle study of sequential combination atezolizumab and Vigil in relapsed ovarian cancer

Vigil® is a personalized vaccine that enhances tumor neoantigen expression. We investigated for the first time safety and efficacy of Vigil in combination with atezolizumab in relapsed ovarian cancer (OC) patients. This is a randomized, Phase 1 study of Vigil, an autologous tumor tissue transfected vaccine encoding for GMCSF and bi-shRNA-furin thereby creating enhanced immune activation and TGFβ expression control. Part 1 is a safety assessment of Vigil (1 × 10e7 cells/mL/21 days) plus atezolizumab (1200 mg/21 days). Part 2 is a randomized study of Vigil first (Vigil-1st) or atezolizumab first (Atezo-1st) for two cycles followed by the combination of both agents. The primary endpoint of the study was the determination of safety. Twenty-four patients were enrolled in the study; three patients to Part 1 and 21 to Part 2. Patients in Part 1 completed combination therapy without dose-limiting toxicity justifying expansion to Part 2. Twenty-one patients were randomized (1:1) to Part 2 to Vigil-1st (n = 11) or Atezo-1st (n = 10). Grade 3/4 treatment-related adverse events of Atezo-1st vs. Vigil-1st were 17.2% vs. 5.1%. Median overall survival (OS) was not reached (NR) (Vigil-1st) vs. 10.8 months (Atezo-1st) (hazard ratio [HR] 0.33). The exploratory subset analysis of BRCA

FAP is critical for ovarian cancer cell survival by sustaining NF-κB activation through recruitment of PRKDC in lipid rafts

Fibroblast activation protein (FAP) is tumor-specific and plays an important role in tumorigenecity. However, agents against its enzymatic activity or extracellular presence were unsuccessful in the clinic for undefined reasons. Here we show that FAP expression is higher in advanced ovarian cancer and is only detected in invasive ovarian cancer cells. Silencing FAP induces apoptosis and FAP's enzymatic activity is dispensable for cell survival. To elucidate the cause of apoptosis, we find that NF-κB activity is diminished when FAP is depleted and BIRC5 (survivin) acts downstream of FAP-NF-κB axis to promote cell survival. To uncover the link between FAP and NF-κB activation, we reveal that PRKDC (DNA-PK, DNA-dependent protein kinase) forms complex with FAP and is required for NF-κB activation and cell survival. Remarkably, FAP-PRKDC interaction occurs only in lipid rafts, and depleting FAP prevents lipid raft localization of PRKDC. Given the known ability of PRKDC to direct NF-κB activation, these results suggest that FAP recruits PRKDC in lipid rafts for NF-κB activation. FAP's non-enzymatic role and functioning from lipid rafts for cell survival also offer an explanation on the failure of past FAP-targeted therapies. Finally, we demonstrate that EpCAM aptamer-delivered FAP siRNA impeded intraperitoneal xenograft development of ovary tumors.

miR-219-5p attenuates cisplatin resistance of ovarian cancer by inactivating Wnt/β-catenin signaling and autophagy via targeting HMGA2

Our previous study confirmed that miR-219-5p inhibits the progression of ovarian cancer (OC) by targeting high mobility group AT-hook 2 (HMGA2), while the role of miR-219-5p on the chemoresistance of OC is unclear. HMGA2 and miR-219-5p expression in OC tumors and various types of OC cells were determined by reverse transcription-quantitative PCR (RT-qPCR) and western blotting. The miRNA profiles in A2780 and cisplatin-resistant A2780 cells were investigated via bulk miRNA sequencing, and the interactions of miR-219-5p and HMGA2 were determined by luciferase reporter activity assay. Cell function was verified through Cell Counting Kit-8, invasion assay, wound-healing, and TUNEL assays. HMGA2 level is highly expressed in cisplatin-resistant OC cell lines compared to normal OC cells, while the expression trend of miR-219-5p is the opposite. In addition, we found that miR-219-5p is one of the miRNAs that have the most significant reduction in levels in the cisplatin-resistant A2780/DDP cell line compared to A2780 cells. Then, we reveal that miR-219-5p directly targets HMGA2 in cisplatin-resistant OC cells, and upregulation of miR-219-5p significantly reduces the resistance of OC cells to cisplatin both in vitro and in vivo. Finally, our results suggest that Wnt/β-catenin signaling and autophagy pathway is involved in the role of miR-219-5p/HMGA2 on resistance of OC cells to cisplatin via gain-of-function experiments. Collectively, the present study shows that miR-219-5p decreases the resistance of OC cells to cisplatin via Wnt/β-catenin signaling and autophagy by regulating HMGA2, which provides a feasible solution for the resistance of OC to chemotherapy.

A targetable MYBL2-ATAD2 axis governs cell proliferation in ovarian cancer

The chromatin-modifying enzyme ATAD2 confers oncogenic competence and proliferative advantage in malignances. We previously identified ATAD2 as a marker and driver of cell proliferation in ovarian cancer (OC); however, the mechanisms whereby ATAD2 is regulated and involved in cell proliferation are still unclear. Here, we disclose that ATAD2 displays a classical G2/M gene signature, functioning to facilitate mitotic progression. ATAD2 ablation caused mitotic arrest and decreased the ability of OC cells to pass through nocodazole-arrested mitosis. ChIP-seq data analyses demonstrated that DREAM and MYBL2-MuvB (MMB), two switchable MuvB-based complexes, bind the CHR elements in the ATAD2 promoter, representing a typical feature and principle mechanism of the periodic regulation of G2/M genes. As a downstream target of MYBL2, ATAD2 deletion significantly impaired MYBL2-driven cell proliferation. Intriguingly, ATAD2 silencing also fed back to destabilize the MYBL2 protein. The significant coexpression of MYBL2 and ATAD2 at both the bulk tissue and single-cell levels highlights the existence of the MYBL2-ATAD2 signaling in OC patients. This signaling is activated during tumorigenesis and correlated with TP53 mutation, and its hyperactivation was found especially in high-grade serous and drug-resistant OCs. Disrupting this signaling by CRISPR/Cas9-mediated ATAD2 ablation inhibited the in vivo growth of OC in a subcutaneous tumor xenograft mouse model, while pharmacologically targeting this signaling with an ATAD2 inhibitor demonstrated high therapeutic efficacy in both drug-sensitive and drug-resistant OC cells. Collectively, we identified a novel MYBL2-ATAD2 proliferative signaling axis and highlighted its potential application in developing new therapeutic strategies, especially for high-grade serous and drug-resistant OCs.

