Cellular and Molecular Life Sciences

Tumor-associated macrophages contribute to cisplatin resistance via regulating Pol η-mediated translesion DNA synthesis in ovarian cancer

Tumor-associated macrophages (TAMs) are known to be involved in the manifestation of aggressive and therapy-resistant phenotypes in solid tumors. Nevertheless, the effects of dynamic intervention by TAMs on the DNA damage response of cancer cells are largely unexplored. Herein, we report that TAMs modulate the DNA damage repair pathways of ovarian cancer cells in response to platinum-(Pt) based therapeutic regimen. We demonstrate that coculture of TAMs with cancer cells directly upregulate Pol η, along with RAD18 and REV1 of the Translesion DNA synthesis (TLS) pathway, while concurrently downregulating components of the high-fidelity nucleotide excision repair (NER) mechanism. Consequently, we observed a better survival probability, DNA repair capacity, and enrichment of stemness properties in ovarian cancer cells. DNA bulky adducts produced by cisplatin are resolved through differential activation NER and TLS pathways. However, we elucidated that TAMs provide favorable conditions for activating the error-prone TLS pathway for lesion bypass over damage resolution. Furthermore, cellular crosstalk in cocultured cancer cells stimulates the nuclear translocation and expression of RelA, which recruits Pol η by acting as a potent transcription factor. In fact, with pristimerin-mediated disruption of p65 (RelA) translocation, the cancer cells become more prone to DNA damage-induced cell death and compromised regenerative potential. In both in vitro cell cultures and in vivo mouse xenograft models, cocultured macrophages exhibited predominantly M2-like phenotype with prevalence in the invasive zone of xenograft tumor margins. Taken together, our investigation revealed multifaceted crosstalk-mediated regulation of DNA damage repair between TAMs and ovarian cancer cells.

Apurinic/apyrimidinic endodeoxyribonuclease 1 (APE1) promotes stress granule formation via YBX1 phosphorylation in ovarian cancer

AbstractAPE1 is an essential gene involved in DNA damage repair, the redox regulation of transcriptional factors (TFs) and RNA processing. APE1 overexpression is common in cancers and correlates with poor patient survival. Stress granules (SGs) are phase-separated cytoplasmic assemblies that cells form in response to environmental stresses. Precise regulation of SGs is pivotal to cell survival, whereas their dysregulation is increasingly linked to diseases. Whether APE1 engages in modulating SG dynamics is worthy of investigation. In this study, we demonstrate that APE1 colocalizes with SGs and promotes their formation. Through phosphoproteome profiling, we discover that APE1 significantly alters the phosphorylation landscape of ovarian cancer cells, particularly the phosphoprofile of SG proteins. Notably, APE1 promotes the phosphorylation of Y-Box binding protein 1 (YBX1) at S174 and S176, leading to enhanced SG formation and cell survival. Moreover, expression of the phosphomutant YBX1 S174/176E mimicking hyperphosphorylation in APE1-knockdown cells recovered the impaired SG formation. These findings shed light on the functional importance of APE1 in SG regulation and highlight the importance of YBX1 phosphorylation in SG dynamics.

Phosphocholine inhibits proliferation and reduces stemness of endometrial cancer cells by downregulating mTOR-c-Myc signaling

Endometrial cancer (EC) represents a serious health concern among women globally. Excessive activation of the protooncogene c-Myc (c-Myc) is associated with the proliferation and stemness of EC cells. Phosphocholine (PC), which is synthesized by choline kinase alpha (CHKA) catalysis, is upregulated in EC tumor tissues. The present study aimed to investigate the effect of PC accumulation on EC cells and clarify the relationship between PC accumulation and c-Myc activity in EC. The c-Myc and CHKA expression in EC tumor tissues were examined using immunohistochemistry. Cell Counting Kit-8 assay, colony formation assay, flow cytometry, western blotting, BrdU staining, and tumorsphere formation assay were used to assess the effect of PC accumulation on EC cells. The mechanism by which PC accumulation inhibits c-Myc was evaluated using RNA-sequencing. Patient-derived organoid (PDO) models were utilised to explore the preclinical efficacy of PC against EC cells. PC accumulation suppressed EC cell proliferation and stemness by inhibiting the activation of the mammalian target of rapamycin (mTOR)-c-Myc signaling. PC accumulation promoted excessive reactive oxygen species production, which reduced the expression of GTPase HRAS. This, in turn, inhibited the mTOR-c-Myc axis and induced EC cell apoptosis. Finally, PC impeded proliferation and downregulated the expression of the mTOR-MYC signaling in EC PDO models. PC accumulation impairs the proliferation ability and stem cell characteristics of EC cells by inhibiting the activated mTOR-c-Myc axis, potentially offering a promising strategy to enhance the efficacy of EC clinical therapy through the promotion of PC accumulation in tumor cells.

