Generation of glioblastoma in mice engrafted with human cytomegalovirus-infected astrocytes.

Mounting evidence is identifying human cytomegalovirus (HCMV) as a potential oncogenic virus. HCMV has been detected in glioblastoma multiforme (GB). Herewith, we present the first experimental evidence for the generation of CMV-Elicited Glioblastoma Cells (CEGBCs) possessing glioblastoma-like traits that lead to the formation of glioblastoma in orthotopically xenografted mice. In addition to the already reported oncogenic HCMV-DB strain, we isolated three HCMV clinical strains from GB tissues that transformed HAs toward CEGBCs and generated spheroids from CEGBCs that resulted in the appearance of glioblastoma-like tumors in xenografted mice. These tumors were nestin-positive mostly in the invasive part surrounded by GFAP-positive reactive astrocytes. The glioblastoma immunohistochemistry phenotype was confirmed by EGFR and cMet gene amplification in the tumor parallel to the detection of HCMV IE and UL69 genes and proteins. Our results fit with an HCMV-induced glioblastoma model of oncogenesis in vivo which will open the door to new therapeutic approaches and assess the anti-HCMV treatment as well as immunotherapy in fighting GB which is characterized by poor prognosis.

Polyploid Giant Cancer Cells Generated from Human Cytomegalovirus-Infected Prostate Epithelial Cells.

BACKGROUND: Prostate cancer is the most commonly diagnosed malignancy and the sixth leading cause of cancer death in men worldwide. Chromosomal instability (CIN) and polyploid giant cancer cells (PGCCs) have been considered predominant hallmarks of cancer. Recent clinical studies have proven the association of CIN, aneuploidy, and PGCCs with poor prognosis of prostate cancer (PCa). Evidence of HCMV transforming potential might indicate that HCMV may be involved in PCa. METHODS: Herein, we underline the role of the high-risk HCMV-DB and -BL clinical strains in transforming prostate epithelial cells and assess the molecular and cellular oncogenic processes associated with PCa. RESULTS: Oncogenesis parallels a sustained growth of "CMV-Transformed Prostate epithelial cells" or CTP cells that highly express Myc and EZH2, forming soft agar colonies and displaying stemness as well as mesenchymal features, hence promoting EMT as well as PGCCs and a spheroid appearance. CONCLUSIONS: HCMV-induced Myc and EZH2 upregulation coupled with stemness and EMT traits in IE1-expressing CTP might highlight the potential role of HCMV in PCa development and encourage the use of anti-EZH2 and anti-HCMV in PCa treatment.

Polyploidy, EZH2 upregulation, and transformation in cytomegalovirus-infected human ovarian epithelial cells.

Human cytomegalovirus (HCMV) infection has been implicated in epithelial ovarian cancer (OC). Polyploidy giant cancer cells (PGCCs) have been observed in high-grade serous ovarian carcinoma (HGSOC); they possess cancer stem cell-like characteristics and give rise to progeny cells expressing epithelial-mesenchymal transition (EMT) markers. EZH2 plays a potential oncogenic role, correlating with high proliferative index and tumor grade in OC. Herein, we present the experimental evidence for HCMV as a reprogramming vector that elicited human ovarian epithelial cells (OECs) transformation leading to the generation of "CMV-transformed Ovarian cells" (CTO). The infection with the two high-risk clinical strains, namely HCMV-DB and BL provoked a distinct cellular and molecular mechanisms in infected OECs. EZH2 upregulation and cellular proliferation were curtailed by using EZH2 inhibitors. The HGSOC biopsies were characterized by an elevated EZH2 expression, possessing a strong positive correlation between the aforementioned marker and HCMV. From HGSOC biopsies, we isolated three HCMV clinical strains that transformed OECs generating CTO cells which displayed proliferative potentials in addition to EZH2 upregulation and PGCCs generation; these features were reduced upon EZH2 inhibition. High-risk HCMV strains transformed OECs confirming an HCMV-induced epithelial ovarian cancer model and highlighting EZH2 tumorigenic properties. Our findings might be highly relevant in the pathophysiology of ovarian tumors thereby nominating new targeted therapeutics.

Polyploid giant cancer cells, cytokines and cytomegalovirus in breast cancer progression.