Extracellular vesicles-encapsulated microRNA-10a-5p shed from cancer-associated fibroblast facilitates cervical squamous cell carcinoma cell angiogenesis and tumorigenicity via Hedgehog signaling pathway

Cancer-associated fibroblast (CAF) secretes extracellular vesicle (EV)-encapsulated microRNAs (miRNAs) which have been underlined great promise for therapeutic target for diseases and cancers. Our study aimed to explore the role of EV-encapsulated miR-10a-5p from CAFs in angiogenesis in cervical cancer. Expression of miR-10a-5p in clinical samples of cervical cancer and cancer cells was detected by in situ hybridization and RT-qPCR. Results demonstrated that miR-10a-5p expression was upregulated in both cancer tissues and cells. CAFs and normal fibroblasts (NFs) from cervical cancer patient tissues were characterized under transmission electron microscopy, followed by EV isolation from CAFs. The EVs labeled with PKH67 were cultured with cervical squamous cell carcinoma (CSCC) cell line (SiHa) and HUVECs. Data indicated that CAF-EVs were internalized by cancer cells and promoted cell proliferation and tube formation. CAF-EVs then were transfected with miR-10a-5p inhibitor and then injected into nude mice. While injection of CAF-EVs promoted tumor growth and increased VEGFR and CD31 expression level, miR-10a-5p inhibitor-treated CAF-EVs resulted in decreased tumor volume and amount of vessel around tumor. Of note, dual-luciferase reporter gene assay and bioinformatic analysis indicated TBX5 as a target gene of miR-10a-5p. Moreover, EV-derived miR-10a-5p promoted angiogenesis in vivo and in vitro through activation of Hedgehog signaling via downregulation of TBX5. Taken altogether, miR-10a-5p in CAF-EVs promoted CSCC cell angiogenesis and tumorigenicity via activation of Hh signaling by inhibition of TBX5, providing insight into novel treatment based on miR-10a-5p against CSCC.

Non-homologous dsODN increases the mutagenic effects of CRISPR-Cas9 to disrupt oncogene E7 in HPV positive cells

Genome editing tools targeting high-risk human papillomavirus (HPV) oncogene could be a promising therapeutic strategy for the treatment of HPV-related cervical cancer. We aimed to improve the editing efficiency and detect off-target effects concurrently for the clinical translation strategy by using CRISPR-Cas9 system co-transfected with 34nt non-homologous double-stranded oligodeoxynucleotide (dsODN). We firstly tested this strategy on targeting the Green Fluorescent Protein (GFP) gene, of which the expression is easily observed. Our results showed that the GFP+ cells were significantly decreased when using GFP-sgRNAs with dsODN, compared to using GFP-sgRNAs without donors. By PCR and Sanger sequencing, we verified the dsODN integration into the break sites of the GFP gene. And by amplicon sequencing, we observed that the indels% of the targeted site on the GFP gene was increased by using GFP-sgRNAs with dsODN. Next, we went on to target the HPV18 E7 oncogene by using single E7-sgRNA and multiplexed E7-sgRNAs respectively. Whenever using single sgRNA or multiplexed sgRNAs, the mRNA expression of HPV18 E7 oncogene was significantly decreased when adding E7-sgRNAs with dsODN, compared to E7-sgRNAs without donor. And the indels% of the targeted sites on the HPV18 E7 gene was markedly increased by adding dsODN with E7-sgRNAs. Finally, we performed GUIDE-Seq to verify that the integrated dsODN could serve as the marker to detect off-target effects in using single or multiplexed two sgRNAs. And we detected fewer on-target reads and off-target sites in multiplexes compared to the single sgRNAs when targeting the GFP and the HPV18 E7 genes. Together, CRISPR-Cas9 system co-transfected with 34nt dsODN concurrently improved the editing efficiency and monitored off-target effects, which might provide new insights in the treatment of HPV infections and related cervical cancer.

Pigment epithelium-derived factor, an anti-VEGF factor, delays ovarian cancer progression by alleviating polarization of tumor-associated macrophages

Ovarian cancer (OC) is one of the most dangerous gynecological malignancies with no effective treatment so far. Pigment epithelium-derived factor (PEDF) has been reported to have ideal anti-tumor effects, but its relationship with the regulation of tumor-associated macrophage polarization is currently unclear. In this study, the mRNA expression of PEDF and macrophage markers were determined in OC tissues from clinic patients and five OC (A2780, SKOV3, CAOV3, OVCAR3, and OVCA433) cell lines through quantitative reverse transcription PCR. Afterwards, tumor growth, cell proliferation and apoptosis, and macrophage polarization in OC tumor-bearing mice with PEDF overexpression were recorded and investigated. Finally, the polarization of macrophages was explored in the presence of lentiviral PEDF overexpression, adipose triglyceride lipase (ATGL) and laminin receptor (LR) knockdown, and mitogen-activated protein kinase (MAPK) pathway inhibition. Our results suggest that PEDF mRNA level is significantly decreased in OC tissues and cells and has a significant negative correlation with OC progression and the level of tumor-related macrophage markers. Furthermore, OC tumors overexpressing PEDF show suppressed growth viability and increased apoptosis rate. The fluorescence activated cell sorting (FACS) analysis reveals that PEDF can promote macrophage polarization in OC tumors towards M1 subtype. Mechanistically, we found that ATGL and extracellular-regulated kinase 1/2 (ERK1/2) signaling are involved in the regulation of macrophage polarization in OC tumors by PEDF. Taken together, these data indicate that the role of PEDF in regulating the polarization of tumor-associated macrophages may make it a potential therapeutic strategy for the treatment of OC in the future.

