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Emerging evidence shows that the efficacy of chemotherapeutic drugs is reliant on their capability to induce immunogenic cell death (ICD), thus transforming dying tumor cells into antitumor vaccines. We wanted to uncover potential therapeutic strategies that target ovarian cancer by having a better understanding of the standard-of-care chemotherapy treatment. Here, we showed in ovarian cancer that paclitaxel induced ICD-associated damage-associated molecular patterns (DAMP, such as CALR exposure, ATP secretion, and HMGB1 release) in vitro and elicited significant antitumor responses in tumor vaccination assays in vivo. Paclitaxel-induced TLR4 signaling was essential to the release of DAMPs, which led to the activation of NF-κB–mediated CCL2 transcription and IkappaB kinase 2–mediated SNARE-dependent vesicle exocytosis, thus exposing CALR on the cell surface. Paclitaxel induced endoplasmic reticulum stress, which triggered protein kinase R–like ER kinase activation and eukaryotic translation initiation factor 2α phosphorylation independent of TLR4. Paclitaxel chemotherapy induced T-cell infiltration in ovarian tumors of the responsive patients; CALR expression in primary ovarian tumors also correlated with patients' survival and patient response to chemotherapy. These findings suggest that the effectiveness of paclitaxel relied upon the activation of antitumor immunity through ICD via TLR4 and highlighted the importance of CALR expression in cancer cells as an indicator of response to paclitaxel chemotherapy in ovarian cancer.

All Supplementary Figures and their respective legends.

Supplementary Table S1

Supplemental Tables S1-S5. Characteristics of the 45 normal tissues included in the PCDH10 expression analysis (S1); Characteristics of the 9 normal tissues included in the MALAT1 expression by ISH analysis (S2); Characteristics at primary treatment of 142 endometrial cancer patients from MoMaTEC* trial included in the MALAT1 investigation (S3); PCDH10 RNA expression from 142 prospectively collected endometrial cancer tumors, 19 metastatic lesions and 18 endometrial hyperplasia samples in relation to clinicopathologic data (S4); Oligos used for qRT-PCR, ChIP-PCR, Cloning and Bisulfate genomic sequencing (S5).

Background Previous studies have shown a major green tea polyphenol (−)-epigallocatechin-3-gallate ((−)-EGCG) as a powerful anti-cancer agent. However, its poor bioavailability and requirement of a high dosage to manifest activity have restricted its clinical application. Recently, our team synthesized a peracetate-protected derivative of EGCG, which can act as a prodrug of (−)-EGCG (ProEGCG) with enhanced stability and improved bioavailability in vitro and in vivo. Herein, we tested the therapeutic efficacy of this novel ProEGCG, in comparison to EGCG, toward human endometrial cancer (EC). Methods In this study, the effects of ProEGCG and EGCG treatments on cell growth, cell survival and modulation of intracellular signaling pathways in RL95–2 and AN3 CA EC cells were compared. The antiproliferative effect was evaluated by cell viability assay. Apoptosis was measured by annexin/propidium iodide staining. Expression of mitogen-activated protein kinases, markers of proliferation and apoptosis were measured by immunoblot analysis. In addition, the effects of ProEGCG and EGCG on tumor growth, vessel formation and gene expression profiles on xenograft models of the EC cells were investigated. Results We found that treatment with ProEGCG, but not EGCG, inhibited, in a time- and dose-dependent manner, the proliferation and increased apoptosis of EC cells. Treatment with low-dose ProEGCG significantly enhanced phosphorylation of JNK and p38 MAPK and inhibited phosphorylation of Akt and ERK which are critical mediators of apoptosis. ProEGCG, but not EGCG, elicited a significant decrease in the growth of the EC xenografts, promoted apoptotic activity of tumour cells in the EC xenografts, and decreased microvessel formation, by differentially suppressing anti-apoptotic molecules, NOD1 and NAIP. Notably, no obvious adverse effects were detected. Conclusions Taken together, ProEGCG at a low dose exhibited anticancer activity in EC cells through its anti-proliferative, pro-apoptotic and anti-tumor actions on endometrial cancer in vitro and in vivo. In contrast, a low dose of EGCG did not bring about similar effects. Importantly, our data demonstrated the efficacy and safety of ProEGCG which manifests the potential of a novel anticancer agent for the management of endometrial cancer.