TRP channel expression correlates with the epithelial–mesenchymal transition and high-risk endometrial carcinoma

AbstractTransient receptor potential (TRP) channels excel in cellular sensing as they allow rapid ion influx across the plasma membrane in response to a variety of extracellular cues. Recently, a distinct TRP mRNA expression signature was observed in stromal cells (ESC) and epithelial cells (EEC) of the endometrium, a tissue in which cell phenotypic plasticity is essential for normal functioning. However, it is unknown whether TRP channel mRNA expression is subject to the phenotypic switching that occurs during epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET), and whether TRP channel mRNA expression is associated with aggressive phenotypes in endometrial cancer (EC). Here, we induced EMT and MET in vitro using in primary EEC and ESC, respectively, and analyzed expression and functionality of TRP channels using RT-qPCR and intracellular Ca2+imaging. The outcome of these experiments showed a strong association between TRPV2 and TRPC1 mRNA expression and the mesenchymal phenotype, whereas TRPM4 mRNA expression correlated with the epithelial phenotype. In line herewith, increased TRPV2 and TRPC1 mRNA expression levels were observed in both primary and metastatic EC biopsies and in primary EC cells with a high EMT status, indicating an association with an aggressive tumor phenotype. Remarkably, TRPV2 mRNA expression in primary EC biopsies was associated with tumor invasiveness and cancer stage. In contrast, increased TRPM4 mRNA expression was observed in EC biopsies with a low EMT status and less aggressive tumor phenotypes. Taken together, this dataset proved for the first time that TRP channel mRNA expression is strongly linked to cellular phenotypes of the endometrium, and that phenotypic transitions caused by either experimental manipulation or malignancy could alter this expression in a predictable manner. These results implicate that TRP channels are viable biomarkers to identify high-risk EC, and potential targets for EC treatment.

LILRB4 shapes an immunosuppressive microenvironment to drive cervical cancer progression through tumor-infiltrating myeloid cell expansion and CD8+ T-cell suppression

Cervical cancer (CC) is the most common gynecological malignancy and is strongly linked to human papillomavirus (HPV) infection. Currently, immune checkpoint blockade therapy has shown limited clinical benefits for CC, highlighting the need to find more effective therapeutic targets. LILRB4, a member of the leukocyte immunoglobulin-like receptor superfamily, is considered a key mediator of cancer immunosuppression. However, its role in the CC immune microenvironment remains unclear. Here, LILRB4 expression was upregulated in CC tissues, and high expression levels were positively associated with advanced disease and immunosuppressive genes in tumors. In an immunocompetent mouse model, LILRB4 expression in CC tumors increased with tumor growth, whereas blocking LILRB4 reduced tumor growth. Flow cytometry analysis revealed that blockade of LILRB4 reduced CD8

Hypoxia drives cervical cancer progression via OCT4/ORAI3-dependent glycolysis and Ca2+ signaling

Cervical cancer is a common cancer among women worldwide. It has been revealed that hypoxia contributes to the progression of cervical cancer. In our study, we discovered that hypoxia indeed promoted the malignant phenotypes of cervical cancer cells by enhancing glycolysis. Loss-of-function experiments showed that hypoxia treatment upregulated the octamer-binding transcriptional factor 4 (OCT4) expression via glycolysis. Through the RNA-sequence and enrichment analyses, we found that hypoxia induced the enrichment of the calcium signaling pathway and upregulation of Calcium Release-Activated Calcium Modulator 3 (ORAI3), which could be abrogated by silencing OCT4. Notably, overexpressing ORAI3 has similar effects on the malignant phenotypes of HeLa and SiHa cells as those of hypoxia. Furthermore, silencing ORAI3 or inactivating calcium signals significantly reversed OCT4-induced malignant progression of cervical cancer both in vitro and in vivo. ChIP and dual-luciferase reporter results confirmed that OCT4 contributed to the transcription of ORAI3. Mechanically, hypoxia upregulated OCT4 expression by facilitating glycolysis, and OCT4 overexpression enhanced the transcription of ORAI3, activating the calcium signaling pathway and ultimately promoting the malignant progression of cervical cancer. Our study reveals novel molecular mechanisms by which hypoxia induces the progression of cervical cancer, providing a new strategy for treating cervical cancer.