BACKGROUND: Breast cancer is the most common cancer among women. Accumulated evidence over the past decades indicates a very high prevalence of human cytomegalovirus (HCMV) in breast cancer. High-risk HCMV strains possess a direct oncogenic effect displayed by cellular stress, polyploid giant cancer cells (PGCCs) generation, stemness, and epithelial-to-mesenchymal transition (EMT) leading to cancer of aggressive phenotype. Breast cancer development and progression have been regulated by several cytokines where the latter can promote cancer cell survival, help in tumor immune evasion, and initiate the EMT process, thereby resulting in invasion, angiogenesis, and breast cancer metastasis. In the present study, we screened cytokines expression in cytomegalovirus-transformed HMECs (CTH cells) cultures infected with HCMV high-risk strains namely, HCMV-DB and BL, as well as breast cancer biopsies, and analyzed the association between cytokines production, PGCCs count, and HCMV presence in vitro and in vivo. METHODS: In CTH cultures and breast cancer biopsies, HCMV load was quantified by real-time qPCR. PGCCs count in CTH cultures and breast cancer biopsies was identified based on cell morphology and hematoxylin and eosin staining, respectively. CTH supernatants were evaluated for the production of TGF-ß, IL-6, IL1-ß, and IL-10 by ELISA assays. The above-mentioned cytokines expression was assessed in breast cancer biopsies using reverse transcription-qPCR. The correlation analyses were performed using Pearson correlation test. RESULTS: The revealed PGCCs/cytokine profile in our in vitro CTH model matched that of the breast cancer biopsies, in vivo. Pronounced cytokine expression and PGCCs count were detected in particularly CTH-DB cultures and basal-like breast cancer biopsies. CONCLUSIONS: The analysis of cytokine profiles in PGCCs present mostly in basal-like breast cancer biopsies and derived from CTH cells chronically infected with the high-risk HCMV strains might have the potential to provide novel therapies such as cytokine-based immunotherapy which is a promising field in cancer treatments.

EZH2-Myc driven glioblastoma elicited by cytomegalovirus infection of human astrocytes.

Mounting evidence is identifying human cytomegalovirus (HCMV) as a potential oncogenic virus. HCMV has been detected in malignant gliomas. EZH2 and Myc play a potential oncogenic role, correlating with the glioma grade. Herewith, we present the first experimental evidence for HCMV as a reprogramming vector, straight through the dedifferentiation of mature human astrocytes, and generation of CMV-Elicited Glioblastoma Cells (CEGBCs) possessing glioblastoma-like traits. HCMV counterparts the progression of the perceived cellular and molecular mechanisms succeeding the transformation and invasion processes with CEGBCs involved in spheroid formation and invasiveness. Glioblastoma multiforme (GBM) biopsies were characterized by an elevated EZH2 and Myc expression, possessing a strong positive correlation between the aforementioned markers in the presence of HCMV. From GBM tissues, we isolated HCMV clinical strains that transformed HAs toward CEGBCs exhibiting upregulated EZH2 and Myc. Spheroids generated from CEGBCs possessed invasion potential and were sensitive to EZH2 inhibitor, ganciclovir, and temozolomide triple therapy. HCMV clinical strains transform HAs and fit with an HCMV-induced glioblastoma model of oncogenesis, and supports the tumorigenic properties of Myc and EZH2 which might be highly pertinent in the pathophysiology of astrocytic brain tumors and thereby paving the way for new therapeutic strategies.

Oncogenic and Stemness Signatures of the High-Risk HCMV Strains in Breast Cancer Progression.

BACKGROUND: Human cytomegalovirus (HCMV) oncomodulation, molecular mechanisms, and ability to support polyploid giant cancer cells (PGCCs) generation might underscore its contribution to oncogenesis, especially breast cancers. The heterogeneity of strains can be linked to distinct properties influencing the virus-transforming potential, cancer types induced, and patient's clinical outcomes. METHODS: We evaluated the transforming potential in vitro and assessed the acquired cellular phenotype, genetic and molecular features, and stimulation of stemness of HCMV strains, B544 and B693, isolated from EZH2HighMycHigh triple-negative breast cancer (TNBC) biopsies. Therapeutic response assessment after paclitaxel (PTX) and ganciclovir (GCV) treatment was conducted in addition to the molecular characterization of the tumor microenvironment (TME). FINDINGS: HCMV-B544 and B693 transformed human mammary epithelial cells (HMECs). We detected multinucleated and lipid droplet-filled PGCCs harboring HCMV. Colony formation was detected and Myc was overexpressed in CMV-Transformed-HMECs (CTH cells). CTH-B544 and B693 stimulated stemness and established an epithelial/mesenchymal hybrid state. HCMV-IE1 was detected in CTH long-term cultures indicating a sustained viral replication. Biopsy B693 unveiled a tumor signature predicting a poor prognosis. CTH-B544 cells were shown to be more sensitive to PTX/GCV therapy. CONCLUSION: The oncogenic and stemness signatures of HCMV strains accentuate the oncogenic potential of HCMV in breast cancer progression thereby leading the way for targeted therapies and innovative clinical interventions that will improve the overall survival of breast cancer patients.