Long non-coding RNA HAND2-AS1 delays cervical cancer progression via its regulation on the microRNA-21-5p/TIMP3/VEGFA axis

Cervical cancer is a common cause of cancer-related mortality in women. Mounting evidence suggests that long non-coding RNAs (lncRNAs) function vitally in many cancers. In this study, we discovered that the regulation of the heart and neural crest derivatives expressed 2-antisense RNA 1 (HAND2-AS1) in cervical cancer. RT-qPCR was conducted to detect the expression of HAND2-AS1 and microRNA-21-5p (miR-21-5p). The relationship of HAND2-AS1 and miR-21-5p was identified by dual-luciferase reporter gene assay. The roles of HAND2-AS1, miR-21-5p and tissue inhibitor of metalloproteinases-3 (TIMP3) in cervical cancer were accessed via gain- and loss-of-function approaches. The expression of related proteins in the vascular endothelial growth factor A (VEGFA) signaling pathway was detected through Western blot analysis. Finally, xenografts of cervical cancer in nude mice were established to assess the effect of HAND2-AS1 on tumorigenesis in vivo. HAND2-AS1 and TIMP3 were downregulated in cervical cancer, which were identified to be associated with a poor prognosis of patients with cervical cancer. Moreover, HAND2-AS1 was upregulated the expression of TIMP3 through competitively binding to miR-21-5p. Overexpressed HAND2-AS1 or downregulated miR-21-5p inhibited cell proliferation, migration, and invasion while promoting cell apoptosis, in association with increased expression of proteins in VEGFA signaling pathway. These changes were reversed by silencing of TIMP3. Overexpressed HAND2-AS1 reduced the tumor formation ability in nude mice. In summary, HAND2-AS1 may exert inhibitory effects on cervical cancer cell growth and cervical cancer development through its regulation on the miR-21-5p/TIMP3/VEGFA axis. This highlights that HAND2-AS1 may serve as a potential target for cervical cancer diagnosis and treatment.

Flotillin-1 palmitoylation turnover by APT-1 and ZDHHC-19 promotes cervical cancer progression by suppressing IGF-1 receptor desensitization and proteostasis

We have shown that insulin-like growth factor-1 (IGF-1) induces palmitoylation turnover of Flotillin-1 (Flot-1) in the plasma membrane (PM) for cell proliferation, after IGF-1 receptor (IGF-1R) signaling activation. However, the enzymes responsible for the turnover have not been identified. Herein, we show that acyl protein thioesterases-1 (APT-1) catalyzes Flot-1 depalmitoylation, and zinc finger DHHC domain-containing protein palmitoyltransferase-19 (ZDHHC-19) repalmitoylation of the depalmitoylated Flot-1 for the turnover in cervical cancer cells. The turnover prevented desensitization of IGF-1R via endocytosis and lysosomal degradation, thereby exerting excessive IGF-1R activation in cervical cancer cells. FLOT1, LYPLA1 and ZDHHC19 were highly expressed, and epithelial-to-mesenchymal transition (EMT)-inducing TIAM1 and GREM1 coordinately upregulated in malignant cervical cancer tissues. And blocking the turnover suppressed the EMT, migration, and invasion of cervical cancer cells. Our study identifies the specific enzymes regulating Flot-1 palmitoylation turnover, and reveals a novel regulatory mechanism of IGF-1-mediated cervical cancer progression.

Editorial Expression of Concern: Herpes simplex virus thymidine kinase/ganciclovir–induced cell death is enhanced by co-expression of caspase-3 in ovarian carcinoma cells

Tumor-derived ARHGAP35 mutations enhance the Gα13-Rho signaling axis in human endometrial cancer

Dysregulated G protein-coupled receptor signaling is involved in the formation and progression of human cancers. The heterotrimeric G protein Gα

Human umbilical cord blood mesenchymal stem cells-derived exosomal microRNA-503-3p inhibits progression of human endometrial cancer cells through downregulating MEST

Endometrial cancer (EC) is a group of epithelial malignant tumors that occur in the endometrium. The specific pathogenesis is not revealed, hence, the goal of this study was to investigate the influence of human umbilical cord blood mesenchymal stem cells (hUMSCs)-derived exosomal microRNA-503-3p (miR-503-3p) on human EC cells by mediating mesoderm-specific transcript (MEST). The binding relationship between MiR-503-3p and MEST was searched. HUMSCs were collected and exosomes (Exos) were isolated and identified. Human EC cell lines HEC-1B and RL95-2 were transfected with elevated miR-503-3p or silenced MEST vector or co-cultured with Exos to figure their roles in biological functions of EC cells. The in vitro effect of miR-503-3p, MEST, and Exos on EC cells was further verified in vivo. MEST was a target of miR-503-3p. Overexpression of miR-503-3p or reduction of MEST suppressed the biological functions of EC cells. Enhanced MEST expression mitigated the role of upregulated miR-503-3p on the growth of EC cells. HUMSCs-derived Exos suppressed EC cell growth, upregulated miR-503-3p-modified HUMSCs-derived Exos had a more obvious inhibitory effect on EC cell growth. The anti-tumor effect of elevated miR-503-3p, silenced MEST, and HUMSCs-derived Exos were verified in nude mice. This study highlights that hUMSCs-derived exosomal miR-503-3p inhibits EC development by suppressing MEST, which is of great benefit to EC therapy.

