Overexpression of the telomerase holoenzyme induces EMT and tumorigenesis of HPV‐immortalized keratinocytes

Cervical cancer is the most frequent malignancy of the female genital tract and is associated with persistent infection of the uterine cervix with high‐risk human papillomaviruses (HPV). The two HPV oncoproteins, E6 and E7, cooperatively immortalize cervical cells and are essential but insufficient for inducing tumorigenicity. During the progression of HPV‐associated cervical dysplasia to carcinoma, the cellular telomerase reverse transcriptase (TERT) gene is activated and the TERC gene amplified. We questioned whether these increases in telomerase components might mediate the acquisition of the tumorigenic phenotype. We therefore transduced the TERT and TERC genes into E6/E7 immortalized keratinocytes that were anchorage‐dependent and nontumorigenic. The resultant cells showed a profound morphological change characteristic of epithelial‐mesenchymal transition as well as a corresponding increase in expression of vimentin, N‐cadherin, Zinc finger E‐Box binding homeobox 1, snail family transcriptional repressor 1 and matrix Metallopeptidase 2 and decrease in keratin and E‐cadherin. More important, the transduced cells were now anchorage‐independent and formed tumors in immunodeficient mice. Our findings indicate that overexpression of the telomerase holoenzyme in HPV‐immortalized cells is sufficient to induce the complete transformed phenotype.

Studies have shown that the expression of viral oncoprotein, HPV E6 and E7, in cells only leads to cellular immortalization. 7 but not cell transformation. Several potential cellular cofactors in cervical carcinogenesis have been identified, including human telomerase reverse transcriptase (hTERT), phosphorylated RAC-α serine/ threonine-protein kinase, wingless/integrated/B-catinen, extracellular signal-regulated kinases/mitogen-activated protein kinase, janus Kinase/signal transducer and activator of transcription, YIN-YANG-1, activator protein 1, nuclear factor kappa-light-chain-enhancer of activated B cells, C-X-C Motif Chemokine Ligand 12, and so forth. 8 The exact roles of these molecules in HPV-mediated cancer progression are still under investigation.
Telomerase, a ribonucleoprotein enzyme complex, extends repetitive telomeric DNA. The holoenzyme includes the catalytic subunit hTERT and the telomerase RNA component, TERC. 9 Although hTERT is usually silenced in almost all somatic cells, it is significantly expressed in almost all human cancers including cervical cancer. The details of the underlying mechanisms of hTERT activation in cervical cancer are still being elucidated, but they mainly include the changes in hTERT promoter's cis and trans elements, the chromatin structure, the mRNA product, and associated RNA regulatory proteins. hrHPV E6 interacts with host cell proteins E6AP, cMyc, HDAC, HAT, mSin3A, NFX1-91, NFX1-123, PABPCs, and mRNA splicing factors to activate hTERT and telomerase activity (Katzenellenbogen 10 ). Studies have suggested that the induction of hTERT by HPV supports cellular immortalization and oncogenesis.
During the progression of cervical cancer, hTERC is amplified. 11 Using fluorescence in situ hybridization, amplification of hTERC was detected in 63% of the CIN2 lesions and 76% of the CIN3 lesions. 12 In a more recent study, the amplification was detected in 31.21% of patients with CIN1 lesions, 53.03% of patients with CIN2/3 lesions, and 85.71% of patients with invasive cervical carcinoma (Liu et al. 13 ).
Whether the amplification of hTERC plays a role in cervical cancer remains unclear.
Telomerase plays a pivotal role in bypassing cellular senescence and maintaining telomere homeostasis, essential properties required for the sustenance and progression of cancer. However, recent investigations have uncovered extra-telomeric properties of telomerase that are independent of its role in telomere extension. 14,15,16 Epithelial-to-mesenchymal transition (EMT), the transdifferentiation of stationary epithelial cells to a mesenchymal, motile phenotype, is a crucial step in cancer progression to metastasis. 17,18 In 1994, it was first demonstrated that the EMT in HPV positive keratinocytes was associated with increased invasiveness. 19 20 It also has been demonstrated that the HPV16 E6/E7 genes cooperate with the activated form of ErbB-2 to induce downregulation of the E-cadherin/catenin complex as well a mesenchyme-like, anchorageindependent, tumorigenic phenotype. 21 In this study we explore the roles of hTERT and hTERC on the transforming activity in HPV-immortalized human epithelial cells.
We found the co-expression of hTERT/hTERT in a HPV16 E6E7 positive cells led to EMT changes and induced transformation. Given the critical role of hTERT/hTERC in human cell transformation, analysis of this important pathway may lead to a better understanding of the requirements for progression to the tumorigenic state in cervical cancer.