Emerging evidence shows that the efficacy of chemotherapeutic drugs is reliant on their capability to induce immunogenic cell death (ICD), thus transforming dying tumor cells into antitumor vaccines. We wanted to uncover potential therapeutic strategies that target ovarian cancer by having a better understanding of the standard-of-care chemotherapy treatment. Here, we showed in ovarian cancer that paclitaxel induced ICD-associated damage-associated molecular patterns (DAMP, such as CALR exposure, ATP secretion, and HMGB1 release) in vitro and elicited significant antitumor responses in tumor vaccination assays in vivo. Paclitaxel-induced TLR4 signaling was essential to the release of DAMPs, which led to the activation of NF-κB–mediated CCL2 transcription and IkappaB kinase 2–mediated SNARE-dependent vesicle exocytosis, thus exposing CALR on the cell surface. Paclitaxel induced endoplasmic reticulum stress, which triggered protein kinase R–like ER kinase activation and eukaryotic translation initiation factor 2α phosphorylation independent of TLR4. Paclitaxel chemotherapy induced T-cell infiltration in ovarian tumors of the responsive patients; CALR expression in primary ovarian tumors also correlated with patients' survival and patient response to chemotherapy. These findings suggest that the effectiveness of paclitaxel relied upon the activation of antitumor immunity through ICD via TLR4 and highlighted the importance of CALR expression in cancer cells as an indicator of response to paclitaxel chemotherapy in ovarian cancer.

Simple Summary Cytokines are a group of small proteins in the body that play an important part in boosting the immune system. Thymic stromal lymphopoietin (TSLP) is a cytokine that plays an important role in the maturation of T cells. Two variants of TSLP, long-form (lfTSLP) and short-form (sfTSLP), have been found, however their roles in cancers are not known. In this study, we discovered that sfTSLP, but not lfTSLP, is predominantly expressed in ovarian and endometrial cancers. The switch that turns the sfTSLP gene on or off is controlled by external modifications of DNA. Our results also found that sfTSLP promotes tumour growth through activating several signal pathways in cancer cells. Abstract Thymic stromal lymphopoietin (TSLP) is an epithelial cell derived cytokine belonging to the IL-7 family and a key initiator of allergic inflammation. Two main isoforms of TSLP, classified as long- (lfTSLP) and short-form (sfTSLP), have been reported in human, but their expression patterns and role(s) in cancers are not yet clear. mRNA expression was examined by isoform-specific RT-PCR and RNA in situ hybridisation. Epigenetic regulation was investigated by chromatin immunoprecipitation-PCR and bisulfite sequencing. Tumour progression was investigated by gene overexpression, cell viability assay, cancer organoid culture and transwell invasion. Signals were investigated by proteome profiler protein array and RNA-sequencing. With the use of isoform-specific primers and probes, we uncovered that only sfTSLP was expressed in the cell lines and tumour tissues of human ovarian and endometrial cancers. We also showed the epigenetic regulation of sfTSLP: sfTSLP transcription was regulated by histone acetylation at promoters in ovarian cancer cells, whereas silencing of the sfTSLP transcripts was regulated by promoter DNA methylation in endometrial cancer cells. In vitro study showed that ectopically overexpressing sfTSLP promoted tumour growth but not invasion. Human phosphokinase array application demonstrated that the sfTSLP overexpression activated phosphorylation of multiple intracellular kinases (including GSK3α/β, AMPKα1, p53, AKT1/2, ERK1/2 and Src) in ovarian cancer cells in a context-dependent manner. We further investigated the impact of sfTSLP overexpression on transcriptome by RNA-sequencing and found that EFNB2 and PBX1 were downregulated in ovarian and endometrial cancer cells, suggesting their role in sfTSLP-mediated tumour growth. In conclusion, sfTSLP is predominantly expressed in ovarian and endometrial cancers and promotes tumour growth.