An organoid-based drug screening identified a menin-MLL inhibitor for endometrial cancer through regulating the HIF pathway

Tumor organoids recapitulate pathological properties and would serve as an excellent ex vivo model for drug discovery. Here, we performed an unbiased drug screening on drivers-defined tumor organoids from mouse endometrial cancer, the most prevalent gynecological malignancy in human, with a small molecule library targeting epigenetic factors. Among them, menin-MLL inhibitors MI-136 and MI-463 scored. The therapeutic capacity of MI-136 was further validated in tumor organoids in vitro and an orthotopic model in vivo. CRISPR/cas9-mediated mutations of major components of the menin-MLL complex, Men1, Kmt2a and Ash2l, inhibited the growth of tumor organoids, suggesting that the complex was the target of MI-136. Transcriptome analysis showed that the hypoxia-inducible factor (HIF) pathway was the most significantly downregulated pathway by MI-136 treatment. Consistently, Men1, Kmt2a, and Ash2l knockout also repressed the expressions of the HIF target genes. Loss of Hif1a or Hif1b partially phenocopied the inhibition of the menin-MLL complex by MI-136 or mutations in term of tumor organoid growth. Further, we found that MEN1 was upregulated in human endometrial cancers, which were tightly correlated with the expression levels of HIF1A, and associated with poor prognosis. Importantly, MI-136 also significantly inhibited the growth of endometrial cancer organoids derived from patients. Thus, our study identified MI-136 as a potential inhibitor for endometrial cancer through regulating the HIF pathway, a novel molecular mechanism distinguished from those in AML and prostate cancer.

TFF3 sensitizes cervical carcinoma cells to cisplatin toxicity by binding to IGF2R

Cisplatin resistance causes ineffectiveness of cisplatin-based treatment for cervical carcinoma. The combination of cisplatin and other chemotherapeutic drugs is an available strategy to overcome this problem. However, chemotherapeutic drugs combined with cisplatin may show tissue toxicity and systemic side effects. Thus, there is a great need of seeking effective substitutes for these chemotherapeutic drugs to improve combination therapy. Here, we found that inactivating IL-6/JAK2/STAT3 signaling pathway sensitized carcinoma cells to cisplatin toxicity by increasing cisplatin accumulation, impairing DNA damage repair, and inhibiting the initiation and development of autophagy, which subsequently caused the increases in DNA damage levels and apoptosis rates in cisplatin-treated cells. We predicted that TFF3 negatively regulated transduction in the IL-6/JAK2/STAT3 pathway based on in silico analysis of the differentially expressed genes (DEGs) between highly trefoil factor 3(TFF3)-encoding mRNA-expressing carcinoma tissues and low-expressing counterparts, and experimentally determined that both ectopic expression of TFF3-encoding gene and TFF3 administration inhibited IL-6-induced STAT3 activation in carcinoma cells. Mechanistically, upon binding to IGF2R, TFF3 stabilized IGF2R by inhibiting the ubiquitin-proteasome degradation pathway to inactivate Akt and thereby STAT3. Moreover, we discovered that TFF3 administration antagonized protective effects of IL-6 stimulation against tumor-killing capacity of cisplatin. Based on these findings, we consider that TFF3 may be employed as a cisplatin sensitizer and have advantages over traditional chemotherapeutic drugs in cisplatin-based combination therapy, since it is a naturally occurring protein in cervical tissue.

Therapeutic targeting of the tryptophan-kynurenine-aryl hydrocarbon receptor pathway with apigenin in MED12-mutant leiomyoma cells

Approximately 77.4% of uterine leiomyomas carry MED12 gene mutations (mut-MED12), which are specifically associated with strikingly upregulated expression and activity of the tryptophan 2,3-dioxygenase (TDO2) enzyme, leading to increased conversion of tryptophan to kynureine. Kynurenine increases leiomyoma cell survival by activating the aryl hydrocarbon receptor (AHR). We used a leiomyoma-relevant model, in which a MED12 Gly44 mutation was knocked in by CRISPR in a human uterine myometrial smooth muscle (UtSM) cell line, in addition to primary leiomyoma cells from 26 patients to ascertain the mechanisms responsible for therapeutic effects of apigenin, a natural compound. Apigenin treatment significantly decreased cell viability, inhibited cell cycle progression, and induced apoptosis preferentially in mut-MED12 versus wild-type primary leiomyoma and UtSM cells. Apigenin not only blocked AHR action but also decreased TDO2 expression and kynurenine production, preferentially in mut-MED12 cells. Apigenin did not alter TDO2 enzyme activity. TNF and IL-1β, cytokines upregulated in leiomyoma, strikingly induced TDO2 expression levels via activating the NF-κB and JNK pathways, which were abolished by apigenin. Apigenin or a TDO2 inhibitor decreased UtSM cell viability induced by TNF/IL-1β. We provide proof-of-principle evidence that apigenin is a potential therapeutic agent for mut-MED12 leiomyomas.