Polyploid giant cancer cells, EZH2 and Myc upregulation in mammary epithelial cells infected with high-risk human cytomegalovirus.

BACKGROUND: Human cytomegalovirus (HCMV) infection has been actively implicated in complex neoplastic processes. Beyond oncomodulation, the molecular mechanisms that might underlie HCMV-induced oncogenesis are being extensively studied. Polycomb repressive complex 2 (PRC2) proteins, in particular enhancer of zeste homolog 2 (EZH2) are associated with cancer progression. Nevertheless, little is known about EZH2 activation in the context of HCMV infection and breast oncogenesis. METHODS: Herein, we identified EZH2 as a downstream target for HCMV-induced Myc upregulation upon acute and chronic infection with high-risk strains using a human mammary epithelial model. FINDINGS: We detected polyploidy and CMV-transformed HMECs (CTH) cells harboring HCMV and dynamically undergoing the giant cells cycle. Acquisition of embryonic stemness markers positively correlated with EZH2 and Myc expression. EZH2 inhibitors curtail sustained CTH cells' malignant phenotype. Besides harboring polyploid giant cancer cells (PGCCs), tumorigenic breast biopsies were characterized by an enhanced EZH2 and Myc expression, with a strong positive correlation between EZH2 and Myc expression, and between PGCC count and EZH2/Myc expression in the presence of HCMV. Further, we isolated two HCMV strains from EZH2HighMycHigh basal-like tumors which replicate in MRC5 cells and transform HMECs toward CTH cells after acute infection. INTERPRETATION: Our data establish a potential link between HCMV-induced Myc activation, the subsequent EZH2 upregulation, and polyploidy induction. These data support the proposed tumorigenesis properties of EZH2/Myc, and allow the isolation of two oncogenic HCMV strains from EZH2HighMycHigh basal breast tumors while identifying EZH2 as a potential therapeutic target in the management of breast cancer, particularly upon HCMV infection. FUNDING: This work was supported by grants from the University of Franche-Comté (UFC) (CR3300), the Région Franche-Comté (2021-Y-08292 and 2021-Y-08290) and the Ligue contre le Cancer (CR3304) to Georges Herbein. Zeina Nehme is a recipient of a doctoral scholarship from the municipality of Habbouch. Sandy Haidar Ahmad is recipient of a doctoral scholarship from Lebanese municipality. Ranim El Baba is a recipient of a doctoral scholarship from Hariri foundation for sustainable human development.

Identification of UL69 Gene and Protein in Cytomegalovirus-Transformed Human Mammary Epithelial Cells.

A growing body of evidence addressing the involvement of human cytomegalovirus (HCMV) in malignancies had directed attention to the oncomodulation paradigm. HCMV-DB infected human mammary epithelial cells (HMECs) in culture showed the emergence of clusters of rapidly proliferating, spheroid-shaped transformed cells named CTH (CMV-Transformed HMECs) cells. CTH cells assessment suggests a direct contribution of HCMV to oncogenesis, from key latent and lytic genes activating oncogenic pathways to fueling tumor evolution. We hypothesized that the presence of HCMV genome in CTH cells is of pivotal importance for determining its oncogenic potential. We previously reported the detection of a long non-coding (lnc) RNA4.9 gene in CTH cells. Therefore, we assessed here the presence of UL69 gene, located nearby and downstream of the lncRNA4.9 gene, in CTH cells. The HCMV UL69 gene in CTH cells was detected using polymerase chain reaction (PCR) and sequencing of UL69 gene was performed using Sanger method. The corresponding amino acid sequence was then blasted against the UL69 sequence derived from HCMV-DB genome using NCBI Protein BLAST tool. A 99% identity was present between the nucleotide sequence present in CTH cells and HCMV-DB genome. UL69 transcript was detected in RNA extracts of CTH cells, using a reverse transcription polymerase chain reaction (RT-PCR) assay, and pUL69 protein was identified in CTH lysates using western blotting. Ganciclovir-treated CTH cells showed a decrease in UL69 gene detection and cellular proliferation. In CTH cells, the knockdown of UL69 with siRNA was assessed by RT-qPCR and western blot to reveal the impact of pUL69 on HCMV replication and CTH cell proliferation. Finally, UL69 gene was detected in breast cancer biopsies. Our results indicate a close link between the UL69 gene detected in the HCMV-DB isolate used to infect HMECs, and the UL69 gene present in transformed CTH cells and tumor biopsies, further highlighting a direct role for HCMV in breast tumor development.