Although the rates of cervical squamous cell carcinoma have been declining, the rates of cervical adenocarcinoma are increasing in some countries. Outcomes for advanced cervical adenocarcinoma remain poor. Precision mapping of genetic alterations in cervical adenocarcinoma may enable better selection of therapies and deliver improved outcomes when combined with new sequencing diagnostics. We present whole-exome sequencing results from 15 cervical adenocarcinomas and paired normal samples from Hong Kong Chinese women. These data revealed a heterogeneous mutation spectrum and identified several frequently altered genes including FAT1, ARID1A, ERBB2 and PIK3CA. Exome sequencing identified human papillomavirus (HPV) sequences in 13 tumors in which the HPV genome might have integrated into and hence disrupted the functions of certain exons, raising the possibility that HPV integration can alter pathways other than p53 and pRb. Together, these provisionary data suggest the potential for individualized therapies for cervical adenocarcinoma based on genomic information.

Immunogenic cell death refers to a phenomenon in which some of the cytotoxic chemotherapeutics kills the tumor cells and induces these dying cells to be immunogenic. The aim of this study is to investigate if the standard-of-care chemotherapy (carboplatin and paclitaxel) induces immunogenic cell death in ovarian cancer. We found that paclitaxel, but not carboplatin, induces multiple molecular determinants of immunogenic cell death (namely calreticulin exposure, ATP secretion, HMGB1 release, ANXA1 expression, and upregulation of CXCL10) in ID8 mouse ovarian cancer cells. By tumor vaccination assay in immunocompetent syngeneic mice, our results showed paclitaxel induces immunogenic cell death in the mice with ID8 ovarian tumor. Since toll-like receptor 4 (TLR4) is a receptor for paclitaxel, we generated isogenic derivatives of ID8 cells with Tlr4 knockout (KO) to investigate if paclitaxel induces immunogenic cell death is dependent on TLR4. Our results showed that the paclitaxel-induced molecular determinants of immunogenic cell death were diminished in the TLR4 KO clones when compared to wild-type controls. Moreover, the tumor vaccination assay showed that paclitaxel was not able to induce immunogenic cell death in mice with ovarian tumor with TLR4 KO. These results indicated that the paclitaxel requires TLR4 to induce immunogenic cell death. To explore the mechanisms of how paclitaxel induces immunogenic cell death through TLR4, we investigated the activation of PERK and phosphorylation of eIF2a in ER stress since they are the first two enumerate elements of the pathways that mediated calreticulin exposure in response to immunogenic anticancer chemotherapeutics. Our results, however, showed that paclitaxel induces activation of PERK and phosphorylation of eIF2a in ovarian cancer cells is independent on TLR4. We then investigate if paclitaxel induces SNARE-dependent exocytosis in mouse ovarian cancer cells because SNARE-dependent exocytosis is the final step of the pathways that mediated calreticulin exposure in immunogenic cell death. Our results showed that paclitaxel induces SNARE-dependent exocytosis of ATP-containing vesicles in mouse ovarian cancer cells via TLR4-mediated IKK2 activation. In summary, our results showed that paclitaxel induces immunogenic cell death in ovarian cancer through TLR4-independent and dependent pathways, in which paclitaxel-induced exocytosis is essential for the release of multiple molecular determinants of immunogenic cell death. Our results provide new evidence that the antitumor effect of paclitaxel occurs in part via activation of an immune response against cancer by inducing immunogenic cell death and provide a rationale for a new combination of paclitaxel and immunotherapies as an anticancer treatment. Citation Format: Tat-San Lau, Loucia Kit-Ying Chan, Gene Chi-Wai Man, Joseph Kwong. Paclitaxel induces immunogenic cell death in ovarian cancer via TLR4-independent and dependent pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1232.