Enhancing anti-tumor immunity by targeting BATF and the STAT1/PD-L1 pathway in cervical carcinoma

Cervical carcinoma remains a leading cause of cancer-related mortality in women worldwide, with poor prognosis often linked to immune evasion mechanisms. The Basic Leucine Zipper Activating Transcription Factor (BATF) has emerged as a critical regulator of T-cell functionality, yet its role in cervical cancer progression and immune modulation remains poorly understood. This study investigates the role of BATF in cervical carcinoma, focusing on its effects on tumor progression, immune modulation, and immune checkpoint regulation, to identify BATF as a therapeutic target to enhance anti-tumor immunity. BATF expression was analyzed in cervical cancer tissues and cell lines. Functional assays, including cell proliferation, migration, and invasion, were performed following BATF knockdown. In vivo studies assessed tumor growth and metastasis in xenograft models. Immune cell populations, cytokine production, and immune checkpoint expression were analyzed using flow cytometry, quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), and immunohistochemistry. Mechanistic studies evaluated BATF regulation of programmed cell death-ligand 1 (PD-L1) through the signal transducer and activator of transcription 1 (STAT1) signaling pathway. Finally, the therapeutic potential of BATF knockdown in combination with PD-L1 blockade was evaluated in vivo. Analysis of patient-derived samples and cell lines revealed that BATF is overexpressed in cervical cancer and correlates with poor prognosis. Functional studies demonstrated that BATF promotes tumor proliferation, epithelial-to-mesenchymal transition (EMT), migration, and invasion. In vivo, BATF knockdown significantly suppressed tumor growth and metastasis in xenograft models. Immune profiling revealed that BATF deficiency enhanced antitumor immunity by increasing CD8⁺ and CD4⁺ T-cell infiltration, increasing the production of CD8⁺ cytotoxic molecules such as granzyme B and IFN-γ while reducing tumor-associated macrophages and immune checkpoint expression, including Programmed death 1 (PD-1), T cell immunoglobulin and mucin-domain-containing-3 (TIM-3), and Lymphocyte activation gene-3 (LAG-3.) Mechanistically, BATF regulated PD-L1 expression via the STAT1 signaling pathway, promoting immune evasion. Combination therapy with BATF knockdown and anti-PD-L1 antibodies significantly enhanced anti-tumor immunity and inhibited tumor growth compared to monotherapy. BATF plays a pivotal role in cervical cancer progression and immune evasion by regulating the STAT1/PD-L1 axis and modulating the tumor immune microenvironment. Targeting BATF, alone or in combination with immune checkpoint inhibitors (PD-L1), represents a promising therapeutic strategy to improve outcomes in cervical cancer patients. Further research is warranted to translate these findings into clinical applications.

PPP1R13L drives cervical cancer progression by suppressing p63-mediated PTEN transcription

Abstract PPP1R13L is a conserved inhibitor of p53, selectively regulating a subset of p53 target genes. Previous studies have reported that PPP1R13L promotes cervical cancer progression, yet its precise mechanism remains unclear and warrants further investigation. In this study, we utilized public databases to reveal the correlation between PPP1R13L and tumor progression pathways. Subsequently, we performed functional assays both in vitro and in xenograft models to assess the impact of PPP1R13L on cervical cancer. Our results demonstrate that PPP1R13L promotes cervical cancer cell proliferation, epithelial-mesenchymal transition, cycle progression, and glycolysis via the PTEN/AKT/mTOR pathway. Mechanistically, PPP1R13L regulates the transcription of PTEN through its Ank-SH3 domain interaction with p53 family, p53 and p63. In 293T cells, p53 originally exhibits significantly higher transcriptional activity than p63. However, in cervical cancer—where E6 continuously degrades p53 and p63 is highly expressed—p63 demonstrates a transcriptional activity for PTEN that is comparable to, or even surpasses, that of p53, depending on E6 expression levels. Additionally, in C33A, an HPV-negative cervical cancer cell line, the p53 R273C mutation causes PPP1R13L to exert an opposite effect, and p63 is shown to be inhibited by PPP1R13L independently of p53. Finally, the response elements of PPP1R13L-regulated p53 family target genes were experimentally validated on p63 for the first time. This provides a sequence basis for the selective regulation of p53 family target genes by PPP1R13L. In summary, we underscore the specificity of the PPP1R13L/p63/PTEN axis in cervical cancer and propose that PPP1R13L holds potential as a therapeutic target for cervical cancer treatment. Graphical abstract