Polyploid giant cancer cells, stemness and epithelial-mesenchymal plasticity elicited by human cytomegalovirus.

A growing body of evidence is recognizing human cytomegalovirus (HCMV) as a potential oncogenic virus. We hereby provide the first experimental in vitro evidence for HCMV as a reprogramming vector, through the induction of dedifferentiation of mature human mammary epithelial cells (HMECs), generation of a polyploid giant cancer cell (PGCC) phenotype characterized by sustained growth of blastomere-like cells, in concordance with the acquisition of embryonic stem cells characteristics and epithelial-mesenchymal plasticity. HCMV presence parallels the succession of the observed cellular and molecular events potentially ensuing the transformation process. Correlation between PGCCs detection and HCMV presence in breast cancer tissue further validates our hypothesis in vivo. Our study indicates that some clinical HCMV strains conserve the potential to transform HMECs and fit with a "blastomere-like" model of oncogenesis, which may be relevant in the pathophysiology of breast cancer and other adenocarcinoma, especially of poor prognosis.

Resveratrol Inhibits HCoV-229E and SARS-CoV-2 Coronavirus Replication In Vitro.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in China at the end of 2019 causing a large global outbreak. As treatments are of the utmost importance, drug repurposing embodies a rich and rapid drug discovery landscape, where candidate drug compounds could be identified and optimized. To this end, we tested seven compounds for their ability to reduce replication of human coronavirus (HCoV)-229E, another member of the coronavirus family. Among these seven drugs tested, four of them, namely rapamycin, disulfiram, loperamide and valproic acid, were highly cytotoxic and did not warrant further testing. In contrast, we observed a reduction of the viral titer by 80% with resveratrol (50% effective concentration (EC50) = 4.6 µM) and lopinavir/ritonavir (EC50 = 8.8 µM) and by 60% with chloroquine (EC50 = 5 µM) with very limited cytotoxicity. Among these three drugs, resveratrol was less cytotoxic (cytotoxic concentration 50 (CC50) = 210 µM) than lopinavir/ritonavir (CC50 = 102 µM) and chloroquine (CC50 = 67 µM). Thus, among the seven drugs tested against HCoV-229E, resveratrol demonstrated the optimal antiviral response with low cytotoxicity with a selectivity index (SI) of 45.65. Similarly, among the three drugs with an anti-HCoV-229E activity, namely lopinavir/ritonavir, chloroquine and resveratrol, only the latter showed a reduction of the viral titer on SARS-CoV-2 with reduced cytotoxicity. This opens the door to further evaluation to fight Covid-19.

Distinct Oncogenic Transcriptomes in Human Mammary Epithelial Cells Infected With Cytomegalovirus.

Human cytomegalovirus is being recognized as a potential oncovirus beside its oncomodulation role. We previously isolated two clinical isolates, HCMV-DB (KT959235) and HCMV-BL (MW980585), which in primary human mammary epithelial cells promoted oncogenic molecular pathways, established anchorage-independent growth in vitro, and produced tumorigenicity in mice models, therefore named high-risk oncogenic strains. In contrast, other clinical HCMV strains such as HCMV-FS, KM, and SC did not trigger such traits, therefore named low-risk oncogenic strains. In this study, we compared high-risk oncogenic HCMV-DB and BL strains (high-risk) with low-risk oncogenic strains HCMV-FS, KM, and SC (low-risk) additionally to the prototypic HCMV-TB40/E, knowing that all strains infect HMECs in vitro. Numerous pro-oncogenic features including enhanced expression of oncogenes, cell survival, proliferation, and epithelial-mesenchymal transition genes were observed with HCMV-BL. In vitro, mammosphere formation was observed only in high-risk strains. HCMV-TB40/E showed an intermediate transcriptome landscape with limited mammosphere formation. Since we observed that Ki67 gene expression allows us to discriminate between high and low-risk HCMV strains in vitro, we further tested its expression in vivo. Among HCMV-positive breast cancer biopsies, we only detected high expression of the Ki67 gene in basal tumors which may correspond to the presence of high-risk HCMV strains within tumors. Altogether, the transcriptome of HMECs infected with HCMV clinical isolates displays an "oncogenic gradient" where high-risk strains specifically induce a prooncogenic environment which might participate in breast cancer development.