Piwil2 is reactivated by HPV oncoproteins and initiates cell reprogramming via epigenetic regulation during cervical cancer tumorigenesis

Abstract

The human papillomavirus (HPV) oncoproteins E6 and E7 are risk factors that are primarily responsible for the initiation and progression of cervical cancer, and they play a key role in immortalization and transformation by reprogramming differentiating host epithelial cells. It is unclear how cervical epithelial cells transform into tumor-initiating cells (TICs). Here, we observed that the germ stem cell protein Piwil2 is expressed in pre-cancerous and malignant lesions of the cervix and cervical cancer cell lines with the exception of the non-HPV-infected C33a cell line. Knockdown of Piwil2 by shRNA led to a marked reduction in proliferation and colony formation, in vivo tumorigenicity, chemo-resistance, and the proportion of cancer stem-like cells. In contrast, Piwil2 overexpression induced malignant transformation of HaCaT cells and the acquisition of tumor-initiating capabilities. Gene-set enrichment analysis revealed embryonic stem cell (ESC) identity, malignant biological behavior, and specifically, activation targets of the cell reprogramming factors c-Myc, Klf4, Nanog, Oct4, and Sox2 in Piwil2-overexpressing HaCaT cells. We further confirmed that E6 and E7 reactivated Piwil2 and that E6 and E7 overexpression resulted in a similar gene-set enrichment pattern as Piwil2 overexpression in HaCaT cells. Moreover, Piwil2 overexpression or E6 and E7 activation induced H3K9 acetylation but reduced H3K9 trimethylation, which contributed to the epigenetic reprogramming and ESC signature maintenance, as predicted previously. Our study demonstrates that Piwil2, reactivated by the HPV oncoproteins E6 and E7, plays an essential role in the transformation of cervical epithelial cells to TICs via epigenetics-based cell reprogramming.

Keywords: HPV oncoprotein; Piwil2; cell reprogramming; cervical cancer; tumor initiating cell.

Figure 1. Characteristic Piwil2 expression in cervical cancer and its precursor stages

a. Piwil2 expression detected by IHC in CIN lesion and cervical cancer tissue. Scale bar, 100 μm. b. Immunoreactive score of Piwil2 staining in CIN lesions and cervical cancer tissue. c. Western blot analysis of Piwil2 expression in CIN lesions and cervical cancer tissue. GAPDH expression is presented as a loading control. d. Western blot analysis of Piwil2 expression in cervical cancer cell lines.

Figure 2. Piwil2 knockdown affects cervical cancer cell line proliferation, invasion, and in vivo tumorigenicity

a. HeLa, SiHa, and CaSki cells were stably transfected with control shRNA or Piwil2 shRNA, and cell viability was measured daily. b. Numbers of invading cells in clones stably transfected with control shRNA and Piwil2 shRNA. c. Equal amounts of lysates from cancer cell lines stably transfected with control shRNA or Piwil2 shRNA were separated by SDS-PAGE, and proteins were analyzed by western blotting with specific antibodies against Piwil2 and molecules regulating cell proliferation. d. Tumor growth over time was measured after the subcutaneous injection of 5×10⁶ of SiHa cells stably transfected with shPiwil2 control shRNA and Piwil2 shRNA. Tumor volume was monitored by caliper measurements twice weekly, and tumor weight was measured after sacrifice at the end of the experiment. The data are presented as the mean±SD. *P < 0.05 and ** P < 0.01 by Student's t-test.

Figure 3. Piwil2 overexpression induces HaCaT cell malignant transformation

a. HaCaT cells were transfected with lentivirus containing Piwil2 or lentiviral vector, and cell numbers were plotted daily. b.-c. Colony formation numbers and invading HaCaT cells infected with lentivirus containing Piwil2 or lentiviral vector. d. Proteins were analyzed by western blotting for c-Myc, E-cadherin, and molecules regulating cell proliferation and apoptosis. e.-f. The expression of EMT markers was determined by qRT-PCR and western blotting in HaCaT cells infected with lentivirus containing Piwil2 or lentiviral vector. The data are presented as the mean±SD. *P < 0.05 and ** P < 0.01 by Student's t-test.

Figure 4. Piwil2 initiates tumorigenicity of HaCaT cells

a. Approximately 5×10⁶ HaCaT cells transfected with Piwil2 or Vector were injected subcutaneously into the oxters of nude mice. Four weeks after injection, tumorigenesis in nude mice was observed. b. Tumor volume was monitored by caliper measurements twice a week, and tumor weight was measured after sacrifice at the end of the experiment. The data are presented as the mean±SD. ** P < 0.01 by Student's t-test.

Figure 5. Piwil2 facilitates the establishment of a cancer stem-like cell gene-set enrichment pattern

a. Gene-set enrichment in HaCaT cells transfected with lentivirus containing Piwil2 compared with cells transfected with lentiviral vector. Red, gene-set enrichment; black, no significant enrichment. b. GSEA analyses demonstrating the gene set expression and the relationship between target gene expression and factor occupancy. c.-d. The expression of 5 transcription factors associated with cell reprogramming was determined by qRT-PCR and western blotting in HaCaT cells transfected with lentivirus containing Piwil2 or lentiviral vector. The data are presented as the mean±SD. *P < 0.05 and ** P < 0.01 by Student's t-test.

Figure 6. The HR-HPV oncoproteins E6 and E7 reactivate Piwil2 expression in HaCaT cells

a.-c. qRT-PCR, RT-PCR and western blotting reveal upregulated expression of Piwil2 in HaCaT cells transfected with lentivirus containing complete HPV16 E6 and E7 sequences compared with those transfected with lentiviral vector. d. Gene-set enrichment pattern of HaCaT cells transfected with lentivirus containing complete HPV16 E6 and E7 sequences. Red, gene-set enrichment; black, no significant enrichment. e. GSEA analyses demonstrating the gene set expression and the relationship between target gene expression and factor occupancy. The data are presented as the mean±SD. *P < 0.05 and ** P < 0.01 by Student's t-test.

Figure 7. Piwil2 initiates cell reprogramming by regulating the balance between the acetylation and trimethylation of H3K9

a. Western blot showing significantly induced H3K9 acetylation but reduced H3K9 trimethylation in HaCaT cells transfected with Piwil2 or E6 and E7 compared with those only transfected with vector. b. Co-immunoprecipitation showing that Piwil2, either overexpressed or induced by E6 and E7, interacted with acetylated H3K9. c. EMT markers upregulated in SiHa cells exhibiting Piwil2 overexpression but downregulated in those cells in which Piwil2 was knocked down, as verified by qRT-PCR. d. The proportion of ALDH-, MSCA-1-, and ABCG2-positive cells, determined by FACS in cells with Piwil2 overexpression or knockdown. e. The LD50 dose of cisplatin, detected by CCK8 assay in cells with Piwil2 overexpression or knockdown. The data are presented as the mean±SD. *P < 0.05 and ** P < 0.01 by Student's t-test.

Figure 8. Model of cell reprogramming and tumor-initiating cell formation secondary to Piwil2 induction