Carboplatin-induced upregulation of pan β-tubulin and class III β-tubulin is implicated in acquired resistance and cross-resistance of ovarian cancer

Resistance to platinum- and taxane-based chemotherapy represents a major obstacle to long-term survival in ovarian cancer (OC) patients. Here, we studied the interplay between acquired carboplatin (CBP) resistance using two OC cell models, MES-OV CBP and SK-OV-3 CBP, and non-P-glycoprotein-mediated cross-resistance to paclitaxel (TAX) observed only in MES-OV CBP cells. Decreased platination, mesenchymal-like phenotype, and increased expression of α- and γ-tubulin were observed in both drug-resistant variants compared with parental cells. Both variants revealed increased protein expression of class III β-tubulin (TUBB3) but differences in TUBB3 branching and nuclear morphology. Transient silencing of TUBB3 sensitized MES-OV CBP cells to TAX, and surprisingly also to CBP. This phenomenon was not observed in the SK-OV-3 CBP variant, probably due to the compensation by other β-tubulin isotypes. Reduced TUBB3 levels in MES-OV CBP cells affected DNA repair protein trafficking and increased whole-cell platination level. Furthermore, TUBB3 depletion augmented therapeutic efficiency in additional OC cells, showing vice versa drug-resistant pattern, lacking β-tubulin isotype compensation visible at the level of total β-tubulin (TUBB) in vitro and ex vivo. In summary, the level of TUBB in OC should be considered together with TUBB3 in therapy response prediction.

Mfn2-mediated mitochondrial fusion promotes autophagy and suppresses ovarian cancer progression by reducing ROS through AMPK/mTOR/ERK signaling

Mitochondrial dynamics are balanced fission and fusion events that regulate mitochondrial morphology, and alteration in these events results in mitochondrial dysfunction and contributes to many diseases, including tumorigenesis. Ovarian cancer (OC) cells exhibit fragmented mitochondria, but the mechanism by which mitochondrial dynamics regulators contribute to OC is considerably less clear. Here, we elucidated the potential role of Mfn2-mediated mitochondrial fusion in OC and present evidence that genetic or pharmacological activation of Mfn2 leads to mitochondrial fusion and reduces ROS generation, which correlates with reduced cell proliferation, invasion, migration, and EMT in OC cells. Also, increased mitochondrial fusion promotes the F-actin remodeling, reduces lamellipodia formation, and thus reduces EMT. Increased expression of Mfn2 triggers AMPK, promotes autophagy, reduces ROS, and suppresses OC progression by downregulating  the p-mTOR (2481 and 2448) and p-ERK axis. OC patients with higher Mfn2 expression have better survival than those with lower Mfn2 levels. Our findings demonstrate that restoration of Mfn2-mediated mitochondrial fusion suppressed OC progression and suggest that this process could be a potential strategy in OC treatment.

PUMILIO-mediated translational control of somatic cell cycle program promotes folliculogenesis and contributes to ovarian cancer progression

Translational control is a fundamental mechanism regulating animal germ cell development. Gonadal somatic cells provide support and microenvironment for germ cell development to ensure fertility, yet the roles of translational control in gonadal somatic compartment remain largely undefined. We found that mouse homolog of conserved fly germline stem cell factor Pumilio, PUM1, is absent in oocytes of all growing follicles after the primordial follicle stage, instead, it is highly expressed in somatic compartments of ovaries. Global loss of Pum1, not oocyte-specific loss of Pum1, led to a significant reduction in follicular number and size as well as fertility. Whole-genome identification of PUM1 targets in ovarian somatic cells revealed an enrichment of cell proliferation pathway, including 48 key regulators of cell phase transition. Consistently granulosa cells proliferation is reduced and the protein expression of the PUM-bound Cell Cycle Regulators (PCCR) were altered accordingly in mutant ovaries, and specifically in granulosa cells. Increase in negative regulator expression and decrease in positive regulators in the mutant ovaries support a coordinated translational control of somatic cell cycle program via PUM proteins. Furthermore, postnatal knockdown, but not postnatal oocyte-specific loss, of Pum1 in Pum2 knockout mice reduced follicular growth and led to similar expression alteration of PCCR genes, supporting a critical role of PUM-mediated translational control in ovarian somatic cells for mammalian female fertility. Finally, expression of human PUM protein and its regulated cell cycle targets exhibited significant correlation with ovarian cancer and prognosis for cancer survival. Hence, PUMILIO-mediated cell cycle regulation represents an important mechanism in mammalian female reproduction and human cancer biology.