TMTC1 promotes invasiveness of ovarian cancer cells through integrins β1 and β4

AbstractOvarian cancer is the most lethal gynecological malignancy and is characterized by peritoneal disseminated metastasis. Although O-mannosyltransferase TMTC1 is highly expressed by ovarian cancer, its pathophysiological role in ovarian cancer remains unclear. Here, immunohistochemistry showed that TMTC1 was overexpressed in ovarian cancer tissues compared with adjacent normal ovarian tissues, and high TMTC1 expression was associated with poor prognosis in patients with ovarian cancer. Silencing TMTC1 reduced ovarian cancer cell viability, migration, and invasion in vitro, as well as suppressed peritoneal tumor growth and metastasis in vivo. Moreover, TMTC1 knockdown reduced cell-laminin adhesion, which was associated with the decreased phosphorylation of FAK at pY397. Conversely, TMTC1 overexpression promoted these malignant properties in ovarian cancer cells. Glycoproteomic analysis and Concanavalin A (ConA) pull-down assays showed that integrins β1 and β4 were novel O-mannosylated protein substrates of TMTC1. Furthermore, TMTC1-mediated cell migration and invasion were significantly reversed by siRNA-mediated knockdown of integrin β1 or β4. Collectively, these results suggest that TMTC1-mediated invasive behaviors are primarily through integrins β1 and β4 and that TMTC1 is a potential therapeutic target for ovarian cancer.

Cell-intrinsic platinum response and associated genetic and gene expression signatures in ovarian cancer

Abstract Ovarian cancers are still largely treated with platinum-based chemotherapy as the standard of care, yet few biomarkers of clinical response have had an impact on clinical decision making. Previous work has relied on poor models of the most common subtypes of epithelial ovarian cancers and necessitates a careful examination of the most suitable in vitro models. We performed extensive drug dose response assays and gene expression profiling on 36 ovarian cancer cell lines across over seven subtypes. This is the largest quantitative database of quantitative cisplatin and carboplatin response in ovarian cancer cell lines. Our results demonstrate that cell lines largely fall either well above or below the clinical maximally achievable dose (Cmax) of each compound. We performed differential expression analysis for high-grade serous ovarian carcinoma cell lines. Further, we generated two platinum-resistant derivatives each for OVCAR3 and OVCAR4. Combined with clinically resistant PEO1/PEO4/PEO6 and PEA1/PEA2 isogenic models, we performed differential expression analysis for seven platinum-resistant isogenic pairs. Common themes in differential expression were innate immunity/STAT activation, epithelial-to-mesenchymal transition (EMT) and stemness, and platinum influx/efflux regulators. We also performed copy number signature analysis and orthogonal measures of homologous recombination deficiency (HRD) scar scores and copy number burden, which is the first report to our knowledge applying field-standard copy number signatures to ovarian cancer cell lines. We also examined markers and functional readouts of stemness that revealed that cell lines are poor models for examination of stemness contributions to platinum resistance, suggesting that this is a transient state. Overall, this study serves as a resource to determine the best cell lines to utilize for ovarian cancer research on certain subtypes and platinum response studies, as well as sparks new hypotheses for future study in ovarian cancer.

HPV E6/E7: insights into their regulatory role and mechanism in signaling pathways in HPV-associated tumor

Human papillomavirus (HPV) is a class of envelope-free double-stranded DNA virus. HPV infection has been strongly associated with the development of many malignancies, such as cervical, anal and oral cancers. The viral oncoproteins E6 and E7 perform central roles on HPV-induced carcinogenic processes. During tumor development, it usually goes along with the activation of abnormal signaling pathways. E6 and E7 induces changes in cell cycle, proliferation, invasion, metastasis and other biological behaviors by affecting downstream tumor-related signaling pathways, thus promoting malignant transformation of cells and ultimately leading to tumorigenesis and progression. Here, we summarized that E6 and E7 proteins promote HPV-associated tumorigenesis and development by regulating the activation of various tumor-related signaling pathways, for example, the Wnt/β-catenin, PI3K/Akt, and NF-kB signaling pathway. We also discussed the importance of HPV-encoded E6 and E7 and their regulated tumor-related signaling pathways for the diagnosis and effective treatment of HPV-associated tumors.

Dual-inhibition of NAMPT and PAK4 induces anti-tumor effects in 3D-spheroids model of platinum-resistant ovarian cancer

Abstract Ovarian cancer follows a characteristic progression pattern, forming multiple tumor masses enriched with cancer stem cells (CSCs) within the abdomen. Most patients develop resistance to standard platinum-based drugs, necessitating better treatment approaches. Targeting CSCs by inhibiting NAD+ synthesis has been previously explored. Nicotinamide phosphoribosyltransferase (NAMPT), which is the rate limiting enzyme in the salvage pathway for NAD+ synthesis is an attractive drug target in this pathway. KPT-9274 is an innovative drug targeting both NAMPT and p21 activated kinase 4 (PAK4). However, its effectiveness against ovarian cancer has not been validated. Here, we show the efficacy and mechanisms of KPT-9274 in treating 3D-cultured spheroids that are resistant to platinum-based drugs. In these spheroids, KPT-9274 not only inhibited NAD+ production in NAMPT-dependent cell lines, but also suppressed NADPH and ATP production, indicating reduced mitochondrial function. It also downregulated of inflammation and DNA repair-related genes. Moreover, the compound reduced PAK4 activity by altering its mostly cytoplasmic localization, leading to NAD+-dependent decreases in phosphorylation of S6 Ribosomal protein, AKT, and β-Catenin in the cytoplasm. These findings suggest that KPT-9274 could be a promising treatment for ovarian cancer patients who are resistant to platinum drugs, emphasizing the need for precision medicine to identify the specific NAD+ producing pathway that a tumor relies upon before treatment.