We have investigated the role of cytokine lymphotoxin in tumour-stromal interactions in human ovarian cancer. We found that lymphotoxin overexpression is commonly shared by the cancer cells of various ovarian cancer subtypes, and lymphotoxin-beta receptor (LTBR) is expressed ubiquitously in both the cancer cells and cancer-associated fibroblasts (CAFs). In monoculture, we showed that ovarian cancer cells are not the major lymphotoxin-responsive cells. On the other hand, our co-culture studies demonstrated that the cancer cell-derived lymphotoxin induces chemokine expression in stromal fibroblasts through LTBR-NF-κB signalling. Amongst the chemokines being produced, we found that fibroblast-secreted CXCL11 promotes proliferation and migration of ovarian cancer cells via the chemokine receptor CXCR3. CXCL11 is highly expressed in CAFs in ovarian cancer biopsies, while CXCR3 is found in malignant cells in primary ovarian tumours. Additionally, the overexpression of CXCR3 is significantly associated with the tumour grade and lymph node metastasis of ovarian cancer, further supporting the role of CXCR3, which interacts with CXCL11, in promoting growth and metastasis of human ovarian cancer. Taken together, these results demonstrated that cancer-cell-derived lymphotoxin mediates reciprocal tumour-stromal interactions in human ovarian cancer by inducing CXCL11 in fibroblasts. Our findings suggest that lymphotoxin-LTBR and CXCL11-CXCR3 signalling represent therapeutic targets in ovarian cancer.

Green tea epigallocatechin-3-gallate (EGCG) can inhibit angiogenesis and development of an experimental endometriosis model in mice, but it suffers from poor bioavailability. A prodrug of EGCG (pro-EGCG, EGCG octaacetate) is utilized to enhance the stability and bioavailability of EGCG in vivo. In this study, the potential of pro-EGCG as a potent anti-angiogenesis agent for endometriosis in mice was investigated. Homologous endometrium was subcutaneously transplanted into mice to receive either saline, vitamin E, EGCG or pro-EGCG treatment for 4 weeks. The growth of the endometrial implants were monitored by IVIS(®) non-invasive in vivo imaging during the interventions. Angiogenesis of the endometriotic lesions was determined by Cellvizio(®) in vivo imaging and SCANCO(®) Microfil microtomography. The bioavailability, anti-oxidation and anti-angiogenesis capacities of the treatments were measured in plasma and lesions. The implants with adjacent outer subcutaneous and inner abdominal muscle layers were collected for histological, microvessel and apoptosis examinations. The result showed that EGCG and pro-EGCG significantly decreased the growth of endometrial implants from the 2nd week to the 4th week of intervention. EGCG and pro-EGCG significantly reduced the lesion size and weight, inhibited functional and structural microvessels in the lesions, and enhanced lesion apoptosis at the end of interventions. The inhibition by pro-EGCG in all the angiogenesis parameters was significantly greater than that by EGCG, and pro-EGCG also had better bioavailability and greater anti-oxidation and anti-angiogenesis capacities than EGCG. Ovarian follicles and uterine endometrial glands were not affected by either EGCG or pro-EGCG. Vitamin E had no effect on endometriosis. In conclusion, pro-EGCG significantly inhibited the development, growth and angiogenesis of experimental endometriosis in mice with high efficacy, bioavailability, anti-oxidation and anti-angiogenesis capacities. Pro-EGCG could be a potent anti-angiogenesis agent for endometriosis.

RNA interference (RNAi) has become one of the most powerful and popular approach on gene silencing in clinical research study especially in virology due to the gene-specific suppression property of small interfering RNA (siRNA). In this report, we demonstrate that expression of vector-mediated small hairpin RNA (shRNA) against human immunodeficiency virus type 1 (HIV-1) integrase (IN), one of the three important enzymes in HIV infection by controlling the integration of viral RNA to host DNA, could suppress the protein synthesis of EGFP-tagged IN in HeLa cell model efficiently. Furthermore, we show that IN shRNA can successfully reduce the HIV particles production in 293T cells at the level similar to the positive control of HIV-1 tat shRNA. These results provide the therapeutic possibility of HIV replication using RNAi against HIV-1 integrase.