RhoC in association with TET2/WDR5 regulates cancer stem cells by epigenetically modifying the expression of pluripotency genes

Emerging evidence illustrates that RhoC has divergent roles in cervical cancer progression where it controls epithelial to mesenchymal transition (EMT), migration, angiogenesis, invasion, tumor growth, and radiation response. Cancer stem cells (CSCs) are the primary cause of recurrence and metastasis and exhibit all of the above phenotypes. It, therefore, becomes imperative to understand if RhoC regulates CSCs in cervical cancer. In this study, cell lines and clinical specimen-based findings demonstrate that RhoC regulates tumor phenotypes such as clonogenicity and anoikis resistance. Accordingly, inhibition of RhoC abrogated these phenotypes. RNA-seq analysis revealed that RhoC over-expression resulted in up-regulation of 27% of the transcriptome. Further, the Infinium MethylationEPIC array showed that RhoC over-expressing cells had a demethylated genome. Studies divulged that RhoC via TET2 signaling regulated the demethylation of the genome. Further investigations comprising ChIP-seq, reporter assays, and mass spectrometry revealed that RhoC associates with WDR5 in the nucleus and regulates the expression of pluripotency genes such as Nanog. Interestingly, clinical specimen-based investigations revealed the existence of a subset of tumor cells marked by RhoC

DCAF13 promotes ovarian cancer progression by activating FRAS1-mediated FAK signaling pathway

Cullin-RING ubiquitin ligase 4 (CRL4) is closely correlated with the incidence and progression of ovarian cancer. DDB1- and CUL4-associated factor 13 (DCAF13), a substrate-recognition protein in the CRL4 E3 ubiquitin ligase complex, is involved in the occurrence and development of ovarian cancer. However, its precise function and the underlying molecular mechanism in this disease remain unclear. In this study, we confirmed that DCAF13 is highly expressed in human ovarian cancer and its expression is negatively correlated with the overall survival rate of patients with ovarian cancer. We then used CRISPR/Cas9 to knockout DCAF13 and found that its deletion significantly inhibited the proliferation, colony formation, and migration of human ovarian cancer cells. In addition, DCAF13 deficiency inhibited tumor proliferation in nude mice. Mechanistically, CRL4-DCAF13 targeted Fraser extracellular matrix complex subunit 1 (FRAS1) for polyubiquitination and proteasomal degradation. FRAS1 influenced the proliferation and migration of ovarian cancer cell through induction of the focal adhesion kinase (FAK) signaling pathway. These findings collectively show that DCAF13 is an important oncogene that promotes tumorigenesis in ovarian cancer cells by mediating FRAS1/FAK signaling. Our findings provide a foundation for the development of targeted therapeutics for ovarian cancer.

The senescent mesothelial matrix accentuates colonization by ovarian cancer cells

Abstract Ovarian cancer is amongst the most morbid of gynecological malignancies due to its diagnosis at an advanced stage, a transcoelomic mode of metastasis, and rapid transition to chemotherapeutic resistance. Like all other malignancies, the progression of ovarian cancer may be interpreted as an emergent outcome of the conflict between metastasizing cancer cells and the natural defense mounted by microenvironmental barriers to such migration. Here, we asked whether senescence in coelom-lining mesothelia, brought about by drug exposure, affects their interaction with disseminated ovarian cancer cells. We observed that cancer cells adhered faster on senescent human and murine mesothelial monolayers than on non-senescent controls. Time-lapse epifluorescence microscopy showed that mesothelial cells were cleared by a host of cancer cells that surrounded the former, even under sub-confluent conditions. A multiscale computational model predicted that such colocalized mesothelial clearance under sub-confluence requires greater adhesion between cancer cells and senescent mesothelia. Consistent with the prediction, we observed that senescent mesothelia expressed an extracellular matrix with higher levels of fibronectin, laminins and hyaluronan than non-senescent controls. On senescent matrix, cancer cells adhered more efficiently, spread better, and moved faster and persistently, aiding the spread of cancer. Inhibition assays using RGD cyclopeptides suggested the adhesion was predominantly contributed by fibronectin and laminin. These findings led us to propose that the senescence-associated matrisomal phenotype of peritoneal barriers enhances the colonization of invading ovarian cancer cells contributing to the metastatic burden associated with the disease.