Boosting cytotoxicity of adoptive allogeneic NK cell therapy with an oncolytic adenovirus encoding a human vIL-2 cytokine for the treatment of human ovarian cancer

AbstractDespite good results in the treatment of hematological malignancies, Natural killer (NK) cells have shown limited effectiveness in solid tumors, such as ovarian cancer (OvCa). Here, we assessed the potential of an oncolytic adenovirus expressing a variant interleukin-2 (vIL-2) cytokine, Ad5/3-E2F-d24-vIL2 (vIL-2 virus), also known as TILT-452, to enhance NK cell therapy efficacy in human OvCa ex vivo. Human OvCa surgical specimens were processed into single-cell suspensions and NK cells were expanded from healthy blood donors. OvCa sample digests were co-cultured ex vivo with NK cells and vIL-2 virus and cancer cell killing potential assessed in real time through cell impedance measurement. Proposed therapeutic combination was evaluated in vivo with an OvCa patient-derived xenograft (PDX) in mice. Addition of vIL-2 virus significantly enhanced NK cell therapy killing potential in treated OvCa co-cultures. Similarly, vIL-2 virus in combination with NK cell therapy promoted the best in vivo OvCa tumor control. Mechanistically, vIL-2 virus induced higher percentages of granzyme B in NK cells, and CD8+ T cells, while T regulatory cell proportions remained comparable to NK cell monotherapy in vivo. Ad5/3-E2F-d24-vIL2 virus treatment represents a promising strategy to boost adoptive NK cell therapeutic effect in human OvCa.

Network based analysis identifies TP53m-BRCA1/2wt-homologous recombination proficient (HRP) population with enhanced susceptibility to Vigil immunotherapy

AbstractThus far immunotherapy has had limited impact on ovarian cancer. Vigil (a novel DNA-based multifunctional immune-therapeutic) has shown clinical benefit to prolong relapse-free survival (RFS) and overall survival (OS) in the BRCA wild type and HRP populations. We further analyzed molecular signals related to sensitivity of Vigil treatment. Tissue from patients enrolled in the randomized double-blind trial of Vigil vs. placebo as maintenance in frontline management of advanced resectable ovarian cancer underwent DNA polymorphism analysis. Data was generated from a 981 gene panel to determine the tumor mutation burden and classify variants using Ingenuity Variant Analysis software (Qiagen) or NIH ClinVar. Only variants classified as pathogenic or likely pathogenic were included. STRING application (version 1.5.1) was used to create a protein-protein interaction network. Topological distance and probability of co-mutation were used to calculated the C-score and cumulative C-score (cumC-score). Kaplan–Meier analysis was used to determine the relationship between gene pairs with a high cumC-score and clinical parameters. Improved relapse free survival in Vigil treated patients was found for the TP53m-BRCAwt-HRP group compared to placebo (21.1 months versus 5.6 months p = 0.0013). Analysis of tumor mutation burden did not reveal statistical benefit in patients receiving Vigil versus placebo. Results suggest a subset of ovarian cancer patients with enhanced susceptibility to Vigil immunotherapy. The hypothesis-generating data presented invites a validation study of Vigil in target identified populations, and supports clinical consideration of STRING-generated network application to biomarker characterization with other cancer patients targeted with Vigil.

Suicide gene strategies applied in ovarian cancer studies

Ovarian cancer represents the most lethal gynecological malignancy among women in developed countries. Despite the recent innovations, the improvements in the 5-year survival rate have been insufficient and the management of this disease still remains a challenge. The fact that the majority of patients experience recurrent or resistant disease have substantiated the necessity of an innovative treatment. Among various strategies investigated, the recent strides made in gene delivery techniques have made gene therapy, including suicide gene strategies, a potential alternative for treating ovarian cancer. Various suicide gene candidates, which are capable of promoting cancer cell apoptosis directly after its entry or indirectly by prodrug administration, can be separated into three systems using enzyme-coding, toxin or pro-apoptotic genes. With this review, we aim to provide an overview of different suicide genes depending on therapeutic strategies, the vectors used to deliver these transgenes specifically to malignant cells, and the combined treatments of these genes with various therapeutic regimens.

CpG-binding protein CFP1 promotes ovarian cancer cell proliferation by regulating BST2 transcription

AbstractEpigenetic alterations have been functionally linked to ovarian cancer development and occurrence. The CXXC zinc finger protein 1 (CFP1) is an epigenetic regulator involved in DNA methylation and histone modification in mammalian cells. However, its role in ovarian cancer cells is unknown. Here, we show that CFP1 protein is highly expressed in human ovarian cancer tissues. Loss of CFP1 inhibited the growth of human ovarian cancer cells, promoted apoptosis, and increased senescence. CFP1 knockdown resulted in reduced levels of SETD1 (a CFP1 partner) and histone H3 trimethylation at the fourth lysine residue (H3K4me3). RNA-sequencing revealed that deletion of CFP1 resulted in mRNA reduction of bone marrow stromal cell antigen 2 (BST2). Bioinformatics analysis and chromatin immunoprecipitation showed that CFP1 binds to the promoter of BST2 and regulates its transcription directly. Overexpression of BST2 rescued the growth inhibitory effect of CFP1 loss. Furthermore, depletion of cullin-RING ubiquitin ligases 4 (CRL4) components ROC1 or CUL4A had significantly inhibited the expression of CFP1 and BST2 similar to MLN4924 treatment that blocked cullin neddylation and inactivated CRL4s. In conclusion, CFP1 promotes ovarian cancer cell proliferation and apoptosis by regulating the transcription of BST2, and the expression of CFP1 was affected by CRL4 ubiquitin ligase complex.

