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. 2023 Oct 15;15(20):4994.
doi: 10.3390/cancers15204994.

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

Fidaa Bouezzedine  1 , Ranim El Baba  1 , Sandy Haidar Ahmad  1 , Georges Herbein  1   2
Affiliations

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

Fidaa Bouezzedine et al. Cancers (Basel). .

Abstract

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.

Keywords: CIN; PGCCs; high-risk HCMV strains; human cytomegalovirus; oncogenesis; prostate cancer; stemness.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Replication of HCMV-DB and -BL strains in PEC cultures. (A) Time course of the viral titer in the supernatant of PECs infected with HCMV-DB and -BL as measured by IE1-qPCR. (B) Confocal microscopic images of IE1 staining in PEC-DB and -BL. UI PECs were used as controls; magnification ×63, scale bar 10 μm. Data are represented as mean ± SD of two independent experiments.
Figure 2
Figure 2
Chronic infection of PECs with the high-risk HCMV strains and detection of morphological heterogeneity in CTP cultures. (A) Microscopic images of distinct cellular morphologies of the giant cell cycle, including (a–f) budding, (g,h) blastomeres and blastocytes, (i–k) mesenchymal cells, (l) lipid droplet-filled cells, (m–r) filopodia; magnification ×100, scale bar 100 μm (for UI PECs) and ×200, scale bar 50 μm (for images a–r). Uninfected PECs were used as a control. (B) Confocal microscopic images of DAPI and phalloidine staining in CTP-DB and -BL cells. Uninfected PECs were used as a negative control; magnification ×63, scale bar 10 μm. Red arrows show the defined morphologies.
Figure 3
Figure 3
Assessing the proliferative potential and detecting polyploidy in CTP-DB and -BL cultures. (A) FACS staining of Ki67Ag in uninfected PECs as well as CTP-DB and -BL cells. (B) Microscopic images of polyploidy detected in CTP-DB and -BL cultures. Cobalt chloride (CoCl2)-treated PECs (300 μM) were used as a positive control, while uninfected PECs were used as a negative control. Magnification ×200, scale bar 50 μm. (C) Propidium iodide (PI) staining for polyploidy detection in CTP-DB and -BL cells by FACS. CoCl2-treated PECs were used as a positive control, and uninfected PECs were used as a negative control. Data are represented as mean ± SD of two independent experiments. * p-value ≤ 0.05. The red line shows the low percentage of P5 (>4 N). Red boxes emphasizes the high percentages of p5 (>4 N).
Figure 4
Figure 4
Transformation potential and phenotypic characterization of CTP-DB and -BL cells. (A) Colony formation in soft agar seeded with CTP-DB and -BL cells; uninfected PECs were used as a negative control. Formed colonies were observed under an inverted light microscope (magnification 200×, scale bar 50 µm). (B) FACS staining of Myc and EZH2 in uninfected PECs as well as CTP-DB and -BL cells. Data are represented as mean ± SD of two independent experiments. (C) Confocal microscopic images of Myc, EZH2, and DAPI staining in CTP-DB and -BL cells. Uninfected PECs were used as controls; magnification ×63, scale bar 10 μm. * p-value ≤ 0.05.
Figure 5
Figure 5
CTP-DB and -BL cells display a stemness phenotype. (A) Spontaneous spheroids were detected under an inverted light microscope in CTP-DB and -BL cultures. Magnification ×200, scale bar 50 μm. (B) Spheroids were observed in the chronically infected CTP-DB and -BL cell cultures in methylcellulose, scale bar 25 μm. (C) Confocal microscopic images of Nestin, Nanog, SOX2, and DAPI staining in CTP-DB and -BL cells. Uninfected PECs were used as controls; magnification ×63, scale bar 10 μm.
Figure 6
Figure 6
EMT in CTP-DB and -BL cultures. (A) Vimentin and E-cadherin expression by FACS in CTP-DB and -BL cells. Uninfected PECs were used as controls. Data are represented as mean ± SD of two independent experiments. (B) Confocal microscopic images of vimentin, E-cadherin, and DAPI staining in CTP-DB and -BL cells. Uninfected PECs were used as controls; magnification ×63, scale bar 10 μm. * p-value ≤ 0.05.
Figure 7
Figure 7
Sustained HCMV replication in CTP-DB and -BL cultures. (A) IE1 protein and gene expression in chronically infected CTP-DB and -BL cell cultures by FACS and qPCR, respectively. Uninfected PECs were used as a negative control. (B) Confocal microscopic images of IE1 and DAPI staining in CTP-DB and -BL cells. Uninfected PECs were used as controls; magnification ×63, scale bar 10 μm.
Figure 8
Figure 8
A schematic diagram illustrating giant cell cycling in CTP-DB and -BL cultures. Microscopic images of distinct cellular morphologies of the giant cell cycle; magnification ×200, scale bar 50 μm (initiation, self-renewal, and termination phases), and ×100, scale bar 100 μm (stability phase). Initiation, self-renewal, termination, and stability represent the four different stages of the giant cell cycle. Post-HCMV infection and via endoreplication, the 2 N PECs go into the initiation phase. Subsequently, polyploid cells (>4 N) and tetraploid cells (4 N) are produced in the self-renewal/dedifferentiation phase. During the termination/differentiation stage, the intermediate cells (2–4 N) will be generated from multinucleated or mononucleated giant cells through budding. Intermediate PECs enter the stability stage and are afterward replaced by diploid small PECs (2 N).
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