AUF1-induced circular RNA hsa_circ_0010467 promotes platinum resistance of ovarian cancer through miR-637/LIF/STAT3 axis

Increasing evidences has indicated that primary and acquired resistance of ovarian cancer (OC) to platinum is mediated by multiple molecular and cellular factors. Understanding these mechanisms could promote the therapeutic efficiency for patients with OC. Here, we screened the expression pattern of circRNAs in samples derived from platinum-resistant and platinum-sensitive OC patients using RNA-sequencing (RNA-seq). The expression of hsa_circ_0010467 was validated by Sanger sequencing, RT-qPCR, and fluorescence in situ hybridization (FISH) assays. Overexpression and knockdown experiments were performed to explore the function of hsa_circ_0010467. The effects of hsa_circ_0010467 on enhancing platinum treatment were validated in OC cells, mouse model and patient-derived organoid (PDO). RNA pull-down, RNA immunoprecipitation (RIP), and dual-luciferase reporter assays were performed to investigate the interaction between hsa_circ_0010467 and proteins. Increased expression of hsa_circ_0010467 is observed in platinum-resistant OC cells, tissues and serum exosomes, which is positively correlated with advanced tumor stage and poor prognosis of OC patients. Hsa_circ_0010467 is found to maintain the platinum resistance via inducing tumor cell stemness, and silencing hsa_circ_0010467 substantially increases the efficacy of platinum treatment on inhibiting OC cell proliferation. Further investigation reveals that hsa_circ_0010467 acts as a miR-637 sponge to mediate the repressive effect of miR-637 on leukemia inhibitory factor (LIF) and activates the LIF/STAT3 signaling pathway. We further discover that AUF1 could promote the biogenesis of hsa_circ_0010467 in OC. Our study uncovers the mechanism that hsa_circ_0010467 mediates the platinum resistance of OC through AUF1/hsa_circ_0010467/miR-637/LIF/STAT3 axis, and provides potential targets for the treatment of platinum-resistant OC patients.

Disabled-2: a protein up-regulated by high molecular weight hyaluronan has both tumor promoting and tumor suppressor roles in ovarian cancer

AbstractAlthough the pro-tumorigenic functions of hyaluronan (HA) are well documented there is limited information on the effects and targets of different molecular weight HA. Here, we investigated the effects of 27 kDa, 183 kDa and 1000 kDa HA on ES-2 ovarian cancer cells overexpressing the stem cell associated protein, Notch3. 1000 kDA HA promoted spheroid formation in ES-2 cells mixed with ES-2 overexpressing Notch3 (1:3). We report disabled-2 (DAB2) as a novel protein regulated by 1000 kDa HA and further investigated its role in ovarian cancer. DAB2 was downregulated in ovarian cancer compared to normal tissues but increased in metastatic ovarian tumors compared to primary tumors. High DAB2 expression was associated with poor patient outcome and positively correlated with HA synthesis enzyme HAS2, HA receptor CD44 and EMT and macrophage markers. Stromal DAB2 immunostaining was significantly increased in matched ovarian cancer tissues at relapse compared to diagnosis and associated with reduced survival. The proportion of DAB2 positive macrophages was significantly increased in metastatic ovarian cancer tissues compared to primary cancers. However, DAB2 overexpression significantly reduced invasion by both A2780 and OVCAR3 cells in vivo. Our research identifies a novel relationship between HA signalling, Notch3 and DAB2. We highlight a complex relationship of both pro-tumorigenic and tumor suppressive functions of DAB2 in ovarian cancer. Our findings highlight that DAB2 has a direct tumor suppressive role on ovarian cancer cells. The pro-tumorigenic role of DAB2 may be mediated by tumour associated macrophages and requires further investigation.