TAF1A and ZBTB41 serve as novel key genes in cervical cancer identified by integrated approaches

AbstractCervical cancer (CC) is the second most common cancer and the leading cause of cancer mortality in women. Numerous studies have found that the development of CC was associated with multiple genes. However, the mechanisms on gene level are enigmatic, hindering the understanding of its functional roles. This study sought to identify prognostic biomarkers of CC, and explore their biological functions. Here we conducted an integrated analysis to screen potential vital genes. Candidate genes were further tested by experiments in clinical specimens and cancer cell line. Then, molecular modeling was used to predict the three-dimensional structure of candidate genes’ proteins, and the interaction pattern was analyzed by docking simulation technique. Among the potential genes identified, we found that TAF1A and ZBTB41 were highly correlated. Furthermore, there was a definite interaction between the proteins of TAF1A and ZBTB41, which was affected by the activity of the p53 signaling pathway. In conclusion, our findings identified TAF1A and ZBTB41 could serve as biomarkers of CC. We confirmed their biological function and deciphered their interaction for the first time, which may be helpful for developing further researches.

METTL3-mediated maturation of miR-126-5p promotes ovarian cancer progression via PTEN-mediated PI3K/Akt/mTOR pathway

Methyltransferase-like 3 (METTL3) functions as an RNA methyltransferase that controls the modification of N(6)-methyladenosine (m6A) to influence the biosynthesis, decay, and translation of mRNAs. This study aims to investigate the regulation of METTL3-mediated promotion of microRNA-126-5p (miR-126-5p) in the progression of ovarian cancer and to identify the mechanisms in relation to phosphatase and tensin homolog (PTEN) and the PI3K/Akt/mTOR pathway. We found high expression of miR-126-5p in ovarian cancer samples compared to paired adjacent samples, and also in ovarian cancer cell lines. Gain-of-function experiments demonstrated that overexpression of miR-126-5p promoted ovarian cancer cell proliferation, migration, and invasion, and inhibited their apoptosis. Luciferase reporter assay identified that miR-126-5p could directly bind to PTEN. By targeting PTEN, miR-126-5p could activate the PI3K/Akt/mTOR pathway. Furthermore, the RNA methyltransferase METTL3 promoted the maturation of miR-126-5p via the m6A modification of pri-miR-126-5p. Finally, in vitro and in vivo experiments substantiated that silencing of METTL3 impeded the progression and tumorigenesis of ovarian cancer by impairing the miR-126-5p-targeted inhibition of PTEN and thus blocking the PI3K/Akt/mTOR pathway. Coherently, knockdown of METTL3 inhibited the effect of miR-126-5p to upregulate PTEN, and thus prevents PI3K/Akt/mTOR pathway activation, thereby suppressing the development of ovarian cancer. These findings highlight potential targets for the future ovarian cancer treatment as well as tumorigenic mechanisms mediated by m6A modification.

Bone marrow stromal cells-derived microRNA-181-containing extracellular vesicles inhibit ovarian cancer cell chemoresistance by downregulating MEST via the Wnt/β-catenin signaling pathway

Cisplatin (DDP)-based strategies are the first-line treatment for cancers; however, resistance to DDP remains a major obstacle to cancer treatment. The current study set out to investigate the effects of microRNA-181c (miR-181c) on the resistance of ovarian cancer cells to DDP. Ovarian cancer-associated miRs as well as the target messenger RNAs were screened using microarray-based analysis followed by determining the expression patterns of miR-181c and mesoderm-specific transcript (MEST) in ovarian cancer tissues with RT-qPCR and Western blot analysis. Subsequently, dual-luciferase reporter gene assay was performed to confirm the targeting relation between miR-181c and MEST. Through gain- or loss-of-function experiments, the study explored the mechanism by which miR-181 regulated MEST to influence the resistance of ovarian cancer cells to DDP via the Wnt/β-catenin signaling pathway. Afterwards, extracellular vesicles (EVs) were isolated from bone marrow stromal cells (BMSCs) and co-cultured with ovarian cancer cells to further investigate the effects of overexpressed miR-181 delivered by BMSCs-derived EVs on ovarian cancer cell resistance to DDP. miR-181c was significantly downregulated, while MEST was up-regulated in ovarian cancer. miR-181c was verified to specifically bind to MEST. Overexpressed miR-181c depleted the expression of MEST to attenuate the resistance of ovarian cancer cells to DDP by inactivating the Wnt/β-catenin signaling pathway. Furthermore, the delivery of overexpressed miR-181c by BMSCs-derived EVs was found to suppress the resistance of ovarian cancer cells to DDP. These findings demonstrate that miR-181c delivered by BMSCs-derived EVs down-regulates MEST, to inactivate the Wnt/β-catenin signaling pathway, thus repressing the resistance of ovarian cancer cells to DDP.

RHPN1-AS1 promotes cell proliferation and migration via miR-665/Akt3 in ovarian cancer

Recent efforts have revealed that long non-coding RNAs exert crucial roles in cancer initiation and progression. RHPN1-AS1 is a 2030 bp transcript from human chromosome 8q24, and involved in tumorigenesis in uveal melanoma and non-small cell lung cancer, but it remains unknown in ovarian cancer. This study focused on the role of RHPN1-AS1 in ovarian cancer and found that RHPN1-AS1 was up-regulated in ovarian cancer tissues and cell lines. Overexpression of RHPN1-AS1 promoted ovarian cancer cell proliferation, migration, and invasion. Mechanistically, overexpression of RHPN1-AS1 decreased the expression of miR-665 and subsequently promoted the expression of Akt3 at posttranscriptional level. Taken together, RHPN1-AS1 positively regulated the expression of Akt3 through sponging miR-665, and exerted an oncogenic role in ovarian cancer progression, and indicates that RHPN1-AS1 may be a potential therapeutic target in ovarian cancer.