The purpose of this study is to investigate underlying mechanism of how cytokine interferon-gamma (IFN-γ) regulates PD-L1 expression in ovarian cancer cells. We treated a panel of human and mouse ovarian cancer cell lines with recombinant human/mouse IFN-γ. Our data showed that IFN-γ up-regulated mRNA and protein expression of PD-L1 significantly in a majority of ovarian cancer cells. The functional IFN-γ receptor is comprised of two ligand-binding IFNGR1 chains associated with two signal-transducing IFNGR2 chains and associated signaling machinery. Here we found that the mRNA expression levels of IFNGR1 and IFNGR2 were abundant in all human ovarian cancer cell lines being tested, while their expressions were not affected by IFN-γ treatment. After knocking down the expression levels of IFNGR1 and IFNGR2 in a ovarian cancer cell line by target gene-specific siRNA, our data showed that the IFN-γ-mediated induction of PD-L1 were diminished in the ovarian cancer cells when compared to those with non-targeting scrambled siRNA controls, indicating the induction of PD-L1 by IFN-γ is dependent on the presence of IFN-γ receptors in the ovarian cancer cells. Although abundant expression of IFNGR1 and IFNGR2 were found in all human ovarian cancer cell lines being tested, the IFN-γ-mediated induction of PD-L1 was not detected in a few of the human ovarian cancer cell lines (namely IGROV-1, TOV21G and SKOV3). We further investigated the integrity of IFN-γ signaling in the human ovarian cancer cell lines by examining activation of STAT1 protein and induction of IRF-1 gene in human ovarian cancer cell lines after IFN-γ treatment. Our data showed that phosphorylated-STAT1 protein and IRF-1 gene expression were up-regulated significantly in a majority of human ovarian cancer cells after IFN-γ treatment, except IGROV-1 and TOV21G cells. These results suggested that IGROV-1 and TOV21G cells might harbor defects in intracellular JAK-STAT1 signaling. We then examined the presence of JAK1 truncating mutations in human ovarian cancer cell lines by Sanger sequencing, and confirmed that IGROV-1 and TOV21G cells, but not the others, have JAK1 truncating mutations. Since our data showed that SKOV3 cells have wild type JAK1, we further investigated other possible defects in IFN-γ signaling in SKOV3 cells. We investigated the IFN-γ-induced STAT3 protein activation in human ovarian cancer cell lines, and defects were found in Y705 STAT3 phosphorylation in SKOV3 as well as in IGROV-1 and TOV21G cells. To summarize, our results showed that IFN-γ induces PD-L1 expression in ovarian cancer cells via IFNGR-JAK-STAT pathway. The failure of IFN-γ-mediated induction of PD-L1 in a minority group of human ovarian cancer cell lines is due to defective IFN-γ signaling, including JAK1 truncating mutations and impaired STAT3 activation. This work is supported by Hong Kong Research Grants Council General Research Fund (467713 and 14109515). Citation Format: Tat-San Lau, Loucia Kit-Ying Chan, Tak-Hong Cheung, So-Fan Yim, Jacqueline Ho-Sze Lee, Joseph Kwong. Interferon-gamma induces PD-L1 expression via IFNGR-JAK-STAT pathway in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 648. doi:10.1158/1538-7445.AM2017-648

Although the rates of cervical squamous cell carcinoma have been declining, the rates of cervical adenocarcinoma are increasing in some countries. Outcomes for advanced cervical adenocarcinoma remain poor. Precision mapping of genetic alterations in cervical adenocarcinoma may enable better selection of therapies and deliver improved outcomes when combined with new sequencing diagnostics. We present whole-exome sequencing results from 15 cervical adenocarcinomas and paired normal samples from Hong Kong Chinese women. These data revealed a heterogeneous mutation spectrum and identified several frequently altered genes including FAT1, ARID1A, ERBB2 and PIK3CA. Exome sequencing identified human papillomavirus (HPV) sequences in 13 tumors in which the HPV genome might have integrated into and hence disrupted the functions of certain exons, raising the possibility that HPV integration can alter pathways other than p53 and pRb. Together, these provisionary data suggest the potential for individualized therapies for cervical adenocarcinoma based on genomic information.

The Protocadherin 10 (PCDH10) is inactivated often by promoter hypermethylation in various human tumors, but its possible functional role as a tumor suppressor gene is not established. In this study, we identify PCDH10 as a novel Wnt pathway regulatory element in endometrioid endometrial carcinoma (EEC). PCDH10 was downregulated in EEC tumor cells by aberrant methylation of its promoter. Restoring PCDH10 levels suppressed cell growth and triggered apoptosis in EEC cells and tumor xenografts. Gene expression profiling revealed as part of the transcriptomic changes induced by PCDH10 a reduction in levels of MALAT1, a long noncoding RNA, that mediated tumor suppression functions of PCDH10 in EEC cells. We found that MALAT1 transcription was regulated by Wnt/β-catenin signaling via TCF promoter binding and PCDH10 decreased MALAT1 by modulating this pathway. Clinically, MALAT1 expression was associated with multiple parameters in patients with EEC. Taken together, our findings establish a novel PCDH10–Wnt/β-catenin–MALAT1 regulatory axis that contributes to EEC development. Cancer Res; 74(18); 5103–17. ©2014 AACR.