Preclinical models of epithelial ovarian cancer: practical considerations and challenges for a meaningful application

AbstractDespite many improvements in ovarian cancer diagnosis and treatment, until now, conventional chemotherapy and new biological drugs have not been shown to cure the disease, and the overall prognosis remains poor. Over 90% of ovarian malignancies are categorized as epithelial ovarian cancers (EOC), a collection of different types of neoplasms with distinctive disease biology, response to chemotherapy, and outcome. Advances in our understanding of the histopathology and molecular features of EOC subtypes, as well as the cellular origins of these cancers, have given a boost to the development of clinically relevant experimental models. The overall goal of this review is to provide a comprehensive description of the available preclinical investigational approaches aimed at better characterizing disease development and progression and at identifying new therapeutic strategies. Systems discussed comprise monolayer (2D) and three-dimensional (3D) cultures of established and primary cancer cell lines, organoids and patient-derived explants, animal models, including carcinogen-induced, syngeneic, genetically engineered mouse, xenografts, patient-derived xenografts (PDX), humanized PDX, and the zebrafish and the laying hen models. Recent advances in tumour-on-a-chip platforms are also detailed. The critical analysis of strengths and weaknesses of each experimental model will aid in identifying opportunities to optimize their translational value.

Hotair promotes the migration and proliferation in ovarian cancer by miR-222-3p/CDK19 axis

AbstractPrevious studies in our laboratory have reported that miR-222-3p was a tumor-suppressive miRNA in OC. This study aims to further understand the regulatory role of miR-222-3p in OC and provide a new mechanism for its prevention and treatment. We first found that miR-222-3p inhibited the migration and proliferation of OC cells. Then, we observed CDK19 was highly expressed in OC and inversely correlated with miR-222-3p. Besides, we observed that miR-222-3p directly binds to the 3′-UTR of CDK19 and inhibits CDK19 translation, thus inhibiting OC cell migration and proliferation in vitro and repressed tumor growth in vivo. We also observed the inhibitory effect of Hotair on miR-222-3p in OC. In addition, Hotair could promote the proliferation and migration of OC cells in vitro and facilitate the growth and metastasis of tumors in vivo. Moreover, Hotair was positively correlated with CDK19 expression. These results suggest Hotair indirectly up-regulates CDK19 through sponging miR-222-3p, which enhances the malignant behavior of OC. This provides a further understanding of the mechanism of the occurrence and development of OC.

The non-apoptotic function of Caspase-8 in negatively regulating the CDK9-mediated Ser2 phosphorylation of RNA polymerase II in cervical cancer

AbstractCervical cancer is the fourth most frequently diagnosed and fatal gynecological cancer. 15–61% of all cases metastasize and develop chemoresistance, reducing the 5-year survival of cervical cancer patients to as low as 17%. Therefore, unraveling the mechanisms contributing to metastasis is critical in developing better-targeted therapies against it. Here, we have identified a novel mechanism where nuclear Caspase-8 directly interacts with and inhibits the activity of CDK9, thereby modulating RNAPII-mediated global transcription, including those of cell-migration- and cell-invasion-associated genes. Crucially, low Caspase-8 expression in cervical cancer patients leads to poor prognosis, higher CDK9 phosphorylation at Thr186, and increased RNAPII activity in cervical cancer cell lines and patient biopsies. Caspase-8 knock-out cells were also more resistant to the small-molecule CDK9 inhibitor BAY1251152 in both 2D- and 3D-culture conditions. Combining BAY1251152 with Cisplatin synergistically overcame chemoresistance of Caspase-8-deficient cervical cancer cells. Therefore, Caspase-8 expression could be a marker in chemoresistant cervical tumors, suggesting CDK9 inhibitor treatment for their sensitization to Cisplatin-based chemotherapy.

Periostin in lymph node pre-metastatic niches governs lymphatic endothelial cell functions and metastatic colonization

AbstractAlthough lymph node (LN) metastasis is an important prognostic parameter in cervical cancer, the tissue remodeling at a pre-metastatic state is poorly documented in LNs. We here identified periostin (POSTN) as a component of non-metastatic LNs by applying proteomic analyses and computerized image quantifications on LNs of patients with cervical cancer. We provide evidence for remarkable modifications of POSTN and lymphatic vessel distributions and densities in non-metastatic sentinel and metastatic human LNs, when compared to distant non-metastatic LNs. POSTN deposition at a pre-metastatic stage was demonstrated in a pre-clinical murine model (the ear sponge assay). Its expression by fibroblastic LN cells was assessed by in situ hybridization and in vitro cultures. In vitro, POSTN promoted lymphatic endothelial cell functions and tumor cell proliferation. Accordingly, the in vivo injection of recombinant POSTN together with VEGF-C boosted the lymphangiogenic response, while the metastatic potential of tumor cells was drastically reduced using a POSTN blocking antibody. This translational study also supports the existence of an unprecedented dialog “in cascade”, between the primary tumor and the first pelvic nodal relay in early cervical cancer, and subsequently from pelvic LN to para-aortic LNs in locally advanced cervical cancers. Collectively, this work highlights the association of POSTN deposition with lymphangiogenesis in LNs, and provides evidence for a key contribution of POSTN in promoting VEGF-C driven lymphangiogenesis and the seeding of metastatic cells.