| Softagar assay
The growth of keratinocytes in soft agar was assayed as described previously. 24 In short, 1 ml of 0.3% agarose containing 400 cells was layered over 1 ml of 0.6% agarose in 35-mm dishes. A sterile 3% agarose stock solution was prepared in Dulbecco's phosphate buffered saline and diluted to the above concentrations by mixing with 3 + 1 medium. Cultures initially were overlaid with 0.5 ml of this medium and were given further additions as necessary to prevent desiccation for a period of 3−4 weeks. Plates were stained with 0.1% Crystal violet for 30 min, and de-stain using dH 2 O rinses. The images of assays were obtained using a scanner. The numbers of the colonies in softagar were counted using software ImageJ. 25

| Real-time quantitative polymerase chain reaction (RT-qPCR)
Total RNAs were isolated from cultured cells using the Total RNA Extraction Kit (Bio-Rad) according to the manufacturer's instructions.

| Western blot (WB)
Total proteins from cultured cells were isolated using RIPA buffer containing protease and phosphatase inhibitor (Thermo Scientific).

| Statistical analysis
Experimental data were expressed as Mean ± SEM (Standard Error of the Mean). Significance was evaluated by Student's t-test (for two groups). ***represents p＜0.001.

| The overexpression of hTERT/hTERC increased cell growth rate
Immortalized HFKs transduced with HPV16 E6 and E7, HFK/E6E7, was described previously. 22 To study the role of the overexpression of hTERT/hTERC, HFK/E6E7 cells were infected with vector retrovirus as a control or with retrovirus co-expressing hTERT and hTERC ( Figure 1A). Retrovirus-infected cells were selected in puromycin for 5 days, and designated HFK/E6E7 + vector and HFK/E6E7 + hTERT/hTERC, respectively. The overexpression of hTERT and hTERC were confirmed at the RNA level ( Figure 1B) and the overexpression of hTERT was also verified at the protein level ( Figure 1C). Immunofluorescence (IF) showed that hTERT was located in the nucleus of cells ( Figure 1D). HFK/E6E7 + hTERT/hTERC had a much higher growth rate at the average of 1.36 days/doubling, and the control had a growth rate at 3.40 days/doubling ( Figure 1E). . HFK/E6E7 cells were infected with vector retrovirus as a control or with retrovirus expressing hTERT and TERC. Cells were selected in puromycin, and designated HFK/E6E7 + vector and HFK/E6E7 + hTERT/ TERC, respectively. The overexpression of hTERT/TERC was confirmed at RNA level using Realtime PCR. Data were normalized to β-actin expression. The data were expressed as relative fold change using the ∆∆ 2 C t method. Bars indicate standard deviation. **represents p < 0.01, ***represents p < 0.001 between two indicated groups. (B) and at the protein level using Western Blots (C). The localization of hTERT was revealed by immunofluorescence (D). Cell growth curve was generated to represent growth rate by plotting population doubling over days in time (E). hTERT, human telomerase reverse transcriptase.

| Other hallmarks of EMT were apparent in the cells with overexpression of hTERT/hTERC
Epithelial cells which undergo EMT display a reduced expression of epithelial adherens junction components, such as the epithelial (E)cadherin, that are replaced with mesenchymal adhesion molecules, like neuronal (N)-cadherin. 27 These EMT changes were confirmed in HFK/E6E7 + hTERT/hTERC cells. Both RNA ( Figure 3A) and protein ( Figure 3C) levels of E-cadherin decreased dramatically in the cells. In contrast, the levels of N-cadherin increased ( Figures 3B,C).

Matrix metalloproteinases (MMPs) also play significant roles in
EMT. MMP-2 is a collagenase that represents the main proteolytic enzyme among MMPs, and is a major promoter of tumor cell invasion and metastasis through breaking down of the basement membrane. 29 The level of matrix Metallopeptidase 2 (MMP2) in HFK/E6E7 + hTERT/hTERC was increased by 9.2-fold compared with the vector control ( Figure 3B).
Since the growth rate of HFK/E6E7 + vector control was about only half of the one of HFK/E6E7 + hTERT/hTERC, we kept another set of soft agar assay to week 10. We did not observe any colony formation for the HFK/E6E7 + vector control during the extended period of incubation.
Furthermore, we conducted xenograft assays to test cell transformation. We injected a total of 10 immunodeficient mice with HFK/E6E7 + hTERT/hTERC or HFK/E6E7 + vector cells. A total of 10 mice injected with HFK/E6E7 + hTERT/hTERC developed measurable tumors as early as 7 days post injection (Figures 5A,B).  increased. This is in agreement with the notion that the aggressive clinical behavior of cervical cancer triggered by the abnormal and prolonged EMT occurring in its lesions. 33 It remains to be seen whether the induction of cellular transformation is a direct result of the hTERT/hTERC induced EMT.
MMPs play a crucial role in determining cell invasiveness. 34