We have investigated the role of cytokine lymphotoxin in tumour-stromal interactions in human ovarian cancer. We found that lymphotoxin overexpression is commonly shared by the cancer cells of various ovarian cancer subtypes, and lymphotoxin-beta receptor (LTBR) is expressed ubiquitously in both the cancer cells and cancer-associated fibroblasts (CAFs). In monoculture, we showed that ovarian cancer cells are not the major lymphotoxin-responsive cells. On the other hand, our co-culture studies demonstrated that the cancer cell-derived lymphotoxin induces chemokine expression in stromal fibroblasts through LTBR-NF-κB signalling. Amongst the chemokines being produced, we found that fibroblast-secreted CXCL11 promotes proliferation and migration of ovarian cancer cells via the chemokine receptor CXCR3. CXCL11 is highly expressed in CAFs in ovarian cancer biopsies, while CXCR3 is found in malignant cells in primary ovarian tumours. Additionally, the overexpression of CXCR3 is significantly associated with the tumour grade and lymph node metastasis of ovarian cancer, further supporting the role of CXCR3, which interacts with CXCL11, in promoting growth and metastasis of human ovarian cancer. Taken together, these results demonstrated that cancer-cell-derived lymphotoxin mediates reciprocal tumour-stromal interactions in human ovarian cancer by inducing CXCL11 in fibroblasts. Our findings suggest that lymphotoxin-LTBR and CXCL11-CXCR3 signalling represent therapeutic targets in ovarian cancer.

Peritoneum is the commonest site for ovarian cancer metastasis. In peritoneum, stromal fibroblasts are the second most common cell types. In this study, we aimed to investigate the role of tumor-stromal interaction in peritoneal metastasis of ovarian cancer. Using three-dimensional (3D) organoid co-culture model, we found that the interaction between metastatic human ovarian cancer cells and normal human stromal fibroblasts promotes cancer colony formation. Using cytokine antibody array, we identified a group of cytokines are de novo produced in the 3D co-culture. Among them, transforming growth factor alpha (TGF-α) was produced anew from normal stromal fibroblasts in the 3D co-culture. In the co-culture, transcription of TGF-α in normal stromal fibroblasts is activated by tumor necrosis factor alpha (TNF-α) that derived by ovarian cancer cells. By epigenetic studies, we discovered that over-expression of TNF-α in human ovarian cancer cells is due to DNA hypomethylation and chromatin remodeling in TNF-α promoter. TGF-α is a ligand of epidermal growth factor receptor (EGFR), and we confirmed EGFR is over-expressed in a significant portion of human ovarian cancer. When applying EGFR-specific inhibitors (AG1478, Gefitini, and Erlotinib) in our 3D organoid models (3D mono-cultures and 3D co-cultures), we further found that the fibroblast-derived TGF-α promotes colony formation of metastatic ovarian cancer cells in vitro through EGFR activation. In fact, TGF-α activates Hippo/YAP signaling pathway in human ovarian cancer cells via EGFR. In addition, our in vivo experiments showed that normal stromal fibroblasts promote peritoneal metastasis of ovarian cancer through EGFR activation in a xenograft model. Finally, the TNFα-TGFα-EGFR interacting loop was detected in cancer-stroma-cancer compartments of omental metastases of ovarian cancer patients by immunohistochemistry. Based on our experimental results, we proposed a cancer-stroma-cancer interacting loop that promotes peritoneal metastasis of ovarian cancer through TNFα-TGFα-EGFR. The proposed mechanism involves: 1) TNF-α is over-expressed in human ovarian cancer cells by promoter DNA hypomethylation and chromatin remodeling; 2) the ovarian cancer cell-derived TNF-α induces de novo TGF-α production in normal human omental stromal fibroblasts; and 3) the fibroblast-derived TGF-α, in turn, promotes colony formation of metastatic ovarian cancer cells through EGFR-mediated Hippo/YAP signaling pathway. Our results thereby suggest that peritoneal metastasis in the ovarian cancer patients after cytoreductive surgery can be prevented by EGFR-targeted therapy. Citation Format: Tat-San Lau, Loucia Kit-Ying Chan, Chi-Hang Wong, Chi-Wai Man, Tak-Hong Cheung, So-Fan Yim, Jacqueline Ho-Sze Lee, Tony Kwok-Hung Chung, Joseph Kwong. A cancer-stroma-cancer interacting loop promotes peritoneal metastasis of ovarian cancer through TNFα-TGFα-EGFR. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1566.