ALKBH5-mediated m6A regulates the alternative splicing events of SRSF10 in ovarian cancer

N6-methyladenosine (m6A) methylation was found to be involved in the tumorigenesis and development of ovarian cancer. Until now, it is not clear to identify the mechanism by m6A demethylase ALKBH5 affects RNA splicing in ovarian cancer. In this study, we examined ALKBH5 protein expression and m6A levels by immunohistochemistry and analyzed their correlation with clinical features and prognosis in patients with ovarian cancer. We identified the elevated expression of ALKBH5 and a general reduction in the level of m6A in ovarian cancer patients. In the ovarian cancer cell line A2780, ALKBH5 depletion was found using the siRNA strategy or the CRISPR/Cas9 knockout (KO) method, which significantly reduced the level of m6A and inhibited cell viability, proliferation, and migration. The MeRIP-seq and RNA-seq showed that ALKBH5-regulated m6A RNA modification mainly affects RNA splicing function in ovarian cancer cells. SRSF10 is a key target gene involved in alternative splicing regulation through ALKBH5-m6A. ALKBH5 knockdown resulted in increased retention of SRSF10 exon 5 and decreased expression of transcript SRSF10-211. In conclusion, the alternative splicing regulation effect by ALKBH5-mediated m6A suggests a novel promising approach for m6A modification in OC and provides novel insights into the mechanisms involved in ovarian cancer therapy.

Synergistic antitumor effect on cervical cancer by rational combination of PD1 blockade and CRISPR-Cas9-mediated HPV knockout

Targeted therapy results in objective responses in cervical cancer. However, the responses are short. In contrast, treatment with immune checkpoint inhibitors results in a lower responses rate, but the responses tend to be more durable. Based on these findings, we hypothesized that HPV16 E6/E7-targeted therapy may synergize with the PD-1 pathway blockade to enhance antitumor activity. To test hypothesis, we described for the first time the effects of the CRISPR/Cas9 that was targeted to the HPV and PD1 in vitro and in vivo. Our data showed that gRNA/cas9 targeted HPV16 E6/E7 induced cervical cancer cell SiHa apoptosis, and suggested that overexpression of PD-L1, induced by HPV16 E6/E7, may be responsible for lymphocyte dysfunction. In established SiHa cell- xenografted humanized SCID mice, Administration of gRNA-PD-1 together with gRNA-HPV16 E6/E7 treatment improved the survival and suppressed the tumor growth obviously. In addition, combination treatment increased the population of dendritic cells, CD8+ and CD4+ T lymphocyte cells. According, it enhanced the expression of Th1-associated immune-stimulating genes while reducing the transcription of regulatory/suppressive immune genes, reshaping tumor microenvironment from an immunosuppressive to a stimulatory state. These results demonstrate potent synergistic effects of combination therapy using HPV16 E6/E7-targeted therapy and immune checkpoint blockade PD1, supporting a direct translation of this combination strategy in clinic for the treatment of cervical cancer.

The application of CRISPR/Cas9 system in cervical carcinogenesis

AbstractIntegration of high-risk HPV genomes into cellular chromatin has been confirmed to promote cervical carcinogenesis, with HPV16 being the most prevalent high-risk type. Herein, we evaluated the therapeutic effect of the CRISPR/Cas9 system in cervical carcinogenesis, especially for cervical precancerous lesions. In cervical cancer/pre-cancer cell lines, we transfected the HPV16 E7 targeted CRISPR/Cas9, TALEN, ZFN plasmids, respectively. Compared to previous established ZFN and TALEN systems, CRISPR/Cas9 has shown comparable efficiency and specificity in inhibiting cell growth and colony formation and inducing apoptosis in cervical cancer/pre-cancer cell lines, which seemed to be more pronounced in the S12 cell line derived from the low-grade cervical lesion. Furthermore, in xenograft formation assays, CRISPR/Cas9 inhibited tumor formation of the S12 cell line in vivo and affected the corresponding protein expression. In the K14-HPV16 transgenic mice model of HPV-driven spontaneous cervical carcinogenesis, cervical application of CRISPR/Cas9 treatment caused mutations of the E7 gene and restored the expression of RB, E2F1, and CDK2, thereby reversing the cervical carcinogenesis phenotype. In this study, we have demonstrated that CRISPR/Cas9 targeting HPV16 E7 could effectively revert the HPV-related cervical carcinogenesis in vitro, as well as in K14-HPV16 transgenic mice, which has shown great potential in clinical treatment for cervical precancerous lesions.

Genomic alterations caused by HPV integration in a cohort of Chinese endocervical adenocarcinomas

AbstractThe association between human papillomavirus (HPV) integration and relevant genomic changes in uterine cervical adenocarcinoma is poorly understood. This study is to depict the genomic mutational landscape in a cohort of 20 patients. HPV+ and HPV− groups were defined as patients with and without HPV integration in the host genome. The genetic changes between these two groups were described and compared by whole-genome sequencing (WGS) and whole-exome sequencing (WES). WGS identified 2916 copy number variations and 743 structural variations. WES identified 6113 somatic mutations, with a mutational burden of 2.4 mutations/Mb. Six genes were predicted as driver genes: PIK3CA, KRAS, TRAPPC12, NDN, GOLGA6L4 and BAIAP3. PIK3CA, NDN, GOLGA6L4, and BAIAP3 were recognized as significantly mutated genes (SMGs). HPV was detected in 95% (19/20) of patients with cervical adenocarcinoma, 7 of whom (36.8%) had HPV integration (HPV+ group). In total, 1036 genes with somatic mutations were confirmed in the HPV+ group, while 289 genes with somatic mutations were confirmed in the group without HPV integration (HPV− group); only 2.1% were shared between the two groups. In the HPV+ group, GOLGA6L4 and BAIAP3 were confirmed as SMGs, while PIK3CA, NDN, KRAS, FUT1, and GOLGA6L64 were identified in the HPV− group. ZDHHC3, PKD1P1, and TGIF2 showed copy number amplifications after HPV integration. In addition, the HPV+ group had significantly more neoantigens. HPV integration rather than HPV infection results in different genomic changes in cervical adenocarcinoma.