The association of telomere maintenance and TERT expression with susceptibility to human papillomavirus infection in cervical epithelium

The role of telomerase reverse transcriptase (TERT) induction and telomere maintenance in carcinogenesis including cervical cancer (CC) pathogenesis has been well established. However, it remains unclear whether they affect infection of high-risk human papillomavirus (hrHPV), an initiating event for CC development. Similarly, genetic variants at the TERT locus are shown to be associated with susceptibility to CC, but it is unclear whether these SNPs modify the risk for cervical HPV infection. Here we show that in CC-derived HeLa cells, TERT overexpression inhibits, while its depletion upregulates expression of Syndecan-1 (SDC-1), a key component for HPV entry receptors. The TCGA cohort of CC analyses reveals an inverse correlation between TERT and SDC-1 expression (R = -0.23, P = 0.001). We further recruited 1330 females (520 non-HPV and 810 hrHPV-infected) without CC or high-grade cervical intraepithelial neoplasia to analyze telomeres in cervical epithelial cells and SNPs at rs2736098, rs2736100 and rs2736108, previously identified TERT SNPs for CC risk. Non-infected females exhibited age-related telomere shortening in cervical epithelial cells and their telomeres were significantly longer than those in hrHPV-infected group (1.31 ± 0.62 vs 1.19 ± 0.48, P < 0.001). There were no differences in rs2736098 and rs2736100 genotypes, but non-infected individuals had significantly a higher C-allele frequency (associated with higher TERT expression) while lower T-allele levels at rs2736108 compared with those in the hrHPV group (P = 0.020). Collectively, appropriate telomere maintenance and TERT expression in normal cervical cells may prevent CC by modulating hrHPV infection predisposition, although they are required for CC development and progression.

Mechanism of LncRNA NORAD regulating ferroptosis in endometrial cancer cells by modifying GPX4 through FTO-mediated m6A methylation

Endometrial cancer (EC) represents the most prevalent gynecologic malignancy. We probed the mechanism of long noncoding RNA activated by DNA damage (lncRNA NORAD) regulating ferroptosis in EC cells (ECCs) by modifying glutathione peroxidase 4 (GPX4) through N6 methyladenosine (m6A) methylation. The relationship between NORAD and EC clinically, and correlations of NORAD with GPX4 and fat mass and obesity-associated protein (FTO) levels were analyzed by Starbase database, Kaplan-Meier curve, and Pearson. Total m6A modification of EC tissues was detected by Dot blotting. Effects of regulating NORAD, GPX4, FTO and YTHDF2 on m6A modification and ferroptosis in EC were explored in ECCs and nude mouse xenograft tumor models. Mechanistically, NORAD-FTO, FTO-YTHDF2 and YTHDF2-GPX4 interactions were detected by RNA pull down, Co-IP and RIP assays. GPX4 mRNA stability was determined by Actinomycin D test. NORAD was down-regulated in EC tissues and cells. Lowly-expressed NORAD in EC tissues predicted EC patients' poor prognoses, and negatively correlated with GPX4. NORAD overexpression promoted GPX4-mediated ferroptosis. NORAD promoted ECC ferroptosis by down-regulating GPX4. NORAD promoted YTHDF2-mediated m6A modification to reduce GPX4 mRNA stability by interacting with FTO. YTHDF2 silencing or FTO overexpression partially averted NORAD-promoted ECC ferroptosis by modulating GPX4. NORAD promoted m6A modification to down-regulate GPX4 by interacting with FTO, and promoted ferroptosis to inhibit tumor growth in vivo. NORAD was down-regulated in EC and affected the EC prognosis. NORAD overexpression facilitated YTHDF2-mediated m6A modification by interacting with FTO to elevate GPX4 degradation, thereby stimulating ECC ferroptosis and hindering EC progression.