MicroRNAs (miRNAs) regulate mRNA stability and protein expression, and certain miRNAs have been demonstrated to act either as oncogenes or tumor suppressors. Differential miRNA expression signatures have been documented in many human cancers but the role of miRNAs in endometrioid endometrial cancer (EEC) remains poorly understood. This study identifies significantly dysregulated miRNAs of EEC cells, and characterizes their impact on the malignant phenotype. We studied the expression of 365 human miRNAs using Taqman low density arrays in EECs and normal endometriums. Candidate differentially expressed miRNAs were validated by quantitative real-time PCR. Expression of highly dysregulated miRNAs was examined in vitro through the effect of anti-/pre-miRNA transfection on the malignant phenotype. We identified 16 significantly dysregulated miRNAs in EEC and 7 of these are novel findings with respect to EEC. Antagonizing the function of miR-7, miR-194 and miR-449b, or overexpressing miR-204, repressed migration, invasion and extracellular matrix-adhesion in HEC1A endometrial cancer cells. FOXC1 was determined as a target gene of miR-204, and two binding sites in the 3'-untranslated region were validated by dual luciferase reporter assay. FOXC1 expression was inversely related to miR-204 expression in EEC. Functional analysis revealed the involvement of FOXC1 in migration and invasion of HEC1A cells. Our results present dysfunctional miRNAs in endometrial cancer and identify a crucial role for miR-204-FOXC1 interaction in endometrial cancer progression. This miRNA signature offers a potential biomarker for predicting EEC outcomes, and targeting of these cancer progression- and metastasis-related miRNAs offers a novel potential therapeutic strategy for the disease.

Tumor colonization involves reciprocal interactions between the metastatic cancer cells and various stromal cells in foreign microenvironment. Fibroblasts are the second most numerous cell types in omentum, which is the commonest site of ovarian cancer metastasis. However, the crosstalk between metastatic ovarian cancer cells and fibroblasts that involved in tumor colonization remains largely unknown. In this study, we used a three-dimensional (3D) co-culture model to study the role of normal fibroblasts on tumor colonization of ovarian cancer cells. Human ovarian cancer cell lines, which were established from peritoneal ascites, were seeded on normal fibroblasts embedded in extracellular matrix (Matrigel). Our results showed that ovarian cancer spheroids formed in Matrigel with fibroblasts were significantly larger and also more invasive than those formed in Matrigel without fibroblasts, suggesting the cancer-fibroblast interaction promotes the colonization of ovarian cancer cells. To search the signal, we compared the cytokine profile of the 3D co-culture with 3D monoculture of ovarian cancer cells/fibroblasts. De novo production of TGF-α was found in the fibroblasts of the co-culture. To determine whether TGF-α promotes the colonization of ovarian cancer cells, we treated the monoculture of ovarian cancer cells in Matrigel with recombinant human TGF-α (rhTGF-α). rhTGF-α significantly promoted the growth and invasion of ovarian cancer spheroids. Additionally, our qRT-PCR results demonstrated that EGFR, a receptor for TGF-α, was over-expressed in primary human ovarian cancer tissues when compared to non-malignant human ovarian tissues. To test whether fibroblast-derived TGF-α promotes tumor colonization of ovarian cancer cells through EGFR, we treated our in-vitro colonization assays with an EGFR inhibitor, AG1478. AG1478 significantly reduced the growth and invasion of rTGFα-induced ovarian cancer spheroids and the cancer spheroids formed in the co-culture, indicating the colonization of human ovarian cancer cells is promoted by cancer-fibroblast interaction via TGFα-EGFR signaling. We also found that Erlotinib and Gefitinb, two clinically approved EGFR-specific tyrosine kinase inhibitors, significantly reduced the growth and invasion of ovarian cancer spheroids in our in-vitro colonization assays. This result implicates the peritoneal metastasis of ovarian cancer can be potentially treated by EGFR-targeted therapy. (This project is supported by CUHK Research Committee Funding). Citation Format: Tat-San Lau, Tony Kwok-Hung Chung, Tak-Hong Cheung, So-Fan Yim, Chi-Hang Wong, Joseph Kwong. Colonization of human ovarian cancer cells is promoted by cancer-fibroblast interaction via TGFα-EGFR signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A66.

microRNAs (miRNAs) regulate gene expression and some have been shown to function as either oncogenes or tumor suppressors (oncomirs). Changes in miRNA expression have been observed in many human cancers but their role in endometrioid endometrial cancer (EEC) remains poorly understood. We studied the expression of 365 human miRNAs using Taqman low density arrays in 98 EECs as well as 60 normal endometria. Potentially interesting miRNAs were then validated by qRT-PCR. Expression of highly deregulated miRNAs on malignant cell properties was also examined in vitro using pre-/anti-miRNA transfection. Predicted mRNA targets for the miRNA of interest were also verified. We identified 16 significantly deregulated miRNAs in EEC and 7 of these are reported for the first time. Of these, miR-7, miR-194 and miR-449b knockdown and miR-204 induction repressed migration, invasion and extracellular matrix-adhesion in HEC1A endometrial cancer cells. For the cancer progression dysregulation of the expression of miR204 mediates migration and invasion of endometrial cancer by regulating FOXC1-related miR-204, FOXC1 was determined as its target gene possessing two miR-204-binding sites at the 3’-untranslated region. FOXC1 expression was inversely related to miR-204 expression in EEC. Functional analysis also revealed the involvement of FOXC1 in migration of HEC1A cells. We have identified dysfunctional miRNAs in endometrial cancer and elucidated the crucial role of miR-204-FOXC1 interaction in endometrial cancer progression. This work provides insight into miRNA-based diagnostic and therapeutic approaches for endometrial cancers.

[Introduction] Endometrial cancer has now become the most common gynaecologic malignancy worldwide. About 80-90 % of endometrial cancer are endometrioid endometrial adenocarcinomas (EEC). MicroRNAs (miRNAs) are short, non-coding RNAs that regulate gene expression by inducing cleavage of the targeted messenger or by inhibiting translation. Our entire study was to mine miRNA signatures involved in EEC tumorigenesis and progression. Here we report a part of the results that is on miRNAs associated with endometrial cancer progression in vitro. [Methods] We examined the expression of 365 mature miRNAs by real-time RT-PCR based plate assays in 52 EEC including 30 advanced- and 22 early-stage tumors, and compared that to 30 normal counterparts. To know about the deregulated miRNAs associated with endometrial cell behavior, miRNA inhibitor and pre-miRNA were used to inhibit up-regulated miRNA and induce down-regulated miRNA in highly invasive endometrial cancer cell line HEC-1A cells. Quantitative TaqMan RT-PCR was performed to measure miRNA levels in cells before and after treatment, respectively. Migration and invasion assays were carried out to evaluate effects of those deregulated miRNAs on invasion of endometrial cancer cells in vitro. [Results] Supervised clustering analysis of global miRNA expression in endometrial caner and normal control identified 17 miRNAs up-regulated and 2 miRNAs down-regulated, respectively, in EEC. When the miRNA expression profiles in advanced stage tumors were compared to those in early stage tumors, 6 miRNAs were significantly over-expressed and one miRNA under-expressed in advanced stage tumors. Cell migration and invasion assay showed the ability of cell migration and invasion was significantly inhibited by anti-miR-7, anti-miR-194, anti-miR-449b and pre-miR-204 in HEC-1A cells in vitro. [Conclusion] Unique miRNA signatures seem to be existed in ECC. Deregulated miRNAs miR-7, miR-194, miR-204 and miR-449b might mediate endometrial cancer progress. These miRNAs are potentially to be developed as novel diagnostic and/or prognostic molecular markers as well as candidate therapeutic targets of EEC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4047.